Search

Study Design Animal and cellular models of spinal cord injury (SCI) were used to explore the role of miR-335 in regulating cell viability and apoptosis. Objective. To investigate the role and the target of miR-335 in SCI. Summary of Background. Based on analysis of the GSE19890 data set, miR-335 was identified as a downregulated microRNA (miRNAs) following SCI. Thus, this study investigated whether downregulation of miR-335 is important in the pathological process of SCI. Materials and Methods. The GSE19890 data set investigating the expression profiles of miRNAs after SCI was downloaded from the GEO database. GSE45006 and GSE4550 data sets were used to identify differentially expressed genes between normal samples and SCI samples. The targets of rno-miR-335 were predicted using the TargetScan database. An experimental model of SCI was established, and agomir-miR-335 was intrathecally injected into rats with SCI. Functional recovery was evaluated by assessment of Basso-Beattie-Bresnahan scores and spinal cord water content and performing hematoxylin-eosin staining. Apoptosis was estimated by TUNEL staining. The H2O2-treated PC12 cells were used as in vitro models of SCI. Cell viability and apoptosis were examined by cell counting kit-8 and flow cytometry. Caspase-3 expression was evaluated by immunofluorescence staining. Levels of miRNAs and mRNAs were measured by reverse transcriptase quantitative polymerase chain reaction. Western blotting was performed to measure Bcl-2, Bax, cleaved caspase-3, and specificity protein 1 (SP1) protein levels. Results. For in vivo studies, miR-335 was downregulated following SCI, and agomir-miR-335 delivery improved functional recovery through suppressing neuronal apoptosis by inactivating the SP1-Bax/Bcl-2/caspase-3 signaling. During in vitro analysis, miR-335 protected PC12 cells against H2O2-induced damage by negatively regulating the SP1-Bax/Bcl-2/caspase-3 signaling axis. Moreover, upregulation of SP1 abolished the apoptosis suppressive effects of miR-335 upregulation. Conclusion. MiR-335 ameliorates locomotor impairment in rats with SCI through the suppression of neuronal apoptosis by inactivating SP1-Bax/Bcl-2/caspase-3 signaling. [ABSTRACT FROM AUTHOR]

Abstract Background The implication of zinc finger protein 148 (ZNF‐148) in pathophysiology of most human cancers has been reported; however, the biological functions of ZNF‐148 in breast cancer remain unclear. This study sought to elucidate the potential molecular mechanism of ZNF‐148 on breast cancer pathology. Methods ZNF148 expression was tested in breast cancer tissues and cells. Then, cells were transfected with ZNF‐148 overexpression or downregulation vector, and the cell proliferation, pyroptosis, apoptosis, and reactive oxygen species (ROS) production were analyzed by MTT, western blot, flow cytometry, and immunofluorescence staining, respectively. Tumor‐bearing nude mouse was used to evaluate tumorigenesis of ZNF‐148. Mechanisms underpinning ZNF‐148 were examined using bioinformatics and luciferase assays. Results We found that ZNF‐148 was upregulated in breast cancer tissues and cell lines. Knockdown of ZNF‐148 suppressed malignant phenotypes, including cell proliferation, epithelial‐mesenchymal transition, and tumorigenesis in vitro and in vivo, while ZNF‐148 overexpression had the opposite effects. Further experiments showed that ZNF‐148 deficiency promoted ROS production and triggered both apoptotic and pyroptotic cell death, which were restored by cotreating cells with ROS scavengers. A luciferase reporter assay revealed that miR‐335 was the downstream target of ZNF‐148 and that overexpressed ZNF‐148 increased superoxide dismutase 2 (SOD2) expression by sponging miR‐335. In parallel, both miR‐335 downregulation and SOD2 overexpression abrogated the antitumor effects of ZNF‐148 deficiency on proliferation and pyroptosis in breast cancer cells. Conclusions Our findings indicated that ZNF‐148 promotes breast cancer progression by triggering miR‐335/SOD2/ROS‐mediated pyroptotic cell death and aid the identification of potential therapeutic targets for breast cancer.

Background: The implication of zinc finger protein 148 (ZNF‐148) in pathophysiology of most human cancers has been reported; however, the biological functions of ZNF‐148 in breast cancer remain unclear. This study sought to elucidate the potential molecular mechanism of ZNF‐148 on breast cancer pathology. Methods: ZNF148 expression was tested in breast cancer tissues and cells. Then, cells were transfected with ZNF‐148 overexpression or downregulation vector, and the cell proliferation, pyroptosis, apoptosis, and reactive oxygen species (ROS) production were analyzed by MTT, western blot, flow cytometry, and immunofluorescence staining, respectively. Tumor‐bearing nude mouse was used to evaluate tumorigenesis of ZNF‐148. Mechanisms underpinning ZNF‐148 were examined using bioinformatics and luciferase assays. Results: We found that ZNF‐148 was upregulated in breast cancer tissues and cell lines. Knockdown of ZNF‐148 suppressed malignant phenotypes, including cell proliferation, epithelial‐mesenchymal transition, and tumorigenesis in vitro and in vivo, while ZNF‐148 overexpression had the opposite effects. Further experiments showed that ZNF‐148 deficiency promoted ROS production and triggered both apoptotic and pyroptotic cell death, which were restored by cotreating cells with ROS scavengers. A luciferase reporter assay revealed that miR‐335 was the downstream target of ZNF‐148 and that overexpressed ZNF‐148 increased superoxide dismutase 2 (SOD2) expression by sponging miR‐335. In parallel, both miR‐335 downregulation and SOD2 overexpression abrogated the antitumor effects of ZNF‐148 deficiency on proliferation and pyroptosis in breast cancer cells. Conclusions: Our findings indicated that ZNF‐148 promotes breast cancer progression by triggering miR‐335/SOD2/ROS‐mediated pyroptotic cell death and aid the identification of potential therapeutic targets for breast cancer. [ABSTRACT FROM AUTHOR]

Aims: The rostral ventrolateral medulla (RVLM) is an essential vasomotor center responsible for regulating the development of stress‐induced hypertension (SIH). Long non‐coding RNAs (lncRNAs) play critical roles in various physiopathology processes, but existing research on the functions of RVLM lncRNAs on SIH has been lacking. In this study, we investigated the roles of RVLM lncRNAs in SIH. Methods: Genome‐wide lncRNA profiles in RVLM were determined by RNA sequencing in a SIH rat model established using electric foot shocks plus noises. The hypotensive effect of lncRNA INPP5F and the underlying mechanisms of lncRNA INPP5F on SIH were explored through in vivo and in vitro experiments, such as intra‐RVLM microinjection and immunofluorescence. Results: We discovered 10,179 lncRNA transcripts, among which the lncRNA INPP5F expression level was significantly decreased in SIH rats. Overexpression of lncRNA INPP5F in RVLM dramatically reduced the blood pressure, sympathetic nerve activity, and neuronal excitability of SIH rats. LncRNA INPP5F overexpression markedly increased Cttn expression and reduced neural apoptosis by activating the PI3K‐AKT pathway, and its inhibition had opposite effects. Mechanistically, lncRNA INPP5F acted as a sponge of miR‐335, which further regulated the Cttn expression. Conclusion: LncRNA INPP5F was a key factor that inhibited SIH progression, and the identified lncRNA INPP5F/miR‐335/Cttn/PI3K‐AKT/apoptosis axis represented one of the possible mechanisms. LncRNA INPP5F could serve as a therapeutic target for SIH. [ABSTRACT FROM AUTHOR]

