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Pancreatic cell dynamics have important contributions to the development of type 2 diabetes and related diseases such as nonalcoholic fatty pancreas disease. The aim of this study was to investigate the effects of prolonged excessive glucose exposure on the functions of pancreatic beta cells and duct cells in single and co-culture conditions. In this study, we focused on the effects of glucotoxicity on insulin secretion which is the main function of beta cells and on progenitor functions of duct cells. Rat primary INS1 beta cells and ARIP duct cells were exposed to glucose (25 mM) for 72 h under single or indirect co-culture conditions. Glucotoxicity stimuli increased insulin secretion and decreased insulin expression in single beta cells while stimulating beta-cell differentiation and adipogenesis in single duct cells. On the other hand, glucotoxicity caused functional loss and increased proliferation and apoptosis in beta cells while increasing proliferation but suppressed beta-cell differentiation and adipogenesis in duct cells under co-culture conditions. The expression level of miR-335, a microRNA known to be upregulated by leptin and target Runx2, was measured. As a result, unlike single-cell culture, glucotoxicity upregulated miR-335, downregulated Runx2, and decreased insulin signaling in beta cells while downregulating miR-335 and upregulating Runx2, and decreased insulin signaling in duct cells under co-culture conditions. When the results of single and co-culture experiments are compared, insulin and miR-335 may be seen as important mediators for setting up the relation between beta and duct cells. Our findings are important for preventing the development of type 2 diabetes and nonalcoholic fatty pancreas disease, even developing new diagnosis and treatment strategies. [ABSTRACT FROM AUTHOR]

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]

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]

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]

MicroRNAs (miRNAs), a series of small noncoding RNAs that regulate gene expression at the post-transcriptional/translational level, are pivotal in cell differentiation, biological development, occurrence, and development of diseases, especially in cancers. Early studies have shown that miRNA-335 (miR-335) is widely dysregulated in human cancers and play critical roles in tumorigenesis and tumor progression. In this review, we aim to summarize the regulation of miR-335 expression mechanisms in cancers. We focus on the target genes regulated by miR-335 and its downstream signaling pathways involved in the biological effects of tumor growth, invasion, and metastasis both in vitro and in vivo, and analyze the relationships between miR-335 expression and the clinical characteristics of tumors as well as its effects on prognosis. The collected evidences support the potential use of miR-335 in prognosis and diagnosis as well as the therapeutic prospects of miR-335 in cancers. [ABSTRACT FROM AUTHOR]

miR-335 and miR-34a are two microRNAs (miRNAs) usually downregulated in gastric cancer (GC). But, their exact regulative roles were not fully elucidated. In this study, we studied the association between miR-335 and/or miR-34a expression and overall survival of GC patients and explored the regulative role of miR-335 and -34a over survivin expression and GC cell growth and invasion. Fifty patients with GC were regularly followed up from 2011 to 2015. miRNA microarray was used to examine the expression trend of miRNAs in eight tumor tissue samples and adjacent normal tissue samples. The possible binding site between miR-335 and survivin messenger RNA (mRNA) was predicted using online database and verified using qRT-PCR, Western blot, and dual luciferase assay. The regulative role of miR-335 and miR-34a over GC cell growth, apoptosis, and invasion was studied using Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and transwell assay, respectively. Among the GC patients, low miR-335 or miR-34a expression is associated with higher clinical stage and lymph node metastasis. Patients with low miR-335 or miR-34a had poor overall survival, while those with combined low miR-335 and miR-34a expression had even poorer overall survival. miR-335 can directly regulate survivin expression through binding to the 3′UTR, while miR-34a has indirect modulating effect. Both miR-335 and miR-34a could inhibit cell proliferation and invasion and enhance cell apoptosis. But, these effects are largely abrogated by overexpression of survivin without 3′UTR. Therefore, besides the targets identified in previous studies, miR-335 and miR-34a can also regulate GC cell growth, apoptosis, and invasion at least partly through survivin. [ABSTRACT FROM AUTHOR]

The involvement of miR-335 in csolorectal cancer (CRC) development remains controversial. Here, we found that miR-335 was highly up-regulated in CRC specimens relative to normal mucosa, and high miR-335 expression level was markedly associated with the tumour size and differentiation of CRC. The overexpression of miR-335 in CRC cells facilitated cell proliferation in vitro and tumour growth in vivo. RASA1 was validated as a target of miR-335 that was downregulation in CRC. Forced expression of miR-335 silenced RASA1 and triggered Ras/ERK cascade in CRC. Together, miR-335-RASA1 contributes to cell growth in CRC, and elucidation of downstream pathway will provide new insights into the molecular mechanisms of CRC progression. [ABSTRACT FROM AUTHOR]

