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Jiarui Zhang, Rui Zhao, Dongliang Xing, Jing Cao, Yan Guo, Liang Li, Yanan Sun, Linli Tian, Ming Liu Department of Otorhinolaryngology, Head and Neck Surgery, The Second Affiliated Hospital, Harbin Medical University, Harbin City, Heilongjiang Province 150086, People’s Republic of ChinaCorrespondence: Linli Tian; Ming LiuDepartment of Otorhinolaryngology, Head and Neck Surgery, The Second Affiliated Hospital, Harbin Medical University, No. 148 Baojian Road, Nangang District, Harbin City, Heilongjiang Province 150086, People’s Republic of ChinaEmail zt0675@163.com; qr9830@163.comBackground: Magnesium isoglycyrrhizinate (MI) was extracted from roots of the plant Glycyrrhiza glabra, which displays multiple pharmacological activities such as anti-inflammation, anti-apoptosis, and anti-tumor. Here, we aimed to investigate the effect of MI on the progression and epithelial–mesenchymal transition (EMT) of laryngeal cancer.Methods: Forty laryngeal cancer clinical samples were used. The role of MI in the proliferation of laryngeal cancer cells was assessed by MTT assay, Edu assay and colony formation assay. The function of MI in the migration and invasion of laryngeal cancer cells was tested by transwell assays. The effect of MI on apoptosis of laryngeal cancer cells was determined by cell apoptosis assay. The impact of MI on tumor growth in vivo was analyzed by tumorigenicity analysis using Balb/c nude mice. qPCR and Western blot analysis were performed to measure the expression levels of gene and protein, respectively.Results: We identified that EMT-related transcription factor Twist was significantly elevated in the laryngeal cancer tissues. The expression of Twist was also enhanced in the human laryngeal carcinoma HEP-2 cells compared with that in the primary laryngeal epithelial cells. The high expression of Twist was remarkably correlated with poor overall survival of patients with laryngeal cancer. Meanwhile, our data revealed that MI reduced cell proliferation, migration and invasion and enhanced apoptosis of laryngeal cancer cells in vitro. Moreover, MI decreased transcriptional activation and the expression levels of NF-κB and Twist, and alleviated EMT in vitro and in vivo. MI remarkably inhibited tumor growth and EMT of laryngeal cancer cells in vivo.Conclusion: MI restrains the progression of laryngeal cancer and induces an inhibitory effect on EMT in laryngeal cancer by modulating the NF-κB/Twist signaling. Our finding provides new insights into the mechanism by which MI inhibits laryngeal carcinoma development, enriching the understanding of the anti-tumor function of MI.Keywords: laryngeal cancer, progression, EMT, magnesium isoglycyrrhizinate, Twist, NF-κB signaling

Homeobox D gene cluster antisense growth-associated long noncoding RNA (HAGLR) functions as a crucial regulator in the progression and development of human cancers. We analyzed effects of HAGLR, microRNA (miR)-143-5p and lysosome-associated membrane glycoprotein (LAMP)3 on esophageal cancer (EC) and the related mechanisms. Microarray analysis was used to screen out EC-related genes and the regulation network among HAGLR, miR-143-5p, and LAMP3. The regulatory mechanisms of HAGLR and miR-143-5p in EC were analyzed following the treatment of miR-143-5p mimic, miR-143-5p inhibitor, HAGLR vector, or small interfering RNA against HAGLR in EC cells. The expression of N-cadherin, vimentin, Twist1, Snail1, and E-cadherin as well as the abilities of cell proliferation, invasion, and migration were measured. The effects of the HAGLR/miR-143-5p/LAMP3 axis were determined in vivo by assessing tumor formation in nude mice. The expression of HAGLR and LAMP3 was increased, whereas that of miR-143-5p was diminished in EC tissues and cells. HAGLR could competitively bind to miR-143-5p, and miR-143-5p targeted LAMP3. Down-regulated HAGLR or up-regulated miR-143-5p increased E-cadherin expression and significantly diminished expression of LAMP3, N-cadherin, vimentin, Twist1, and Snail1. Moreover, down-regulated HAGLR inhibited cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT), and tumor growth. Moreover, down-regulation of HAGLR inhibited LAMP3 expression by sponging miR-143-5p, thereby suppressing the progression of EC. Taken together, our results suggest HAGLR acts as a competing endogenous RNA of miR-143-5p to increase the expression of LAMP3, thus promoting EMT, proliferation, invasion, and migration in EC cells.-Yang, C., Shen, S., Zheng, X., Ye, K., Sun, Y., Lu, Y., Ge, H. Long noncoding RNA HAGLR acts as a microRNA-143-5p sponge to regulate epithelial-mesenchymal transition and metastatic potential in esophageal cancer by regulating LAMP3.