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types. [ABSTRACT FROM AUTHOR]

Rostral ventrolateral medulla (RVLM) is thought to serve as a major vasomotor center that participates in controlling the progression of stress-induced hypertension (SIH). Circular RNAs (circRNAs) perform important functions in the regulation of diverse physiological and pathological processes. However, information concerning the functions of RVLM circRNAs on SIH remains limited. RNA sequencing was performed to profile circRNA expression in RVLMs from SIH rats, which were induced by electric foot shocks and noises. The functions of circRNA Galntl6 in reducing blood pressure (BP) and its potential molecular mechanisms on SIH were investigated via various experiments, such as Western blot and intra-RVLM microinjection. A total of 12,242 circRNA transcripts were identified, among which circRNA Galntl6 was dramatically downregulated in SIH rats. The upregulation of circRNA Galntl6 in RVLM effectively decreased the BP, sympathetic outflow, and neuronal excitability in SIH rats. Mechanistically, circRNA Galntl6 directly sponged microRNA-335 (miR-335) and restrained it to reduce oxidative stress. Reintroduction of miR-335 observably reversed the circRNA Galntl6-induced attenuation of oxidative stress. Furthermore, Lig3 can be a direct target of miR-335. MiR-335 inhibition substantially increased the expression of Lig3 and suppressed oxidative stress, and these favorable effects were blocked by Lig3 knockdown. CircRNA Galntl6 is a novel factor that impedes SIH development, and the circRNA Galntl6/miR-335/Lig3 axis represents one of the possible mechanisms. These findings demonstrated circRNA Galntl6 as a possibly useful target for the prevention of SIH.

MicroRNAs are small non-coding RNAs that control gene expression during development, physiology, and disease. Transcription is a key factor in microRNA abundance and tissue-specific expression. Many databases predict the location of microRNA transcription start sites and promoters. However, these candidate regions require functional validation. Here, dCas9 fused to transcriptional activators or repressors - CRISPR activation (CRISPRa) and inhibition (CRISPRi)- were targeted to the candidate promoters of two intronic microRNAs, mmu-miR-335 and hsa-miR-3662, including the promoters of their respective host genes Mest and HBS1L. We report that in mouse embryonic stem cells and brain organoids, miR-335 was downregulated upon CRISPRi of its host gene Mest. Reciprocally, CRISPRa of Mest promoter upregulated miR-335. By contrast, CRISPRa of the predicted miR-335-specific promoter (located in an intron of Mest) did not affect miR-335 levels. Thus, the expression of miR-335 only depends on the promoter activity of its host gene Mest. By contrast, miR-3662 was CRISPR activatable both by the promoter of its host gene HBS1L and an intronic sequence in HEK-293T cells. Thus, CRISPRa and CRISPRi are powerful tools to evaluate the relevance of endogenous regulatory sequences involved in microRNA transcription in defined cell types.

Abstract Background Corneal neovascularization (CRNV) is a severe threat to the vision of people. MicroRNA-335 (miR-335) has the function of facilitating angiogenesis. However, whether miR-335 regulates the progression of CRNV remains unclear. Methods The miR-335 expressions in CRNV rats induced by corneal suture and HUVECs induced by b-FGF were detected by quantitative real-time PCR. For the miR-335 function, wound healing and tube formation assays were performed. For the miR-335 mechanism, a dual-luciferase reporter gene assay was conducted. Besides, for the epidermal growth factor receptor (EGFR) function, Cell Counting Kit-8 and wound healing assays were performed. Meanwhile, the rescue assay was used to assess the miR-335/EGFR function in the migration and angiogenesis of b-FGF-treated HUVECs. Results Functionally, the miR-335 knockdown weakened the migration and angiogenesis of b-FGF-treated HUVECs, while the miR-335 overexpression showed an opposite trend. Mechanistically, miR-335 interacted with EGFR and negatively regulated the expression of EGFR. The rescue assay illustrated that miR-335 regulated the migration and angiogenesis of b-FGF-treated HUVECs through EGFR. Conclusions In general, our data confirmed that miR-335 facilitated the process of CRNV by targeting EGFR.

Abstract Lung adenocarcinoma (LAC) is one of the most common pulmonary adenocarcinomas with a high peak of mortality, and metastasis is the main culprit of LAC deaths. microRNAs play important role in cancer metastasis, and thus are regarded as potential diagnostic and prognostic markers for human cancers. However, many miRNAs exhibit dual roles in diverse cellular contexts. Here, we revealed that hsa‐miR‐335, a previously reported tumor suppressor, exhibited an oncogenic role in LAC. Overexpression of miR‐335 enhanced the abilities of A549 and H1299 cells to invade and migrate by regulating epithelial‐mesenchymal transition, while inhibition of miR‐335 exhibited an opposite effect in vitro and in vivo. Mechanically, miR‐335 inhibited the expression of Copine‐1 (CPNE1), an NF‐κB suppressor, through interacting with its mRNA 3′UTR, while mutating the binding sites abolished this inhibitory effect. This finding not only highlights the suppressive effect of CPNE1 on cell motility, but also provides new insight into miR‐335 in promoting LAC metastasis.

Dihydroartemisinin (DHA) is a traditional antimalarial drug. DHA plays a crucial role in preventing pulmonary hypertension (PH); however, its regulatory function on microRNAs (miRNAs) in PH remains unclear. This study aimed to investigate whether DHA exerts its protective functions by regulating miR-335 in PH. Hypoxia-induced PH models were induced both in vitro and in vivo. Mice were treated with various concentrations of DHA, and pulmonary arterial smooth muscle cells (PASMCs) were treated with DHA, miR-335 inhibitor, miR-335 mimic, or Van Gogh-like 2 (Vangl2) plasmid. The expression of miR-335 and Vangl2, pulmonary arterial remodeling index; right ventricular hypertrophy index; and proliferation and migration indexes were measured. DHA improved pulmonary vascular remodeling and alleviated PH in vivo. miRNA sequencing and real-time PCR results further show that the increase in hypoxia-induced miR-335 was avoided by DHA administration, and miR-335 increased the hypoxia-induced PASMC proliferation and migration. MiRNA databases and dual-luciferase reporter assay show that miR-335 directly targets Vangl2, and Vangl2 decreased the hypoxia-induced PASMC proliferation and migration. The miR-335 inhibitor failed to inhibit hypoxia-induced proliferation and migration upregulation in Vangl2 knockdown PASMCs, and the effect of DHA can be blocked by miR-335 upregulation. In hypoxic PH, MiR-335 is increased, whereas Vangl2 is decreased. MiR-335 can significantly promote the hypoxia-induced proliferation and migration of PASMCs by targeting the Vangl2 gene. DHA effectively reverses the hypoxia-induced upregulation of miR-335 expression, avoiding the miR-335-mediated downregulation of Vangl2 and thereby promoting the expression of Vangl2 to prevent PH. [ABSTRACT FROM AUTHOR]

Abstract Background X inactivation-specific transcript (XIST) is the long non-coding RNA (lncRNA) related to cancer, which is involved in the development and progression of various types of tumor. However, up to now, the exact role and molecular mechanism of XIST in the progression of ovarian cancer are not clear. We studied the function of XIST in ovarian cancer cells and clinical tumor specimens. Methods RT-qPCR was performed to detect the expression levels of miR-335 and BCL2L2 in ovarian cancer cells and tissues. MTT and transwell assays were carried out to detect cell proliferation, migration, and invasion abilities. Western blot was performed to analyze the expression level of BCL2L2. The interaction between miR-335 and XIST/BCL2L2 was confirmed using a luciferase reporter assay. Results The inhibition of XIST can inhibit the proliferation invasion and migration of human ovarian cancer cells. In addition, the miR-335/BCL2L2 axis was involved in the functions of XIST in ovarian cancer cells. These results suggested that XIST could regulate tumor proliferation and invasion and migration via modulating miR-335/BCL2L2. Conclusion XIST might be a carcinogenic lncRNA in ovarian cancer by regulating miR-335, and it can serve as a therapeutic target in human ovarian cancer.