Increasing evidence has demonstrated that small non-coding microRNAs (miRNAs) play important roles in cancer development and progression. Recent studies have shown that microRNA-335 (miR-335) functions as an oncogene or a tumor suppressor in various human cancer types, but its role in clear cell renal cell carcinoma (ccRCC) remains poorly understood. In our study, we firstly found that the expression level of miR-335 was significantly downregulated in ccRCC tissues versus corresponding non-tumor tissues and the low expression of miR-335 was significantly associated with lymph node metastasis, larger tumor size, and poor T stage. Then, we found that overexpression of miR-335 significantly suppressed the proliferation and invasion of 786-O and CaKi-1 ccRCC cell lines. We subsequently found that miR-335 could interact with the 3′-untranslated regions (3′UTR) of B-cell CLL/lymphoma 2 like 2 ( BCL- W or BCL2L2) messenger RNA (mRNA) and repress its expression. In addition, re-expression of BCL-W (without the 3′UTR) could partially abrogate the miR-335-induced 786-O and CaKi-1 ccRCC cell proliferation and invasion inhibition. Furthermore, we found that expression patterns of miR-335 were inversely correlated with those of BCL- W mRNA in ccRCC tissues. Taken together, these results indicate that miR-335 acts as a novel tumor suppressor to regulate ccRCC cell proliferation and invasion through downregulation of BCL-W expression. [ABSTRACT FROM AUTHOR]

Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to tumor progression. Previous miRNA microarray analysis illustrated that miR-335 is downregulated in various cancers; however, the role of miR-335 on hepatocellular carcinoma (HCC) has not been well elucidated. In this study, we investigated the biological functions and molecular mechanisms of miR-335 in human HCC in vitro, discussing whether it could be a therapeutic biomarker of HCC in the future. Four HCC cell lines and samples from 62 patients with HCC were analyzed for the expression of miR-335 by quantitative RT-PCR. Overexpression of miR-335 was established by transfecting mimics into HepG2 and HuH7 cells. Cell proliferation and cell migration were assessed by cell viability assay and transwell assay. Luciferase reporter assay and Western blot were to verify ROCK1 as a novel target gene of miR-335. We observed that miR-335 was downregulated in human HCC tissues and in all four HCC cell lines. The MTT assay revealed that overexpression of miR-335 subsequently inhibited cell growth. Furthermore, the transwell assay also showed significant cell migration inhibition in miR-335 transfectant. The expression of ROCK1 was decreased evidently after overexpression of miR-335, indicating that ROCK1 is a target gene for miR-335. Our data revealed that miR-335 could inhibit the proliferation and migration invasion of HCC cells via regulating ROCK1, suggesting that miR-335 could be a therapeutic biomarker of HCC in the future. [ABSTRACT FROM AUTHOR]

Metastasis is the leading cause of death in patients with breast cancer and aberrantly expressed microRNAs (miRNAs) are highly associated with this process. A previous study has shown that miR-335 is downregulated in breast cancer and can suppress tumor invasion and metastasis. Emerging evidences indicate that c-Met is implicated in cell scattering, migration, and invasion. However, little is known about the relationship between miR-335 expression and c-Met alteration in breast cancer. In the present study, we found that miR-335 expression was downregulated and c-Met protein expression was upregulated in two human breast cell lines. MiR-335 was found to negatively regulate c-Met protein level by directly targeting its 3′ untranslated region (UTR). Forced expression of miR-335 decreased c-Met expression at protein levels and consequently diminished hepatocyte growth factor (HGF)-induced phosphorylation of c-Met and subsequently inhibited HGF promotion of breast cancer cell migration in a c-Met-dependent manner. MiR-335 expression was increased after 5-aza-2′-deoxycytidine (5-AZA-CdR) treatment, and 5-AZA-CdR treatment resulted in the same phenotype as the effect of miR-335 overexpression. Taken together, these results demonstrate that miR-335 suppresses breast cancer cell migration by negatively regulating the HGF/c-Met pathway. [ABSTRACT FROM AUTHOR]

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation ( P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC. [ABSTRACT FROM AUTHOR]