Liver injury is an important cause of serious liver disease. This study aims to explore the effects of miR-217 targeting NAT2 on hepatocyte proliferation, apoptosis, and autophagy following carbon tetrachloride (CCL4)-induced liver injury. Rat models of CCL4-induced liver injury were established. Healthy Wistar rats were randomized into the normal, blank, negative control (NC), microRNA-217 (miR-217) mimic, miR-217 inhibitor, small interfering RNA (siRNA)-N-acetyltransferase 2 (NAT2), and miR-217 inhibitor + siRNA-NAT2 groups. NAT2 activity was evaluated with reversed-phase high-performance liquid chromatographic method. Immunohistochemistry was used to detect NAT2 protein positive rate. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to examine expressions of miR-217, NAT2, Bcl-2, Bax, p35, LC3-II, Becline-1, and the ratio of caspase-3/cleaved caspase-3. Autophagy, proliferation, and cell cycle distribution were determined by electron microscope, CCK-8, and flow cytometry. NAT2 protein positive rate and miR-217, NAT2, Bcl-2, and p35 expressions were higher and Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3 lower in the normal group than the other six groups. Compared with the blank and NC groups, in the miR-217 mimic and siRNA-NAT2 groups, Bax, LC3-II, and Becline-1 expressions and the ratio of caspase-3/cleaved caspase-3, and hepatocyte apoptosis and autophagy increased, while NAT2, Bcl-2, and p35 expressions and hepatocyte proliferation decreased; opposite results were observed in the miR-217 inhibitor group. Collectively, miR-217 targeting NAT2 inhibits proliferation and promotes apoptosis and autophagy of hepatocytes in CCL4-induced liver injury.

Backgrounds/Aims: MicroRNAs (miRs) often contribute to the progression of non-small cell lung cancer (NSCLC) via regulation of mRNAs that are involved in lung homeostasis. We conducted a study aimed at exploring the roles of miR-183 in the proliferation, epithelial-mesenchymal transition (EMT), invasion and migration of human NSCLC cells via targeting MTA1. Methods: NSCLC and adjacent normal tissues were collected from 194 patients with NSCLC. Positive expression of MTA1 protein was detected by immunohistochemistry. The highest levels of expression of miR-183 were detected using RT-qPCR in SPC-A-1 cells, which were selected and assigned to the following groups: blank, negative control (NC), miR-183 mimic, miR-183 inhibitor, siRNA-MTA1, and miR-183 inhibitor + siRNA-MTA1. The expression of miR-183 and the mRNA and protein expression of MTA1, E-cadherin, Vimentin, Snail, PCNA, Bax and Bcl-2 in tissues and transfected cells were measured using RT-qPCR and western blot analysis. Cell proliferation, apoptosis, migration and invasion were evaluated by CCK-8, flow cytometry, scratch tests and Transwell assays. Tumor xenografts were conducted in nude mice to determine tumor growth. Results: SPC-A-1 cells with the highest levels of miR-183 expression were selected. Compared with adjacent normal tissues, the expression of miR-183 and the mRNA and protein expression of E-cadherin and Bax were decreased in NSCLC tissues, while mRNA and protein expression of MTA1, Vimentin, snail, PCNA and Bcl-2 were increased. MiR-183 was over-expressed in the miR-183 mimic group and under-expressed in the miR-183 inhibitor and miR-183 inhibitor + siRNA-MTA1 groups. In the miR-183 mimic and siRNA-MTA1 groups, the mRNA and protein expression of E-cadherin and Bax, as well as cell apoptosis, were enhanced, while the expression levels of MTA1, Vimentin, snail, PCNA and Bcl-2 mRNA and protein, cell proliferation, migration, invasion and tumor growth were reduced relative to the blank and NC groups. The miR-183 inhibitor group exhibited an opposite trend. Conclusion: Our study indicates that miR-183 down-regulates MTA1 to inhibit the proliferation, EMT, migration and invasion of human NSCLC cells.