Background: Corneal neovascularization (CRNV) is a severe threat to the vision of people. MicroRNA-335 (miR-335) has the function of facilitating angiogenesis. However, whether miR-335 regulates the progression of CRNV remains unclear.Methods: The miR-335 expressions in CRNV rats induced by corneal suture and HUVECs induced by b-FGF were detected by quantitative real-time PCR. For the miR-335 function, wound healing and tube formation assays were performed. For the miR-335 mechanism, a dual-luciferase reporter gene assay was conducted. Besides, for the epidermal growth factor receptor (EGFR) function, Cell Counting Kit-8 and wound healing assays were performed. Meanwhile, the rescue assay was used to assess the miR-335/EGFR function in the migration and angiogenesis of b-FGF-treated HUVECs.Results: Functionally, the miR-335 knockdown weakened the migration and angiogenesis of b-FGF-treated HUVECs, while the miR-335 overexpression showed an opposite trend. Mechanistically, miR-335 interacted with EGFR and negatively regulated the expression of EGFR. The rescue assay illustrated that miR-335 regulated the migration and angiogenesis of b-FGF-treated HUVECs through EGFR.Conclusions: In general, our data confirmed that miR-335 facilitated the process of CRNV by targeting EGFR. [ABSTRACT FROM AUTHOR]

The rostral ventrolateral medulla (RVLM) is known as the vasomotor center that plays a crucial role in mediating the development of stress‐induced hypertension (SIH). MicroRNAs (miRNAs) are involved in many different biological processes and diseases. However, studies that evaluated the roles of miRNAs in the RVLM during SIH do not exist. Here, we performed RNA sequencing to explore the genome‐wide miRNA profiles in RVLM in an SIH rat model established by administering electric foot‐shocks and noises. The function of miRNAs in blood pressure regulation was determined in vivo via the intra‐RVLM microinjection of the agomir or antagomir. Furthermore, the underlying mechanisms of miRNAs on SIH were investigated through in vitro and in vivo experiments, like gain‐of‐function. We discovered 786 miRNA transcripts among which 4 were differentially expressed. The over‐expression of miR‐335 and miR‐674‐3p in RVLM dramatically increased the heart rate (HR), arterial blood pressure (ABP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) levels of normotensive rats, whereas the knockdown of miR‐335 and miR‐674‐3p in RVLM markedly reduced the HR, ABP, SBP, DBP, and MAP levels of SIH rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that miR‐335 and miR‐674‐3p participated in regulating the development of SIH from different aspects, like apoptosis‐multiple species pathway. Sphk1, whose expression was markedly decreased in SIH, was identified as a novel target of miR‐335. MiR‐335 over‐expression substantially reduced the expression of Sphk1 and promoted neural apoptosis, and its inhibition had opposite effects. Re‐introduction of Sphk1 dramatically abrogated the apoptosis induced by miR‐335. This study provides the first systematic dissection of the RVLM miRNA landscape in SIH. MiR‐335 and miR‐674‐3p act as SIH promoters, and the identified miR‐335/Sphk1/apoptosis axis represents one of the possible mechanisms. These miRNAs can be exploited as potential targets for the molecular‐based therapy of SIH. [ABSTRACT FROM AUTHOR]

Early diagnosis of breast cancer decreases mortality rate; therefore, novel diagnostic methods are urgently required. In this study, authors aimed to investigate the role of serum-derived miR-335 in breast cancer, and the expression of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) and evaluating their feasibility and clinical utility as biomarkers for the early detection of breast cancer. Blood samples were collected from a total of 210 individuals who were enrolled in this study. The participants were divided into newly diagnosed breast cancer patients (n = 115), patients with benign breast lesions (n =55) and healthy individuals as control group (n =40). The expression profile of miR-335, MMP2 and MMP9 were determined using quantitative polymerase chain reaction (qPCR). MiR 335 expression level was down-regulated in primary breast cancer group as compared to benign breast group and healthy individuals with 98% and 94.9% sensitivity and specificity, respectively. MMP2 and MMP9 showed significantly higher expression levels in breast cancer group as compared to both benign and healthy group and reporting 92.7% and 93% sensitivity, respectively. The relations between investigated markers and pathologic types, staging, grading, and lymph node involvement were significant with these factors. Expression level of miR-335 was decreased with increased MMP2 and MMP9 at significant level. MiR-335, MMP2, and MMP9 can be used as diagnostic markers in breast cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect. Methods The BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss®) and then implanted into the femoral bone defect of athymic rats. Results mDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo. Conclusion mDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335.

Ischemic stroke (IS) has become a cerebrovascular disease which seriously threatens the elderly people. It has been reported that circRNAs participate in multiple diseases, including IS. However, the role of circHECTD1 in IS remains largely unknown. To mimic IS in vitro, human cerebral microvascular endothelial cells (HCMECs) were treated with oxygen glucose deprivation/reperfusion (OGD/R). Meanwhile, MCAO mouse model was established to detect the expression of circHECTD1 in IS. qRT-PCR and western blot were used to test gene and protein expressions, respectively. CCK-8 assay was used to investigate the cell viability. Moreover, cell migration and tube formation were assessed by transwell and tube formation assays. In addition, RIP and luciferase assay were performed to explore the association among circHECTD1, miR-335 and NOTCH2. CircHECTD1 was significantly upregulated in IS. OGD/R significantly induced EndoMT in HCMECs, while knockdown of circHECTD1 notably reversed this phenomenon. In addition, silencing of circHECTD1 remarkably reversed OGD/R-induced promotion of HCMEC tube formation and migration. Meanwhile, circHECTD1 upregulated the level of NOTCH2 through binding with miR-335. Furthermore, miR-335 inhibited the process of EndoMT in IS via targeting NOTCH2. In summary, circHECTD1 knockdown significantly alleviated EndoMT process in HCMECs via mediation of miR-335/NOTCH2 axis. Thus, circHECTD1 might act as a potential target against IS. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis is a hallmark of various heart diseases and ultimately leads to heart failure. Although long noncoding RNA (lncRNA) SNHG20 has been reported to play important roles in various cancers, its function in cardiac fibrosis remains unclear. The expression of SNHG20 and microRNA 335 (miR-335) in heart tissues of angiotensin II-induced mice and angiotensin II-stimulated mouse cardiomyocyte cell line HL-1 were detected by quantitative real-time PCR (qRT-PCR). Cell viability was evaluated by cell counting kit-8 assay. The expression of galectin-3, fibrosis-related proteins (fibronectin, collagen IaI, and a-SMA), and apoptosis-related proteins [cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP)] was detected by Western blotting. Bioinformatics prediction, luciferase reporter assay, and RNA pulldown assay were performed to determine the relationship between SNHG20 and miR-335 as well as miR-335 and Galectin-3. Gain- and loss-function assays were performed to determine the role of SNHG20/miR-335/Galectin-3 in cardiac fibrosis. SNHG20 was significantly upregulated and miR-335 was downregulated in heart tissues of angiotensin II-treated mice and angiotensin II-stimulated HL-1 cells. Downregulation of SNHG20 effectively enhanced cell viability and decreased cell size of HL-1 cells and the expression levels of fibrosis-related proteins (fibronectin, collagen IaI, and a-SMA) and apoptosis-related proteins (cleaved caspase-3 and cleaved PARP), which were induced by angiotensin II treatment. Furthermore, SNHG20 elevated the expression levels of Galectin-3 by directly regulating miR-335. Our study revealed that downregulation of SNHG20 improved angiotensin IIinduced cardiac fibrosis by targeting the miR-335/Galectin-3 axis, suggesting that SNHG20 is a therapeutic target for cardiac fibrosis and hypertrophy. [ABSTRACT FROM AUTHOR]