Octamer-binding transcription factor 4 (OCT4) was closely related to pancreatic cancer progression, but its regulation in pancreatic cancer by microRNA (miRNA) is not fully clear. OCT4-positive and OCT4-negative pancreatic cells were isolated by flow cytometry, and it was found that OCT4-positive cells are enriched in transplanted pancreatic cancer cells compared with the primary ones and showed increasing proliferation and sphere formation. The data of miRNA array assay showed that miR-335 in OCT4-positive pancreatic cancer cells was lower than that in the negative ones. The results were confirmed in pancreatic cancer tissue and cell lines. Through expression analysis, it was found that miR-335 was underexpressed in OCT4(+) pancreatic cancer cells purified from primary tumors. Enforced expression of miR-335 in OCT4(+) pancreatic cancer cells inhibited clonogenic expansion and tumor development. miR-335 re-expression in OCT4(+) pancreatic cancer cells was blocked. Systemically delivered miR-335 inhibited pancreatic cancer metastasis and extended animal survival. Of significance, OCT4 was identified and validated as a direct and functional target of miR-335. Taken together, our results provide evidence that miR-335 might inhibit progression and stem cell properties of pancreatic cancer targeting OCT4. [ABSTRACT FROM AUTHOR]

MicroRNAs have been suggested to play a vital role in regulate tumor progression and invasion. However, the expression of miR-335 in colorectal cancer (CRC) and its clinical significance are not known. Here, we report that miR-335 is a tumor suppressor by regulating expression of ZEB2. In this study, we showed that downregulated miR-335 levels in highly invasive CRC cell lines and tissues. Kaplan-Meier survival analysis indicated that patients with reduced miR-335 had a poor overall survival. Furthermore, enhancing the expression of miR-335 inhibited CRC cell migration and invasion in vitro and lung and liver metastasis in vivo, while silencing its expression resulted in increased migration and invasion. Additionally, we identified a novel miR-335 target, ZEB2, and the direct interaction between them was verified by 3′-untranslated region dual-luciferase reporter assay. In conclusion, our results demonstrate that miR-335 functions as a tumor suppressor and play a role in inhibiting metastasis of CRC cells through targeting ZEB2. These findings suggest that miR-335 may be useful as a new potential therapeutic target for CRC. [ABSTRACT FROM AUTHOR]

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recently, miR-335 has been identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-335 in osteosarcoma is unclear at this point. In this study, we found that the expression of miR-335 in osteosarcoma tissues and cell lines was much lower than that in normal control, respectively, and the downregulated miR-335 was significantly associated with lymph-node metastasis. Transfection of miR-335 mimics could significantly inhibit the cell migration and invasion in MG-63 and U2OS osteosarcoma cell lines. Moreover, we also showed that ROCK1 was negatively regulated by miR-335 at the posttranscriptional level, via a specific target site within the 3′UTR by luciferase reporter assay. The expression of ROCK1 was inversely correlated with miR-335 expression in osteosarcoma tissues, and knockdown of ROCK1 by siRNA-inhibited osteosarcoma cells migration and invasion resembling that of miR-335 overexpression. Thus, our findings suggest that miR-335 acts as tumor suppressor by targeting the ROCK1 gene and inhibiting osteosarcoma cells migration and invasion. The findings of this study contribute to current understanding of the functions of miR-335 in osteosarcoma. [ABSTRACT FROM AUTHOR]

MicroRNAs are small non-coding RNAs that may also function as oncogenes and tumor-suppressor genes, as the abnormal expression of microRNAs is associated with various human tumors. However, the effect of miR-335 on the lung cancer cells remains unclear. The aim of the paper was to study the expression of miR335 in non-small cell lung cancer (NSCLC) and miR335's relation to the metastasis, invasion, and apoptosis in lung cancer cells A549 and H1299. qRT-PCR was used to identify the miR-335 expression. The effects of miR-335 on cell proliferation, apoptosis, and invasion were further analyzed. Luciferase reporter assay and Western blot were to verify Bcl-w and SP1 as potential major target genes of miR-335. Finally, the effect of Bcl-w on miR-335-induced cell survival was determined. Our results showed that miR-335 expression was significantly lower in NSCLC tissue, which was significantly associated with lymph node metastasis. In contrast to cells in blank and negative control groups, incidence of apoptosis was significantly higher ( P < 0.05) and the number of cells migrating through matrigel was significantly lower ( P < 0.05) in miR-335 mimics transfected group. Western blot and luciferase reporter assay demonstrated that miR-335 could bind to the putative binding sites in Bcl-w (or SP1) mRNA 3′-untranslated region to visibly lower the expression of Bcl-w (or SP1). The introduction of Bcl-w cDNA without 3′-untranslated region abrogated miR-335-induced cell survival. These results indicated that upregulation of miR-335 can simultaneously suppress the invasiveness and promote apoptosis of lung cancer cell A549 and H1299 by targeting Bcl-w and SP1. Therefore, miR-335 may be a potential therapeutic target in NSCLC treatment. [ABSTRACT FROM AUTHOR]