Nan Li,1 Liang Yang,2 Xinna Deng,3 Yanan Sun4 1Department of Gynecology, Second Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of China; 2Department of Neurosurgery, Second Hospital of Hebei Medical University, Shijiazhuang, People’s Republic of China; 3Department of Oncology & Immunotherapy, Hebei General Hospital, Shijiazhuang, People’s Republic of China; 4Department of Obstetrics and Gynecology, Bethune International Peace Hospital of PLA, Shijiazhuang, People’s Republic of China Background: Ovarian cancer is one of the most fatal gynecologic malignancies, with most patients diagnosed at the late stage due to insidious onset and lack of early onset specific symptoms. Previous studies have implied that isoliquiritigenin (ILQ) is a promising chemopreventive agent against oral cancer. Aim: This study aimed to investigate effects of ILQ and elucidate the related mechanism. Materials and methods: Ovarian cancer cell lines, SKOV3 and OVCAR3, were treated with various concentrations of ILQ to detect the dose-dependent effects of ILQ and select the suitable concentration. CCK8 assay and clone formation efficiency assays were used to detect viability and proliferation. The cell migration, invasion, and apoptosis were evaluated by wound healing assays, transwell, and flow cytometry assays. The expression of apoptosis-related proteins (Caspase-3, Caspase3-p17, Bcl-2, Bax, and Bim) and related-signaling pathway proteins were also detected by Western blot. Results: It was observed that the treatment of ILQ inhibited the survival and proliferation of SKOV3 and OVCAR3 cells. ILQ treatment inhibited migration and invasion, and induced apoptosis in SKOV3 and OVCAR3 cells. Also, the ILQ treatment increased the Bax/Bcl-2 ratio in SKOV3 and OVCAR3 cells, suggesting that a mitochondrial apoptotic pathway was triggered. It was also observed that, after treated with ILQ, the phosphorylated form of Akt and mTOR decreased and the expression of GSK3β increased, while P70/S6K decreased. ILQ treatment also decreased the expression of Wnt3a and, therefore, caused the decrease of phosphorylated ERK. ILQ also suppressed the PI3K/Akt/mTOR pathway by reduced the expression level of p-Akt, p-mTOR, P70/S6K and Cyclin D1 in Ishikawa and ES-2 cells. Conclusion: The data suggested that ILQ inhibited viability, proliferation, and invasion, and induced apoptosis of SKOV3 and OVCAR3 cells through the PI3K/Akt/mTOR pathway. Together, the data revealed that ILQ treatment may be used as a novel strategy for ovarian cancer therapy. Keywords: isoliquiritigenin (ILQ), ovarian cancer, SKOV3, OVCAR3, apoptosis, PI3K/Akt/mTOR pathway

// Linli Tian 1 , Jiarui Zhang 1 , Xiuxia Ren 1 , Xinyu Liu 1 , Wei Gao 1 , Chen Zhang 1 , Yanan Sun 1 and Ming Liu 1 1 Department of Otorhinolaryngology, Head and Neck Surgery, The Second Affiliated Hospital, Harbin Medical University, Harbin, China, 150086 Correspondence to: Ming Liu, email: haerbinmingliu@21cn.com Keywords: laryngeal cancer, Hep-2/R, miR-26b, ATF2, cisplatin Received: July 10, 2017 Accepted: August 09, 2017 Published: September 08, 2017 ABSTRACT Cisplatin is a common used chemotherapeutic drug for the treatment of laryngeal cancer. However, drug-resistance is a major obstacle in platinum-based chemotherapy for laryngeal cancer. Recent studies have demonstrated that dysregulation of microRNAs (miRNAs) is responsible for chemoresistance in multiple cancers including laryngeal cancer, but the potential mechanisms are required to be explored. In the present study, we constantly exposed the laryngeal cancer cell line Hep-2 with cisplatin to establish a cisplatin-resistant laryngeal cancer cell model (Hep-2/R). We found that Hep-2/R cells exhibited obvious resistance to cisplatin compared to the Hep-2 cells. However, overexpression of miR-26b significantly decreased the half maximal inhibitory concentration (IC50) of cisplatin to Hep-2/R. Mechanically, miR-26b in Hep-2/R decreased the expression of ATF2, and thus inhibiting the phosphorylation of ATF2 and formation of cellular ATF2-c-Jun complex induced by cisplatin. As the results, Hep-2/R cells failed to overexpress the Bcl-xl which is a key anti-apoptotic protein under the cisplatin treatment. Therefore, overexpression of miR-26b was found to be able to promote mitochondrial apoptosis induced by cisplatin.