MiR-10b, miR-335, and miR-21 are classes of microRNAs (miRNAs) that are overexpressed in breast cancer. Thus, in our study we aimed to test the hypothesis that miRNAs may have direct interactions with proteins and the possibility to inhibit/activate the functional site of proteins and enzymes. For this purpose, we choose three miRNAs involved in breast cancer to study interactions between some proteins and genes, including BRCA1 and PTEN, by processing the docking and matching tools using the Hex8 and HADDOCK server. Mathematically, the hidden Markov models were created by using MATLAB script according to the algorithm in order to study and validate the interactions and bonds between proteins and miRNAs. The main results demonstrate the ability of miR-10b, miR-335, and miR-21 to create direct interactions with 3D protein structures. Furthermore, these results may lead to another pathway of research, i.e. the direct interaction between proteins and their sub-units, to highlight the data obtained previously and demonstrate that proteins may directly interact with ncRNA instead of mRNA. Moreover, our study suggests developing research on different pathways of association proteins-miRNAs as a part of epigenetic extra-nuclear regulation. Taken together, our study provides the first evidence of direct interactions between miRNAs and proteins.

Abstract Background Metastasis is a leading cause of breast cancer mortality. The induction of epithelial-to-mesenchymal transition (EMT) and complex oncogenic signaling is a vital step in the evolution of highly metastatic and therapeutically-intractable breast cancer; necessitating novel target discovery or development of therapeutics that target metastatic breast cells (MBCs). Methods To achieve this, this study employs a combination of in silico bioinformatics analyses, protein and transcript analyses, drug sensitivity assays, functional assays and animal studies. Results The present study identified CDH11 as an inductor and/or facilitator of metastatic signaling, and biomarker of poor prognosis in MBCs. Furthermore, we showed that in the presence of CDH11-rich cancer-associated fibroblasts (CAFs), MCF7 and MDA-MB-231 MBC cell lines acquired enhanced metastatic phenotype with increased CDH11, β-catenin, vimentin, and fibronectin (FN) expression. We also demonstrated, for the first time to the best of our knowledge that exposure to anti-CDH11 antibody suppresses metastasis, reduces CDH11, FN and β-catenin expression, and abrogate the cancer stem cell (CSC)-like traits of MBC cells. Interestingly, ectopic expression of miR-335 suppressed CDH11, β-catenin and vimentin expression, in concert with attenuated metastatic and CSC potentials of the MBC cells; conversely, inhibition of miR-335 resulted in increased metastatic potential. Finally, corroborating the in silica and in vitro findings, in vivo assays showed that the administration of anti-CDH11 antibody or miR-335 mimic suppressed tumorigenesis and inhibited cancer metastasis. Conclusions These findings validate our hypotheses that miR-335 mediates anti-CDH11 antibody therapy response and that an enhanced miR-335/CDH11 ratio elicits marked suppression of the MBC CSC-like and metastatic phenotypes, thus revealing a therapeutically-exploitable inverse correlation between CDH11-enhanced CSC-like and metastatic phenotype and miR-335 expression in MBCs. Thus, we highlight the therapeutic promise of humanized anti-CDH11 antibodies or miR-335-mimic, making a case for their clinical application as efficacious therapeutic option in patients with MBC.

Parkinson’s disease (PD) is mainly driven by dopaminergic neuronal degeneration in the substantia nigra pars compacta accompanied by chronic neuroinflammation. Despite being mainly sporadic, approximately 10% of all cases are defined as heritable forms of PD, with mutations in the leucine-rich repeat kinase (LRRK2) gene being the most frequent known cause of familial PD. MicroRNAs (miRNAs or miRs), including miR-335, are frequently deregulated in neurodegenerative diseases, such as PD. Here, we aimed to dissect the protective role of miR-335 during inflammation and/or neurodegenerative events in experimental models of PD. Our results showed that miR-335 is significantly downregulated in different PD-mimicking conditions, including BV2 microglia cells stimulated with lipopolysaccharide (LPS) and/or overexpressing wild-type LRRK2. Importantly, these results were confirmed in serum of mice injected with 1-methyl-1-4-phenyl-1,2,3,6-tetrahydripyridine hydrochloride (MPTP), and further validated in patients with idiopathic PD (iPD) and those harboring mutations in LRRK2 (LRRK2-PD), thus corroborating potential clinical relevance. Mechanistically, miR-335 directly targeted LRRK2 mRNA. In the BV2 and N9 microglia cell lines, miR-335 strongly counteracted LPS-induced proinflammatory gene expression, and downregulated receptor interacting protein 1 (RIP1) and RIP3, two important players of necroptotic and inflammatory signaling pathways. Further, miR-335 inhibited LPS-mediated ERK1/2 activation. LRRK2-Wt-induced proinflammatory gene expression was also significantly reduced by miR-335 overexpression. Finally, in SH-SY5Y neuroblastoma cells, miR-335 decreased the expression of pro-inflammatory genes triggered by α-synuclein. In conclusion, we revealed novel roles for miR-335 in both microglia and neuronal cells that strongly halt the effects of classical inflammatory stimuli or LRRK2-Wt overexpression, thus attenuating chronic neuroinflammation.

