Yuwen Du

MiR-144 inhibits proliferation and induces apoptosis and autophagy in lung cancer cells by targeting TIGAR.

Background: MiRNAs are noncoding RNAs of 20-24 nucleotides that function as post-transcriptional negative regulators of gene expression. MiRNA genes are usually transcribed by RNA polymerase II in the nucleus. Their initial products are pre-miRNAs which have cap sequences and polyA tails. The p53-induced glycolysis and apoptosis regulator (TIGAR) was discovered through microarray analysis of gene expression following activation of p53. However, little is known about the effect of miR-144 on cell proliferation and apoptosis and how it interacts with TIGAR. Methods: We performed real-time PCR, western blotting, CCK8, colony formation, tumor growth, flow cytometry, Caspase3/7 activity, Hoechst 33342 staining, MDC staining of autophagic cells and luciferase reporter assays to detect the influence of miR-144 to lung cancer cells. Results: miR-144 targeted TIGAR, inhibited proliferation, enhanced apoptosis, and increased autophagy in A549 and H460 cells. Conclusions: Our study improves our understanding of the mechanisms underlying lung cancer pathogenesis and may promote the development of novel targeted therapies.

MicroRNA-124 inhibits cellular proliferation and invasion by targeting Ets-1 in breast cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that, by targeting certain messenger RNAs (mRNAs) for translational repression or cleavage, can regulate the expression of these genes. In addition, miRNAs may also function as oncogenes and tumor-suppressor genes, as the abnormal expression of miRNAs is associated with various human tumors. However, the effects of the expression of miR-124 in breast cancer remain unclear. The present study was conducted to study the expression of miR-124 in breast cancer, paying particular attention to miR-124’s relation to the proliferation, invasion, and apoptosis in breast cancer cell MCF-7 and MDA-MB-231. Real-time quantitative RT-PCR (qRT-PCR) was performed to identify miR-124 that was down-regulated in breast cancer tissues. We also showed E26 transformation specific-1 (Ets-1) and miR-124 expression levels in breast cancer tissues that were associated with lymph node metastases. With transfected synthetic miR-124 agomir into MCF-7 and MDA-MB-231, a significant reduction (P < 0.05) in MCF-7 and MDA-MB-231 cell proliferation and colony forming potential was observed after treatment with miR-124. Apoptosis and migration rates were found to be significantly higher in two breast-derived cell lines transfected with a miR-124 agomir (P < 0.05). Luciferase reporter assay and Western blot were used to verify Ets-1 as a potential major target gene of miR-124, and the result showed that miR-124 can bind to putative binding sites within the Ets-1 mRNA 3′ untranslated region (UTR) to reduce its expression. Based on these findings, we propose that miR-124 and Ets-1 may serve as a therapeutic agent in breast cancer.

miR-663 attenuates tumor growth and invasiveness by targeting eEF1A2 in pancreatic cancer

BackgroundmiR-663 is associated with many important biologic processes, such as the evolution, development, viral infection, inflammatory response, and carcinogenesis among vertebrates. However, the molecular function and mechanism of miR-663 in pancreatic cancer growth and invasion is still unclear.MethodsWestern blot and real-time PCR were used to study the expression level of eEF1A2 protein and miR-663 in pancreatic cancer tissues and cell lines. The Pearson χ2 test was used to determine the correlation between miR-663 expression and clinicopathologic features of patients. Patients’ survival was analyzed using the Kaplan–Meier method, using the log-rank test for comparison. The biological function of miR-663 was examined by measuring cell growth, cell invasion and apoptosis analysis in vitro and in vivo. miR-663 target gene and signaling pathway was identified by luciferase activity assay and western blot.ResultsWe found that, in pancreatic cancer, eEF1A2 was significantly upregulated but miR-663 was significantly downregulated. Further results showed that the expression level of eEF1A2 and miR-663 was strongly associated with TNM stage and node metastasis status of the patients. miR-663 and eEF1A2 were inversely correlated with each other, and the changes in the expression levels of each can also predict the survival of patients with pancreatic cancer. We identified miR-663 as a tumor attenuate molecular that attenuated the proliferation and invasion of pancreatic cancer cells both in vitro and in vivo. Finally, we confirmed that the expression of eEF1A2 can partially restore the pro-apoptotic and anti-invasion functions of miR-663.ConclusionsmiR-663 attenuated the proliferation and invasion of pancreatic cells both in vitro and in vivo by directly targeting eEF1A2. miR-663 and eEF1A2 might be potential targets for the treatment of pancreatic cancer in the future.

MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2

Our previous studies have revealed that miR-148a is downregulated in pancreatic cancer. Bioinformatics analysis has shown cholecystokinin-B receptor (CCKBR) and B cell lymphoma (Bcl-2) to be potential targets of miR-148a. But the pathophysiologic role of miR-148a and its relevance to the growth and development of pancreatic cancer are yet to be investigated. The purpose of this study is to elucidate the molecular mechanisms where miR-148a acts as a tumor suppressor in pancreatic cancer. Our results showed significant downregulation of miR-148a in 28 pancreatic cancer tissue samples and five pancreatic cancer cell lines, compared with their non-tumor counterparts by qRT-PCR. MiR-148a was found to not only inhibit the proliferation of pancreatic cancer cells (PANC-1 and AsPC-1) in vitro by MTT assay and colony formation assay, but also to promote cells apoptosis in vitro by Annexin V-FITC apoptosis detection and caspase activity assay. Using western blot and luciferase activity assay, CCKBR and Bcl-2 were identified as targets of miR-148a. Moreover, we also found that the expression of Bcl-2 lacking in 3′UTR could abrogate the pro-apoptosis function of miR-148a. These findings suggest the importance of miR-148a’s targeting of CCKBR and Bcl-2 in the regulation of pancreatic cancer growth and apoptosis.

Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer

MicroRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, and can function either as oncogenes or tumor suppressors in tumor progression. The effects of the expression of miR-96 in non-small cell lung cancer (NSCLC) remain unclear. In our study, qRT-PCR (quantitative reverse transcription PCR) was performed to identify the miR-96 expression level in 68 paired NSCLC and adjacent normal lung tissues. Trans-well, cell counting kit-8, and apoptosis assays were used to evaluate the effects of miR-96 expression on cell invasion, proliferation, and apoptosis. Dual-luciferase reporter assay and Western blotting were used to verify whether FOXO3 was a potential major target gene of miR-96. Finally, the effect of FOXO3 on miR-96-induced cell survival was determined by transfection of the genes expressing FOXO3 lacking 3′UTR and miR-96. The expression level of miR-96 in NSCLC tissues was higher than that in adjacent normal lung tissues, and this increased expression was significantly associated with lymph node metastasis. In contrast to the cells in the blank and negative control groups, the number of cells migrating through the matrigel was significantly lower and the incidence of apoptosis was significantly higher in cells transfected with a miR-96 inhibitor. Western blotting and dual-luciferase reporter assays demonstrated that miR-96 can bind to the putative seed region in FOXO3 mRNA 3′UTR, and can significantly lower the expression of FOXO3. The introduction of FOXO3 cDNA without 3′UTR restored miR-96 induced cell apoptosis and invasion. MiR-96 is up-regulated in NSCLC tissues. Downregulation of miR-96 inhibits invasion and promotes apoptosis in NSCLC cells A549 and SPC-A-1 by targeting FOXO3. Therefore, our study improves our understanding of the mechanisms underlying NSCLC pathogenesis and may promote the development of novel targeted therapies.

MiR-495 regulates proliferation and migration in NSCLC by targeting MTA3

Our previous studies have showed that metastasis-associated protein 3 (MTA 3) is overexpressed in non-small cell lung cancer (NSCLC) tissue, and increased MTA3 mRNA levels is a risk factor of lymph node metastasis. Using bioinformatics analyses, we found that MTA3 was a potential target of miR-495. However, the pathophysiological role of miR-495 and its relevance to the growth and development of NSCLC have yet to be investigated. The purpose of this study was to elucidate the molecular mechanisms by which miR-495 acts as a tumor suppressor in NSCLC. qRT-PCR data showed significant downregulation of miR-495 in 56 NSCLC tissue samples and 5 lung cancer cell lines, compared with their adjacent normal tissue; furthermore, western blotting analysis revealed MTA3 protein was overexpressed in the tumor samples compared with the matched adjacent normal tissue. MiR-495 was shown to not only inhibit the proliferation of lung cancer cells (A549 and Calu-3) but also to inhibit cell migration in vitro. Using western blotting and luciferase assays, MTA3 was identified as a target of miR-495. These findings suggest the importance of miR-495 targeting of MTA3 in the regulation of lung cancer growth and migration.

Expression analysis of serum microRNAs in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology with considerable morbidity and mortality. Seeking informative diagnostic markers with greater clinical significance is essential for the early diagnosis of IPF. microRNAs (miRNAs or miRs) have emerged as novel serum diagnostic biomarkers for various diseases. In this study, we performed microarray analysis of the miRNA expression profile in the serum of patients with IPF compared to that of control subjects. We then performed a preliminary analysis of biological functions for the most differentially expressed miRNAs. Some of the microarray results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results from this study provide evidence to link the biological role of miRNAs to IPF, and suggest that miRNAs may undertake a variety of functions. Additionally, we found that the altered expression levels of miR-21, miR-155 and miR-101-3p were associated with forced vital capacity (FVC) and radiological features in IPF. Our data may serve as a basis for further investigation, preferably in large prospective studies, before miRNA can be used as a non-invasive screening tool for IPF in routine clinical practice.