Decreased bone volume and strength with aging and enhanced risk of fractures are in part due to reduced number of bone-forming mesenchymal stem cells (MSCs) and cellular dysfunction. In a previous study, we found that osteogenic differentiation of the multipotent and omnipotent preosteoblasts are accompanied by the alterations of intracellular NAD metabolism in which nicotinamide phosphoribosyltransferase (Nampt) plays a regulatory role. The increased Nampt during osteoblast differentiation, the enzyme catalyzing NAD resynthesis from nicotinamide was noted. However, whether Nampt will also be able to affect osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BM-MSCs), it is still uncertain. Here we report the role of Nampt in regulating osteoblast differentiation in primary mouse BM-MSCs. We found that Nampt expression was progressively elevated during BM-MSCs osteogenic differentiation. The Nampt inhibitor FK866 or knock-down of Nampt in BM-MSCs led to declined osteoblastogenesis, including attenuated ALP activity, diminished matrix mineralization and down-regulated osteoblast specific marker genes. In addition, declined osteoblastogenesis by Nampt deficiency or addition of FK866 was related to lower intracellular NAD concentration and decreased Sirt1 activity. The present findings demonstrate that osteogenic differentiation in MSCs can be modulated by intracellular NAD metabolism, in which Nampt may serve as an applicable marker for the osteoblast determination.

Obstructive sleep apnoea-hypopnoea syndrome (OSAHS) is a condition causing apnea and hypopnea. LncRNA-ROR revealed properties in regulating hypoxia response. Our study explored the roles of ROR in CoCl2-induced hypoxia injury in HK-2 cells. HK-2 cells were treated with CoCl2 to induce hypoxia injury. Cell viability, cell apoptosis and apoptotic proteins were detected using CCK-8, flow cytometry and western blot, respectively. The alter expression of ROR and miR-145 was achieved through transfection. Moreover, the expressions of HIF-α and ERK and MAPK related factors were examined using western blot. We found that CoCl2 decreased cell viability and increased apoptosis as well as increased the expression of ROR. ROR overexpression increased cell viability, decreased cell apoptosis. ROR overexpression upregulated anti-apoptotic proteins Bcl-2 and decreased p53, Bax and cleaved-Caspase-3. ROR overexpression also increased the expression of HIF-α. On the opposite, ROR silence led to the opposite results as relative to ROR overexpression. ROR overexpression decreased expression of miR-145. Co-transfection with ROR overexpression and miR-145 impaired the promoting effects of ROR in CoCl2 treated cells. ROR increased phosphorylation of ERK while decreased phosphorylation of MAPK. In conclusion, lncRNA ROR alleviated CoCl2-induced hypoxia injury through regulation of miR-145 as well as modulating ERK and MAPK signalling. Highlights CoCl2 induces ROR upregulation; Overexpression of ROR reduces CoCl2-induced HK-2 cell injury; Silence of ROR promotes CoCl2-induced HK-2 cell injury; Overexpression of ROR decreases miR-145 expression; ROR overexpression modulates ERK and MAPK signalling pathways through regulation of miR-145.

MicroRNAs (miRNAs) are reported to be important regulators of cancer-related processes, and function either as oncogenes or as tumor-suppressor genes. It was found that miR-375 was downregulated in samples of laryngeal squamous cell carcinomas (LSCCs) as compared to the level noted in adjacent non-tumor tissues, and it was inversely correlated with T grade, lymph node metastases and clinical tumor stage. Overexpression of miR-375 led to a decreased protein level of Kruppel-like factor 4 (KLF4) and marked suppression of the proliferation and invasion, and induced apoptosis of LSCC cell line Hep-2 using Cell Counting Kit-8, Transwell chamber and cell cycle assays. In addition, we examined the influence of the upregulation of miR-206 alone and upregulation of both miR-375 and miR-206 on the expression of KLF4 and Hep-2 cell behavior. The results showed that compared with the function of miR-375 in tumor suppression by regulating KLF4, co-transfection of miR-375 and miR-206 exhibited a less effective inhibitory effect not only on tumor cell proliferation and invasion, but also on tumor cell apoptosis. Taken together, miR-375 is possibly a tumor suppressor in LSCC by regulating KLF4. In addition, simple overexpression of several miRNAs did not entail higher efficacy than a single miRNA, similar to co-transfecions of miR-375 and miR-206.