Background: X inactivation-specific transcript (XIST) is the long non-coding RNA (lncRNA) related to cancer, which is involved in the development and progression of various types of tumor. However, up to now, the exact role and molecular mechanism of XIST in the progression of ovarian cancer are not clear. We studied the function of XIST in ovarian cancer cells and clinical tumor specimens. Methods: RT-qPCR was performed to detect the expression levels of miR-335 and BCL2L2 in ovarian cancer cells and tissues. MTT and transwell assays were carried out to detect cell proliferation, migration, and invasion abilities. Western blot was performed to analyze the expression level of BCL2L2. The interaction between miR-335 and XIST/BCL2L2 was confirmed using a luciferase reporter assay. Results: The inhibition of XIST can inhibit the proliferation invasion and migration of human ovarian cancer cells. In addition, the miR-335/BCL2L2 axis was involved in the functions of XIST in ovarian cancer cells. These results suggested that XIST could regulate tumor proliferation and invasion and migration via modulating miR-335/BCL2L2. Conclusion: XIST might be a carcinogenic lncRNA in ovarian cancer by regulating miR-335, and it can serve as a therapeutic target in human ovarian cancer. [ABSTRACT FROM AUTHOR]

Background: Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect. Methods: The BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss®) and then implanted into the femoral bone defect of athymic rats. Results: mDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo. Conclusion: mDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335. [ABSTRACT FROM AUTHOR]

Identifying patient’s cellular radiosensitivity before radiotherapy (RT) in breast cancer (BC) patients allows proper alternations in routinely used treatment programs and reduces the adverse side effects in exposed patients. This study was conducted on blood samples taken from 60 women diagnosed with Invasive Ductal Carcinoma (IDC) BC (mean age: 47±9.93) and 30 healthy women (mean age: 44.43±6.7). The standard G2 assay was performed to predict cellular radiosensitivity. To investigate miR-22 and miR-335 expression levels in peripheral blood mononuclear cells (PBMCs), qPCR was performed. The sensitivity and specificity of the mentioned miRNAs were assessed by plotting the Receiver Operating Characteristic (ROC) curve. Binary logistic regression was performed to identify the miRNA involvement in BC and cellular radiosensitivity (CR) of BC patients. The frequency of spontaneous and radiation-induced chromatid breaks (CBs) was significantly different between control and patient groups (p<0.05). A cut-off value was determined to differentiate the patients with and without cellular radiosensitivity. miR-22 and miR-335 were significantly downregulated in BC patients. miRNAs expression levels were directly associated with CR. ROC curve assessment identified that both miRNAs had acceptable specificity and sensitivity in the prediction of BC and CR of BC patients. Binary logistic regression showed that both miRNAs could also predict BC successfully. Although only miR-22 was shown potent to predict CR of BC patients, both miR-22 and miR-335 might act as tumor suppressor miRNAs in BC. miR-22 and miR-335 may be promising potential biomarkers in BC prediction along with other important biomarkers. Moreover, mirR-22 might be a potential biomarker for the prediction of CR in BC patients. [ABSTRACT FROM AUTHOR]

The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS.

Recent development of integrative therapy against melanoma combines surgery, radiotherapy, targeted therapy, and immunotherapy; however, the clinical outcomes of advanced stage and recurrent melanoma are poor. As a skin cancer, melanoma is generally resistant to radiotherapy. Hence, there is an urgent need for evaluation of the mechanisms of radioresistance. The present study identified miR-335 as one of the differential expression of miRNAs in recurrent melanoma biopsies post-radiotherapy. The expression of miR-335 declined in melanoma tissues compared to the adjacent tissues. Moreover, miR-335 expression correlated with advanced stages of melanoma negatively. Consistent with the prediction of STARBASE and miRDB database, miR-335 targeted ROCK1 via binding with 3′-UTR of ROCK1 directly, resulting in attenuation of proliferation, migration, and radioresistance of melanoma cells. The authors validated that overexpression of miR-335 enhanced X-ray-induced tumor regression by B16 mouse models. Briefly, the present findings gained insights into miR-335/ROCK1-mediated radiosensitivity and provided a promising therapeutic strategy for improving radiotherapy against melanoma.

The modification of methyltransferase-like (METTL) enzymes plays important roles in various cellular responses by regulating microRNA expression. However, how m6A modification is involved in stress granule (SG) formation in the early stage of acute ischemic stroke by affecting the biogenesis processing of microRNAs remains unclear. Here, we established a middle cerebral artery occlusion (MCAO) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in primary cortical neurons and PC12 cells to explore the potential mechanism between m6A modification and SG formation. The in vivo results showed that the level of infarction and apoptosis increased while SG formation decreased significantly within the ischemic cortex with improved reperfusion time after 2 h of ischemia. Consistent with the in vivo data, an inverse association between the apoptosis level and SG formation was observed in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Both in vivo and in vitro results showed that the expression of METTL3 protein, m6A and miR-335 was significantly decreased with the reperfusion period. Overexpression of the METTL3 and METTL3 gene-knockdown in PC12 cells were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. Overexpression or knockdown of METTL3 in oxygen-glucose deprivation of PC12 cells resulted in functional maturation of miR-335, SG formation and apoptosis levels. In addition, we found that miR-335 enhanced SG formation through degradation of the mRNA of the eukaryotic translation termination factor (Erf1). In conclusion, we found that METTL3-mediated m6A methylation increases the maturation of miR-335, which promotes SG formation and reduces the apoptosis level of injury neurons and cells, and provides a potential therapeutic strategy for AIS. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) has high morbidity and mortality, but current therapeutic methods cannot effectively improve patient's prognosis. FOXD3-AS1, a new identified long noncoding RNA, is dysregulated in several cancers and functions as a carcinogenic or tumor-suppressor factor. However, the function of FOXD3-AS1 in HCC has not been reported. Materials and Methods: Quantitative real time-polymerase chain reaction was applied to evaluate the expression of FOXD3-AS1 in HCC tissues and cell lines. miRDB and TargetScan websites were utilized to predict the interaction network of FOXD3-AS1 as a competing endogenous RNA. The interaction was confirmed by luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay. The effect of FOXD3-AS1 on HCC cells (Huh6) were measured by cell counting kit (CCK)-8, BrdU cell proliferation assay, Transwell invasion assay, and wound healing assay. Results: FOXD3-AS1 was overexpressed in HCC, and HCC patients with the high level of FOXD3-AS1 had a poor prognosis. In addition, FOXD3-AS1 knockdown considerably inhibited the proliferation, migration, and invasion of Huh6 cells. Besides, FOXD3-AS1 functioned as a sponge of miR-335, and RICTOR was a direct target gene of miR-335. Furthermore, FOXD3-AS1 could enhance the level of RICTOR through sponging miR-335. Moreover, the knockdown of FOXD3-AS1 could competitively bind with miR-335 to suppress RICTOR expression, thereby inhibiting the growth of Huh6 cells through the deactivation of AKT signaling pathway. Conclusions: FOXD3-AS1 is crucial for the tumorigenesis and progression of HCC. The interaction among FOXD3-AS1, miR-335, and RICTOR provides a novel insight for understanding the molecular mechanism of HCC, and FOXD3-AS1, miR-335, and RICTOR can be regarded as the potential targets for HCC treatment. [ABSTRACT FROM AUTHOR]

An association was found between reduced expression of miR-34a, miR-146a with both metastasis to regional lymph nodes (relative risk RR=10.50 and RR=5.25, respectively) and the development of distant metastases (RR=9.50 and RR=4, 40, respectively) in gastric cancer. They are excellent classifiers: AUC>0.9 for both miRNAs. The association of miR-335 expression with metastasis to the lymph nodes is much weaker, but it is also a good classifier for identifying a group with distant metastasis (RR=5.90). A correlation was found between the expression of miR-34a and miR-146a during metastasis, which is absent in non-metastatic tumors. Thus, miR-34a, miR-146a, and miR-335 miRNAs can be proposed as candidates for biomarkers of the risk of gastric cancer metastasis. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNAs (lncRNAs) are known as key regulators in many cancer types, but their biological functions in nasopharyngeal carcinoma (NPC) remain largely unknown. In the present study, we aim to explore the role of the lncRNA ZNRD1-AS1 in NPC tumor development. Methods: The role of ZNRD1-AS1 in NPC tissues and cells was explored by using quantitative real-time PCR assay. Cellular behavioral experiments were used in testing NPC cell proliferation, invasion, and migration. Luciferase reporter assay, RNA-binding protein immunoprecipitation, and Western blot analysis were used in estimating the associations among ZNRD1-AS1, miR-335, and ROCK1. Results: ZNRD1-AS1 expression was elevated in the NPC tissues and cells, and ZNRD1-AS1 overexpression was positively correlated with advanced TNM stage and the presence of lymph node metastasis. Our biological experiments indicated that ZNRD1-AS1 knockdown reduces NPC cell invasion and metastasis. Further analyses revealed that ZNRD1-AS1 as a ceRNA promotes the migration and invasion of NPC cells by sponging miR-335. We provided evidence that ZNRD1-AS1 facilitates the invasion and metastasis of NPC cells via the miR-335–ROCK1 axis. Conclusion: Our data shed light on the oncogenic role of ZNRD1-AS1 in NPC tumor development, and a promising therapeutic target for NPC was identified. [ABSTRACT FROM AUTHOR]