N-Myc downstream-regulated gene 2 (NDRG2) has recently revealed as a candidate tumor suppressor gene. To inhibit tumor growth and decrease morbidity of esophageal cancer (EC), this study aims to test the hypothesis that the upregulation of NDRG2 may suppress proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of EC cells by regulating the AKT/XIAP signaling pathway. Immunohistochemistry was conducted for the identification of NDRG2, protein kinase B (p-AKT), X-linked inhibitor of apoptosis protein (XIAP) in EC tissues. To identify the regulatory mechanism of NDRG2 on the AKT/XIAP signaling pathway and EMT in EC, over-expressed lentiviral vector and shRNA were applied for up-regulating and interfering NDRG2 expression, and a series of determinations on the biological behavior of EC cells were performed to validate this regulation action. The results of immunohistochemistry showed NDRG2 was lowly expressed in EC tissues while p-AKT and XIAP are highly expressed. Over-expression of NDRG2 suppresses the proteins related to AKT/XIAP signaling pathway and EMT. Besides, a series of determinations shows the proliferation, migration and invasion of TE-13 cells were suppressed by over-expressed NDRG2, while the cell cycle progression was blocked and cell apoptosis was promoted. And in vivo experiment also demonstrated NDRG2 could inhibit tumor growth. Our findings demonstrate over-expression of NDRG2 works as tumor suppressive role in EC through its effects on inhibition of cell migration, invasion, and EMT by inhibiting the AKT/XIAP signaling pathway.

Manganese oxide nanoparticles (MnO NPs) have been regarded as a new class of T1-positive contrast agents. The cytotoxicity of silica coated MnO NPs (MnO@SiO2 NPs) was investigated in human cervical carcinoma cells (HeLa) and mouse fibroblast cells (L929). The changes of cell viability, cell morphology, cellular oxidative stress, mitochondrial membrane potential and cell cycle induced by MnO@SiO2 NPs were evaluated. Compared to HeLa cells, L929 cells showed lower cell viability, more strongly response to oxidative stress and higher percentage in the G2/M phase of cell cycle. The appearance of sub-G1 peak, double staining with Annexin V-FITC/PI and the increase of Caspase-3 activity further confirmed apoptosis should be the major form of cell death. Moreover, the apoptotic pathway was clarified as follows. Firstly, reactive oxygen species (ROS) is generated induced by MnO@SiO2 NPs, then p53 is activated followed by an increase in the bax and a decrease in the bcl-2, ultimately leading to G2/M phase arrest, increasing the activity of caspase-3 and inducing apoptosis.

Background Present study aimed to better understand the potential apoptotic pathways that involved in docosahexaenoic acid (DHA)-induced apoptosis of prostate cancer cells. Methods Human prostate cancer DU145 cells were treated with different concentrations of fish oil, omega-3 PUFA (DHA, and Eicosapentaenoic acid, EPA), or omega-6 PUFA (Arachidonic acid, AA). Cell viability and apoptosis were evaluated by MTT assay and Hoechst staining. Pathway-focused gene expression profiling of DU145 cells was analyzed with the RT2 Profile PCR Array System. The results were verified by real time quantitative polymerase chain reaction (RT-qPCR). Results AA exposure showed no obvious effect on viability of DU145 cells. However, exposure with fish oil, EPA, or DHA for 24 h significantly affected cell viability. The growth inhibition of DHA was more pronounced than that of EPA and showed a time-dependent increase. DHA exposure caused typical apoptotic characteristics. Ten genes were more expressed, while 5 genes were less expressed following DHA exposure. RT-qPCR confirmed the time dependent effect of DHA on the expression of these differentially expressed genes. KEGG pathway analysis showed that DHA may induce the apoptosis of cancer cells preferentially through mediating P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Conclusion Our study demonstrated the beneficial action of DHA on human prostate carcinoma cell line DU145. The pro-apoptotic effect of DHA on DU145 cells may involve mediation various pathways, especially P53, MAPK, TNF, PI3K/AKT, and NF-κB signaling pathways. Molecular mechanisms of DHA on apoptosis of cancer cells still need to be further clarified. Electronic supplementary material The online version of this article (doi:10.1186/s12944-017-0442-5) contains supplementary material, which is available to authorized users.

Homeobox (HOX) transcript antisense RNA (HOTAIR) is a long intergenic noncoding RNA (lincRNA) that is significantly overexpressed in breast and hepatocellular cancers. It remains unclear, however, whether HOTAIR plays an oncogenic role in human laryngeal squamous cell cancer (LSCC). We therefore investigated the expression and functional role of HOTAIR in LSCC. HOTAIR levels were significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with poor histological grade or advanced clinical stage had higher HOTAIR expression. Log-rank test showed a significant association between high levels of HOTAIR and poor prognosis in LSCC patients. Multivariate Cox analysis suggested that HOTAIR is an independent prognostic factor of LSCC. siRNA-mediated knockdown of HOTAIR led to reduced invasion and increased apoptosis of Hep-2 cells in vitro and significantly reduced growth of LSCC xenograft tumors in mice. Moreover, PTEN methylation was significantly reduced in Hep-2 cells depleted of HOTAIR. Taken together, these results suggest that the oncogenic role of HOTAIR in LSCC is related to promotion of PTEN methylation. HOTAIR could serve as a marker for LSCC prognosis and a potential target for therapeutic intervention.

Background Various microRNAs (miRNAs) negatively modulate genes that are involved in cellular proliferation, differentiation, invasion, and apoptosis. In many types of cancer, the expression profiles of these miRNAs are altered. Recently, miR-101 was identified as a tumour suppressor and was found to be expressed at low levels in various types of tumours, including prostate, breast, endometrium, and bladder cancers. However, the function(s) of miR-101 in laryngeal carcinoma remain unknown. Methods The expression levels of miR-101 in laryngeal squamous cell carcinoma (LSCC) tissues and cells were detected by qPCR. Cell proliferation, migration, cell cycle, and apoptosis assay were applied to assess the function(s) of miR-101 in vitro. Nude mice subcutaneous tumour model was used to perform in vivo study. Moreover, we identified Cyclin-dependent kinase 8 (CDK8) as the target of miR-101 by a luciferase assay. The possible downstream effectors of CDK8 were investigated in Wnt/β-catenin signaling pathway. Changes of CDK8, β-catenin, and cyclin D1 protein levels were analyzed by western blotting and immunohistochemical staining. The prognostic effect of miR-101 was evaluated using the Kaplan–Meier method. Results Expression of miR-101 was down-regulated in the LSCC tissues compared with the adjacent normal tissues. Furthermore, downregulation of miR-101 correlated with T3–4 tumour grade, lymph node metastasis, and an advanced clinical stage in the LSCC patients examined (P

MiRNAs are small, noncoding RNA molecules that emerge as important regulators of cancer-related processes. The miR-21 microRNA is overexpressed in a wide variety of cancers and has been causally related to cellular proliferation and apoptosis. In this study, we found that miR-21 is overexpressed in Laryngeal squamous cell carcinoma (LSCC) and correlated with advanced stage. Inhibition of miR-21 by antisense oligonucleotides (ASO) led to decreased protein level of Ras and profound suppression of cell proliferation and invasion. Hep-2 cells exposed to miR-21 ASO exhibited cell cycle arrest at G1 phase and increased apoptosis. Furthermore, growth of LSCC xenograft tumours was significantly suppressed by repeated injection of ASO-miR-21 lentivirus and the Ras protein expression in LSCC xenograft tumours was also downregulate by ASO-miR-21. Taken together, our data suggest that miR-21 may play an oncogenic role in the cellular processes of LSCC and represent a novel target for effective therapies.

Dysregulation of a numerous microRNAs (miRNAs) has been implicated in laryngeal squamous cell carcinoma (LSCC). Among those miRNAs, miR‑21 and miR‑221 are co‑overexpressed and commonly target the phosphatase and tensin homolog protein (PTEN) that is located in the PTEN‑Akt signaling pathway. The present study investigated whether co‑inhibition of miR‑21 and miR‑221 induced synergistic apoptosis of human LSCC cells. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl‑transferase‑mediated deoxynucleotide triphosphate nick end labeling (TUNEL) assays were used to observe the potential effect of miR‑21 and miR‑221 on cell viability and apoptosis in cells co‑transfected with anti‑miRNA oligonucleotide (AMO)‑21 and AMO‑221. The protein expression levels of PTEN, Akt and p53 were determined by western blotting. The cellular abundance of 6 pro‑apoptotic miRNAs transcribed by p53 mediation, consisting of miR‑15a, miR‑16‑1, miR‑26a, miR‑34a, miR‑143 and miR‑203, was measured with using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). MTT results indicate that in vitro co‑transfection of AMO‑21 and AMO‑221 leads to a decline in cell viability, compared with the transfection of AMO alone. This result was verified by the detection of apoptosis using TUNEL assays. Co‑transfection of AMO‑21 and AMO‑221 resulted in a marked reduction in Akt phosphorylation and enhanced expression of PTEN and p53 were observed; consequently, leading to an amplification of the transcription of 6 pro‑apoptotic miRNAs. The present findings confirmed that co‑inhibition of miR‑21 and miR‑221 synergistically triggers cell apoptosis in vitro. The altered PTEN‑Akt signaling and p53‑mediated amplification of the transcription of pro‑apoptotic miRNAs may be involved in the observed synergistic effect. The present study provides novel insights into the mechanism underlying apoptosis‑associated miRNA‑miRNA mutual regulation in LSCC.