Decreased miR-335 has been reported in a variety of cancers. We previously showed that miR-335 played an important role in ovarian cancer metastasis and prognosis. However, miR-335 is down-regulated in ovarian cancer by mechanisms that remain unclear. In silico analysis identified putative transcription factor specificity protein 1 (SP1) transcription factor binding sites in the miR-335 promoter. To investigate the relation between SP1 and miR-335, qRT-PCR was performed. Our results showed both Sp1 knockdown and mithramycin A increased miR-335 expression in ovarian cancer cell lines. Luciferase reporter assays indicated that Sp1 knockdown increased miR-335 transcriptional activity. ChIP experiments showed that Sp1 bound directly to miR-335 promoter. Moreover, transwell migration and wound-healing assays showed that Sp1 knockdown resulted in inhibited cell migration, which was in turn mitigated by miR-335 inhibitor. We propose that miR-335 was negatively regulated by SP1, which in turn contributes to miR-335 deregulation and tumor cells migration. • Mir-335 is negatively regulated by Sp1. • Sp1 promotes ovarian cancer migration via down-regulating miR-335. • Knockdown of Sp1 reduces mRNA expression of DNMTs in EOC cells. [ABSTRACT FROM AUTHOR]

Recent development of integrative therapy against melanoma combines surgery, radiotherapy, targeted therapy, and immunotherapy; however, the clinical outcomes of advanced stage and recurrent melanoma are poor. As a skin cancer, melanoma is generally resistant to radiotherapy. Hence, there is an urgent need for evaluation of the mechanisms of radioresistance. The present study identified miR-335 as one of the differential expression of miRNAs in recurrent melanoma biopsies post-radiotherapy. The expression of miR-335 declined in melanoma tissues compared to the adjacent tissues. Moreover, miR-335 expression correlated with advanced stages of melanoma negatively. Consistent with the prediction of STARBASE and miRDB database, miR-335 targeted ROCK1 via binding with 3′-UTR of ROCK1 directly, resulting in attenuation of proliferation, migration, and radioresistance of melanoma cells. The authors validated that overexpression of miR-335 enhanced X-ray-induced tumor regression by B16 mouse models. Briefly, the present findings gained insights into miR-335/ROCK1-mediated radiosensitivity and provided a promising therapeutic strategy for improving radiotherapy against melanoma. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Cancer Biotherapy is the property of Editorial Office of Chinese Journal of Cancer Biotherapy and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Cervical cancer (CC) ranks as the second most common malignancy in women, accounting for more two 2 million deaths every year in the world. Recently, circular RNAs (circRNAs) have been reported to regulate the progression of multiple human tumors; however, whether it involves in CC remains largely elusive. Materials and Methods: Two GEO circRNA expression profiles (GSE102686, GSE113696) were downloaded to analyze the differentially expressed circRNAs using bioinformatics methods. Expression of circ_103973, miR-335 and PPP6C in CC tissues and cell lines were examined by qRT-PCR. Cell apoptosis was assessed with PI/Annexin-V double staining followed by the analysis of flow cytometry. Cell proliferation was evaluated by MTT and colony formation assays. Interaction between circ_103973 and miR-335, as well as miR-335 and PPP6C, were verified by dual-luciferase reporter assay. Results: Circ_103973 was found to be highly expressed in both GSE102686 and GSE113696 datasets as well as in CC tissue samples and cell lines. Higher levels of circ_103973 were correlated to a worse outcome of CC patients. Knockdown of circ_103973 significantly promoted CC cell apoptosis and inhibited CC cell proliferation in vitro. Mechanistically, we demonstrated that circ_103973 served as a sponge of miR-335, which directly targeted PPP6C in CC cells. miR-335 was found to be decreased in CC, while PPP6C was found to be increased in CC. Moreover, anti-miR-335 could reverse the inhibitory effects of circ_103973 knockdown on CC cell proliferation, and this phenomenon could be blocked by si-PPP6C. Conclusion: Circ_103973 promoted CC cell proliferation in vitro by physically binding miR-335, which further targeted and regulated PPP6C. [ABSTRACT FROM AUTHOR]

Abstract Background Multiple aberrant microRNA expression has been reported in gastric cancer. Among them, microRNA-335-5p (miR-335), a microRNA regulated by DNA methylation, has been reported to possess both tumor suppressor and tumor promoter activities. Results Herein, we show that miR-335 levels are reduced in gastric cancer and significantly associate with lymph node metastasis, depth of tumor invasion, and ultimately poor patient survival in a cohort of Amerindian/Hispanic patients. In two gastric cancer cell lines AGS and, Hs 746T the exogenous miR-335 decreases migration, invasion, viability, and anchorage-independent cell growth capacities. Performing a PCR array on cells transfected with miR-335, 19 (30.6%) out of 62 genes involved in metastasis and tumor invasion showed decreased transcription levels. Network enrichment analysis narrowed these genes to nine (PLAUR, CDH11, COL4A2, CTGF, CTSK, MMP7, PDGFA, TIMP1, and TIMP2). Elevated levels of PLAUR, a validated target gene, and CDH11 were confirmed in tumors with low expression of miR-335. The 3′UTR of CDH11 was identified to be directly targeted by miR-335. Downregulation of miR-335 was also demonstrated in plasma samples from gastric cancer patients and inversely correlated with DNA methylation of promoter region (Z = 1.96, p = 0.029). DNA methylation, evaluated by methylation-specific PCR assay, was found in plasma from 23 (56.1%) out of 41 gastric cancer patients but in only 9 (30%) out of 30 healthy donors (p = 0.029, Pearson’s correlation). Taken in consideration, our results of the association with depth of invasion, lymph node metastasis, and poor prognosis together with functional assays on cell migration, invasion, and tumorigenicity are in accordance with the downregulation of miR-335 in gastric cancer. Conclusions Comprehensive evaluation of metastasis and invasion pathway identified a subset of associated genes and confirmed PLAUR and CDH11, both targets of miR-335, to be overexpressed in gastric cancer tissues. DNA methylation of miR-335 may be a promissory strategy for non-invasive approach to gastric cancer.