MicroRNAs (miRNAs) have been recognised to regulate cancer development and progression in carcinogenesis as either oncogenes or tumour suppressor genes. However, whether miR-203 plays a crucial role in human laryngeal squamous cell carcinoma (LSCC) remains largely unclear. In the study, we have found that miR-203 expression was significantly lower in LSCC tissues than that in corresponding adjacent non-neoplastic tissues and was negatively correlated with ASAP1 expression level. Lower expression of miR-203 was significantly related to poor differentiation, advanced clinical stages, T3–4 tumour grade, lymph node metastasis and decreased 5-year overall survival. Transfection with miR-203 inhibited proliferation, reduced invasion, induced apoptosis and caused G1 phase cell cycle arrest of Hep-2 cells in vitro, suggesting that miR-203 functioned as a tumour suppressor. We have also tested that over-expression of miR-203 may both suppress the growth of xenograft tumours in mice and downregulate the expressions of ASAP1 in vivo. Furthermore, miR-203 may regulate the expressions of mesenchymal transition (EMT) marker of E-cadherin and cancer stem cells (CSCs) marker of CD44. These findings suggest that miR-203 plays a role as a tumour suppressor in LSCC, likely by regulating ASAP1, probably in relation to EMT and CSCs and may serve as a potential target for therapeutic intervention.

MicroRNAs were reported to be involved in the modulation of tumor development. The aim of our study was to investigate the effect of miR-205 on proliferation and apoptosis of laryngeal squamous cell carcinoma (LSCC) and seek associations between miR-205 and Bcl-2 using in vitro and in vivo methods. Real-time qPCR was used to analyze the expression of miR-205 in LSCC samples and Hep-2 cell line. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to test the apoptosis and proliferation of LSCC cells after miR-205 transfection. Bcl-2 expression in cells was assessed with Western blotting. The tumorigenicity of LSCC cells was evaluated in nude mice model. MiR-205 was significantly down-regulated in LSCC tissues compared to adjacent normal tissues. Lower expression of miR-205 was indicated to be statistically related with advanced clinical stage and T3–4 grades. We found that restoration of miR-205 down-regulated the proliferative markers of dihydrofolate reductase and proliferating cell nuclear antigen and apoptotic regulator of Bcl-2. The findings in vitro and in vivo showed miR-205 could suppress cell proliferation and induce cell apoptosis. In addition, Bcl-2 was identified as one of the direct targets of miR-205 in LSCC cells. These results suggest that miR-205 may play as a tumor suppressor in LSCC, probably by targeting Bcl-2 and serve as a potential target for therapeutic intervention.

miRNAs regulate gene expression and are key mediators of tumourigenesis. miR-129 has diverse effects in tumours, but its role in laryngeal squamous cell carcinoma (LSCC) remains unknown. This article focuses on the role of miR-129-5p in LSCC. We show miR-129-5p is upregulated in primary LSCC tumours and correlated with advanced disease. Down-regulating miR-129-5p suppressed cell proliferation and migration, and caused cell cycle arrest in Hep-2 cell lines. Downregulation of miR-129-5p alone is sufficient to induce apoptosis both in vivo and in vitro. Moreover, the growth of LSCC xenograft exposed to miR-129-5p antisense oligonucleotides (ASO) in BALB/c mice was markedly inhibited. In addition, we found that miR-129-5p targeted adenomatous polyposis coli (APC) to release inhibition of Wnt signalling causing cell growth and tumourigenesis. Our results suggest miR-129-5p functions as an oncogene in LSCC by repressing APC and is a potential therapeutic target for LSCC.

The objective of this study was to investigate the role of alpha v-integrin subunit (ITGAV, CD51) in invasion and metastasis of the laryngeal and hypopharyngeal squamous cell carcinomas and to evaluate whether an antisense oligonucleotide sequence (ASONs) targeting ITGAV gene can result in proliferative inhibition and induce to apoptosis of laryngeal carcinoma cell lines (Hep-2). Firstly, a tissue microarray contained 75 primary carcinomas, 29 non-cancerous normal tissues and 20 metastatic lymph nodes was constructed and used to detect the expression of ITGAV by immunohistochemistry. The changes of ITGAV expression from each group were assessed and correlated to the clinical parameters of the patients. Secondly, the ASONs targeting ITGAV gene was transfected into Hep-2 cells in vitro. The proliferative ability of the cells after transfection was measured by MTT assay and the apoptosis was detected using flow cytometry. Results showed that the expression of ITGAV was significantly correlated with differentiation and lymph node metastasis of these cancers. In vitro test showed that the proliferative ability of Hep-2 cells was significantly inhibited by ASONs in a way of concentration- and time-depending mode, and a significant apoptosis of Hep-2 cells was also observed after ASONs transfection. In conclusion, the expression of ITGAV was significantly correlated with differentiation and metastasis of the laryngeal and hypopharyngeal carcinomas; down-regulation of ITGAV gene could inhibit proliferation of Hep-2 cells and induce to its apoptosis. These results suggest that ITGAV gene may become a promising prognostic marker and new treatment target for these cancers.