Multiple myeloma (MM) is a common hematological malignancy and remains incurable. MiRNA-335 is a classic tumor suppressor, yet its expression pattern and biological role in MM is unclear. The aim of the present study was to determine the expression pattern, biological role, and mechanism of miR-335 in MM. In this study, we found that miR-335 expression was decreased in MM. The promoter of miR-335 was also hypermethylated in MM. It was found that over-expression of miR-335 or 5-azacytidine treatment suppressed migration of MM cells and down-regulated the expression of IGF-1R. MiR-335 thus acts as a metastatic suppressor by targeting IGF-1R in MM. Moreover, aberrant promoter hyper-methylation is critical for miR-335 silencing in MM. We also found that miR-335 assisted in predicting both the prognosis and progression of disease in MM patients. Observations might offer a new complementary diagnostic and therapeutic target in MM. [ABSTRACT FROM AUTHOR]

LncRNA LINC01510 (LINC01510) was a newly identified tumor-related lncRNA whose dysregulation and potential function have been reported in several tumors. However, the expression, clinical significances, and action mechanisms of LINC01510 in papillary thyroid carcinoma (PTC) are still unclear. In this study, we firstly reported that LINC01510 was highly expressed in both PTC tissues and cell lines. Additionally, we used dual-luciferase reporter assay and confirmed that SOX2 could bind directly to the LINC01510 promoter region, activating its transcription. Functional assays with a series of cell experiments indicated that knockdown of LINC01510 suppressed the proliferation, migration and invasion of SW1736 and TPC-1 cells. Moreover, down-regulation of LINC01510 resulted in accelerated apoptosis by promoting the expression of Caspase3/9. In particular, LINC01510 acted as an endogenous sponge by directly binding miR-335, resulting in the suppression of miR-335 expressions. Besides, we confirmed that SHH was a target of miR-335 and miR-335 over-expression distinctly reduced SHH expression in PTC cells. Finally, in the cytoplasm, we provided evidenced that LINC01510 acted as a sponge for miR-335, reducing its ability to promote SHH expression. In addition, the results of Western blot indicated that knockdown of LINC01510 inhibited the expression of SHH and GLI1, suggesting that Hedgehog pathway was suppressed. Taken together, our findings revealed that the newly identified LINC01510/miR-335/SHH axis could be a therapeutic target for PTC. • LINC01510 expression induced by SOX2 was upregulated in both papillary thyroid carcinoma tissues and cell lines. • Knockdown of LINC01510 suppressed the proliferation and metastasis of papillary thyroid carcinoma cells. • LINC01510 acted as a sponge for miR-335, modulating miR-335/SHH and Hedgehog pathway. [ABSTRACT FROM AUTHOR]

Growth arrest‐specific transcript 5 (GAS5), along non‐coding RNA (LncRNA), is highly expressed in hypoxia/reoxygenation (H/R)‐cardiomyocytes and promotes H/R‐induced apoptosis. In this study, we determined whether down‐regulation of GAS5 ameliorates myocardial ischaemia/reperfusion (I/R) injury and further explored its mechanism. GAS5 expression in cardiomyocytes and rats was knockdown by transfected or injected with GAS5‐specific small interfering RNA or adeno‐associated virus delivering small hairpin RNAs, respectively. The effects of GAS5 knockdown on myocardial I/R injury were detected by CCK‐8, myocardial enzyme test, flow cytometry, TTC and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. qRT‐PCR and luciferase reporter assay were carried out to analyse the relationship between GAS5 and miR‐335. The regulation of GAS5 on Rho‐associated protein kinase 1 (ROCK1) expression, the activation of PI3K/AKT/GSK‐3β pathway and mitochondrial permeability transition pore (mPTP) opening was further evaluated. The results indicated that GAS5 knockdown enhanced the viability, decreased apoptosis and reduced the levels of lactate dehydrogenase and creatine kinase‐MB in H/R‐treatment cardiomyocytes. Meanwhile, down‐regulation of GAS5 limited myocardial infarct size and reduced apoptosis in I/R‐heart. GAS5 was found to bind to miR‐335 and displayed a reciprocal inhibition between them. Furthermore, GAS5 knockdown repressed ROCK1 expression, activated PI3K/AKT, thereby leading to inhibition of GSK‐3β and mPTP opening. These suppressions were abrogated by miR‐335 inhibitor treatment. Taken together, our results demonstrated that down‐regulation of GAS5 ameliorates myocardial I/R injury via the miR‐335/ROCK1/AKT/GSK‐3β axis. Our findings suggested that GAS5 may be a new therapeutic target for the prevention of myocardial I/R injury. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Gallbladder carcinoma is a malignant tumor in the bile duct with poor prognosis. Although aberrant expression of miR-335 has been reported in the tumor tissues of gallbladder carcinoma, the biological role of miR-335 was still largely unknown. This study was intended to explore the role of miR-335 in the progression of gallbladder carcinoma. PATIENTS AND METHODS: The gallbladder carcinoma cell lines GBC-SD and SGC-996 were used in our study. MiR-335 mimic, miR-335 inhibitor, and si-myocyte enhancer factor 2D (MEF2D) were transfected into gallbladder carcinoma cells, respectively. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay analysis was used to determine cell viability. The colony formation was also analyzed. Cell cycle progression was determined using flow cytometer. To verify the target gene of miR-335, the luciferase assay was used. RESULTS: MiR-335 overexpression inhibited cell viability and colony formation of GBC-SD and SGC-996 cells. The percentage of cells in first gap phase (G1)/resting phase (G0) was significantly increased, and the expression of cell division cycle 2 (cdc2) and cell division cycle 25 (cdc25) was decreased after miR-335 was overexpressed, indicating its role in inducing the cell cycle arrest of GBC-SD and SGC-996 cells. MEF2D was up-regulated in gallbladder cancer and associated with tumor size and clinical stage. Down-regulation of MEF2D inhibited cell viability and colony formation, induced cell cycle arrest in G1/G0 phase, and decreased the expression of cdc2 and cdc25 in GBC-SD and SGC-996 cells. Bioinformatics analysis by TargetScan and luciferase assay verified that MEF2D could be targeted by miR-335. Importantly, the effects of miR-335 inhibitor on cell growth were rescued by small interfering RNA of MEF2D (siMEF2D) in GBC-SD and SGC-996 cells. Besides, miR-335 overexpression increased cell sensitivity to 5-Fluoracil (Fu) treatment and decreased the expression levels of ATP-binding cassette transporter B1 (ABCB1) and ATP-binding cassette G2 (ABCG2) in GBC-SD and SGC-996 cells. CONCLUSIONS: MiR-335 participates in the progression of gallbladder carcinoma by targeting MEF2D. MiR-335 may be a potential therapeutic target for gallbladder carcinoma. [ABSTRACT FROM AUTHOR]

MiR-10b, miR-335, and miR-21 are classes of microRNAs (miRNAs) that are overexpressed in breast cancer. Thus, in our study we aimed to test the hypothesis that miRNAs may have direct interactions with proteins and the possibility to inhibit/activate the functional site of proteins and enzymes. For this purpose, we choose three miRNAs involved in breast cancer to study interactions between some proteins and genes, including BRCA1 and PTEN, by processing the docking and matching tools using the Hex8 and HADDOCK server. Mathematically, the hidden Markov models were created by using MATLAB script according to the algorithm in order to study and validate the interactions and bonds between proteins and miRNAs. The main results demonstrate the ability of miR-10b, miR-335, and miR-21 to create direct interactions with 3D protein structures. Furthermore, these results may lead to another pathway of research, i.e. the direct interaction between proteins and their sub-units, to highlight the data obtained previously and demonstrate that proteins may directly interact with ncRNA instead of mRNA. Moreover, our study suggests developing research on different pathways of association proteins-miRNAs as a part of epigenetic extra-nuclear regulation. Taken together, our study provides the first evidence of direct interactions between miRNAs and proteins. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are expressed at high levels in the brain and are involved in various CNS diseases. However, the potential role of circRNAs in ischemic stroke-associated neuronal injury remains largely unknown. Here, we investigated the important functions of circRNA TLK1 (circTLK1) in this process. The levels of circTLK1 were significantly increased in brain tissues in a mouse model of focal cerebral ischemia and reperfusion. Knockdown of circTLK1 significantly decreased infarct volumes, attenuated neuronal injury, and improved neurological deficits. Furthermore, circTLK1 functioned as an endogenous miR-335-3p sponge to inhibit miR-335-3p activity, resulting in the increase of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase expression and a subsequent exacerbation of neuronal injury. Clinical studies confirmed increased levels of circTLKl in the plasma of patients with acute ischemic stroke (59 males and 12 females). Our findings reveal a detrimental role of circTLKl in ischemic brain injury. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are key regulators in the development and progression of osteosarcoma (OS). Based on our previous microarray data, we found that miR-335 expression was downregulated in OS tissue relative to normal tissue. Herein, we further found that miR-335 was downregulated in OS cell lines relative to the osteoblastic cell line hFOB 1.19. Further functional experiments found that upregulation of miR-335 suppressed the proliferation, invasion and migration of OS cells in vitro and tumor growth and lung metastasis in vivo. In addition, the expression of a total of 750 mRNAs was decreased upon the upregulation of miR-335, and SNIP1 was found to be the direct target of miR-335. Restoration of SNIP1 expression attenuated the suppressive effect of miR-335 on OS cells. Additionally, miR-335 suppressed the mRNA and protein expression of SNIP1, MMP-2, and MMP-7 in vitro and in vivo. MiR-335 also suppressed c-Myc and NFκb p65 in vitro and in vivo. In conclusion, our data suggest that miR-335 plays a significant role in the tumorigenesis and metastasis of OS and may serve as a therapeutic target for OS treatment. [ABSTRACT FROM AUTHOR]

OBJECTIVE: microRNAs (miRNAs) abnormal expression was proved to regulate the bladder cancer (BC) development. Here, we aimed to investigate the role of miR-335 played in BC. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to examine the miR-335 and CRKL (CT10 regulator of kinase-like protein) expression level in BC. Methyl thiazolyl tetrazolium (MTT) and RT-qPCR were used to examine cell viability of BC cells. Cell transwell assay was used to assess the migratory ability of BC cells. The direct target of miR-335 in BC was verified by luciferase reporter assay. RESULTS: The results showed that the expression of miR-335 and CRKL in normal and adjacent tissues showed no significant differences. Whereas, miR-335 expression in BC was signifi- cantly lower and CRKL expression was observably higher than normal. CRKL was verified as a specific target of miR-335 in BC cells and the relationship between CRKL and miR-335 expression was negatively correlated in BC tissues. Furthermore, CRKL siRNA group in BC cells remarkably inhibited cell proliferation and migration. MiR-335 mimic in BC cells remarkably curbed cell proliferation and migration and CRKL could reverse the proliferative and migratory ability of BC cells regulated by miR-335. CONCLUSIONS: miR-335 could suppress BC cell proliferation and migration by upregulating of CRKL. [ABSTRACT FROM AUTHOR]

At the nuclear periphery, the genome is anchored to A- and B-type nuclear lamins in the form of heterochromatic lamina-associated domains. A-type lamins also associate with chromatin in the nuclear interior, away from the peripheral nuclear lamina. This nucleoplasmic lamin A environment tends to be euchromatic, suggesting distinct roles of lamin A in the regulation of gene expression in peripheral and more central regions of the nucleus. The hot-spot lamin A R482W mutation causing familial partial lipodystrophy of Dunnigan-type (FPLD2), affects lamin A association with chromatin at the nuclear periphery and in the nuclear interior, and is associated with 3-dimensional (3D) rearrangements of chromatin. Here, we highlight features of nuclear lamin association with the genome at the nuclear periphery and in the nuclear interior. We address recent data showing a rewiring of such interactions in cells from FPLD2 patients, and in adipose progenitor and induced pluripotent stem cell models of FPLD2. We discuss associated epigenetic and genome conformation changes elicited by the lamin A R482W mutation at the gene level. The findings argue that the mutation adversely impacts both global and local genome architecture throughout the nucleus space. The results, together with emerging new computational modeling tools, mark the start of a new era in our understanding of the 3D genomics of laminopathies.

MicroRNAs (miRNAs) are small noncoding RNAs that exert their functions by targeting specific mRNA sequences. Many studies have demonstrated that miRNAs are crucial for cancer progression, during which they can act as either oncogenes or tumor suppressors. Previous research has shown that miR-335 is downregulated in breast cancer, and it has been shown to be a breast cancer suppressor. In addition, emerging evidence indicates that erythropoietin-producing hepatocellular A4 (EphA4) is implicated in cancer cell proliferation, migration, and invasion. However, little is known about the relationship between miR-335 and EphA4 in breast cancer. In the present study, we used bioinformatic and biochemical analyses to demonstrate that EphA4 is a direct downstream target of miR-335 in human breast cancer MCF-7 and MDA-MB-23 cells and revealed that miR-335 negatively regulates the expression of EphA4 in these cells. Further investigation revealed that miR-335 overexpression inhibits MCF-7 and MDA-MB-231 cell proliferation and that this inhibition is attenuated by EphA4 coexpression. Similarly, miR-335 overexpression also inhibited growth and downregulated EphA4 expression in tumors in nude mice. Moreover, our results demonstrated that miR-335 overexpression suppresses migration and invasion in MCF-7 and MDA-MB-231 cells, an effect that was reversed by EphA4 overexpression. These findings confirmed that EphA4 is a direct target gene of miR-335 and that miR-335 suppresses breast cancer cell proliferation and motility in part by directly inhibiting EphA4 expression. [ABSTRACT FROM AUTHOR]

Accumulating evidence has suggested that the dysregulation of miRNA is an important factor in the pathogenesis of lung cancer. Here, we demonstrate that miR‐335 expression is reduced in non‐small cell lung cancer (NSCLC) tumors relative to non‐cancerous adjacent tissues, while the expression of Tra2β is increased. In addition, clinical data revealed that the increased Tra2β and decreased miR‐335 expression observed in NSCLC cells was associated with poor patient survival rates. In vitro experimentation showed that the overexpression of miR‐335 inhibited the growth, invasion and migration capabilities of A459 lung cancer cells, by targeting Tra2β. In contrast, inhibition of miR‐335 or overexpression of the Tra2β target gene stimulated the growth, invasion and migratory capabilities of A459 lung cancer cells in vitro. Furthermore, overexpression of miR‐335 or inhibition of Tra2β decreased the phosphorylation of Rb‐S780 and Rb‐AKT. Overall, these findings suggest that the downregulation of miR‐335 in A459 lung cancer cells promoted cell proliferation through upregulation of Tra2β, mediated via activation of the AKT/mTOR signaling pathway, and suggest that miR‐335 may have potential as a novel therapeutic target for NSCLC. [ABSTRACT FROM AUTHOR]