Objective: The purpose of this study was to explore the inhibiting role of adenoviral-mediated (Ad)-tissue factor pathway inhibitor (TFPI)-2 gene in the growth of laryngeal squamous cell carcinoma (LSCC). Study Design: The nude mice bearing LSCC were prepared by intracutaneous injection of Hep-2 cell. Gene therapy was performed by injecting adenoviruses carrying TFPI-2 gene around tumors in the animal model. Methods: Eighteen nude mice bearing Hep-2 cell tumor were randomly separated into the treated group and control group. The former were injected with recombinant adenovirus Ad-TFPI-2 around the tumor, and the later were injected with equivalent Ad-LacZ. After treatment, differences of tumor weight, volume, and ultrastructure of tumor cells between these two groups were observed by measuring and using transmission electron microscope. Apoptosis in Hep-2 xenotransplants was detected using the terminal deoxy-transferase-mediated dUTP nick end labeling. In addition, the expressions of TFPI-2 protein and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected using Western blot and immunohistochemistry, respectively. Results: The average weight and volume of tumor in the treated group were significantly lower than that in the control group (P < .01). The PCNA index was obviously lower in the tumors treated group than that in the control group (P < .01). In addition, cell apoptosis was observed in xenotransplants of the treated group but not in the control group. Conclusion: Peritumor injection of Ad-TFPI-2 can inhibit growth of LSCC in nude mice model, and TFPI-2 might be a desirable gene for gene therapy in LSCC.

CXCR4 and its ligand CXCL12 can promote the proliferation, survival, and invasion of cancer cells. They have been shown to play an important role in regulating metastasis of breast cancer to specific organs. High CXCR4 expression was also correlated to poor clinical outcome. Previous study also showed that tumor cells express a high level of CXCR4 and that tumor metastasis target tissues (lung, liver, and bone) express high levels of the ligand CXCL12, allowing tumor cells to directionally migrate to target organs via a CXCL12-CXCR4 chemotactic gradient. However, the exact mechanisms of how CXCR4 and CXCL12 enhance metastasis and/or tumor growth and their full implications on breast cancer progression are unknown. Yet it is likely that chemokine receptor signaling may provide more than just a migrational advantage by also helping the metastasized cells establish and survive in secondary environments. In this study, we investigated CXCR4 and CXCL12 expression in breast cancer and analyzed its association with clinicopathological factors by immunohistochemistry first. Then, we detected the mRNA and protein expression of CXCR4 and CXCL12 in breast cancer cell lines by Western blot and RT-PCR. The MDA-MB-231 has CXCR4 expression and very weak CXCL12 expression. So, we constructed the functional CXCL12 expression in MDA-MB-231 using a gene transfection technique. Further experiments were conducted to evaluate the effect of CXCL12 transfection on the biological behaviors of MDA-MB-231. The cell proliferation of MDA-MB-231–CXCL12 was accessed by MTT assay; the apoptosis was analyzed by an AnnexinV-FITC/propidium iodide double staining of flow cytometry method; and the cell invasive ability was examined by Matrigel invasion assay. Immunohistochemical analysis showed the co-expression of CXCR4 and CXCL12 correlated with lymph node metastasis and TNM stage (p

Background Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays key role in the progression of some human cancers. However, the role of NEAT1 in human laryngeal squamous cell cancer (LSCC) is still unknown. We therefore investigated the expression and function of NEAT1 in LSCC. Methods NEAT1 level in LSCC and adjacent non-neoplastic tissues were detected by qRT-PCR. NEAT1 was knockdown in LSCC cells and cell proliferation, apoptosis and cell cycle were examined. The growth of xenografts with NEAT1 knockdown LSCC cells was analyzed. Results NEAT1 level was significantly higher in LSCC than in corresponding adjacent non-neoplastic tissues, and patients with neck nodal metastasis or advanced clinical stage had higher NEAT1 expression. Moreover, siRNA mediated NEAT1 knockdown significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at G1 phase in LSCC cells. The growth of LSCC xenografts was significantly suppressed by the injection of NEAT1 siRNA lentivirus. Furthermore, NEAT1 regulated CDK6 expression in LSCC cells which was mediated by miR-107. Conclusion NEAT1 plays an oncogenic role in the tumorigenesis of LSCC and may serve as a potential target for therapeutic intervention.