Up-regulation of microRNA-138 induce radiosensitization in lung cancer cells

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to identify the association between miR-138 and Sentrin/SUMO-specific protease 1 (SENP1) as a radiosensitization-related gene and characterize the biological function by which SENP1 was regulated by miR-138 to influence radiosensitization in lung cancer cells. In this study, we showed that miRNA-138 is reduced in both lung cancer clinical specimens and cell lines and is effective to inhibit SENP1 expression. Moreover, high levels of miR-138 are associated with lower levels of lung cancer cell proliferation and colony formation. Then, we investigated the underlying mechanisms responsible for the increase in the radiosensitivity of lung cancer cells when SENP1 is inhibited by miR-138. We further show that the increased radiosensitivity may be the result of an increased γ-H2AX expression, an increased rate of apoptosis, and changes in the cell cycle. In conclusion, our data demonstrate that the miR-138/SENP1 cascade is relative to radiosensitization in lung cancer cells and is a potential radiotherapy target.

Mir-655 up-regulation suppresses cell invasion by targeting pituitary tumor-transforming gene-1 in esophageal squamous cell carcinoma

BackgroundMicroRNAs (miRNAs) can act as either oncogenes or tumor suppressor genes under different conditions and thus can play a significant role in cancer development. We investigated miR-655 expression in a cohort of esophageal squamous cell carcinoma (ESCC) to assess the impact of this miRNA on ESCC cell invasion and metastasis.MethodsA qRT-PCR assay was used to quantify miR-655 expression levels in 34 paired ESCC samples and adjacent non-tumor tissues. Wound healing and transwell assays were used to evaluate the effects of miR-655 expression on the invasiveness of ESCC cells. Luciferase reporter and western blot assays were used to determine whether the mRNA encoding pituitary tumor-transforming gene-1 (PTTG1) is a major target of miR-655.ResultsThe expression level of miR-655 in ESCC tissues was found to be lower than in adjacent non-tumor tissues (P < 0.05). This relatively low expression level was significantly associated with the occurrence of lymph node metastases (P < 0.05). Migration rates were significantly lower for two ESCC-derived cell lines (EC9706 and KYSE150) transfected with miR-429 mimics (P < 0.05). Subsequent western blot and luciferase reporter assays demonstrated that miR-655 could bind to putative binding sites within the PTTG1 mRNA 3’-untranslated region (3’-UTR) and thus reduce the expression.ConclusionsmiR-655 is expressed at low levels in primary ESCC tissues, and up-regulation of miR-655 inhibits ESCC cell invasiveness by targeting PTTG1. Our findings suggest that PTTG1 may act as a major target of miR-655. This study improves our understanding of the mechanisms underlying ESCC pathogenesis and may promote the development of novel targeted therapies.

Knockdown of long non-coding RNA TP73-AS1 inhibits cell proliferation and induces apoptosis in esophageal squamous cell carcinoma.

Recent studies have shown that long non-coding RNAs (lncRNAs) are involved in a variety of biological processes and diseases in humans, including cancer. Our study serves as the first comprehensive analysis of lncRNA TP73-AS1 in esophageal cancer. We utilized a lncRNA microarray to analyze the expression profile of lncRNAs in esophageal squamous cell carcinoma. Our results show that lncRNA TP73-AS1 and BDH2 levels are generally upregulated in esophageal cancer tissues and are strongly correlated with tumor location or TNM stage in clinical samples. LncRNA TP73-AS1 knockdown inhibited BDH2 expression in EC9706 and KYSE30 cells, whereas BDH2 knockdown repressed esophageal cancer cell proliferation and induced apoptosis via the caspase-3 dependent apoptotic pathway. Overexpression of BDH2 in lncRNA TP73-AS1 knockdown cells partially rescued cell proliferation rates and suppressed apoptosis. In mouse xenografts, tumor size was reduced in lncRNA TP73-ASI siRNA-transfected tumors, suggesting that downregulation of lncRNA TP73-AS1 attenuated EC proliferation in vitro and in vivo. In addition, BDH2 or lncRNA TP73-AS1 knockdown enhanced the chemosensitivity of esophageal cancer cells to 5-FU and cisplatin. Our results suggest that lncRNA TP73-AS1 may be a novel prognostic biomarker that could serve as a potential therapeutic target for the treatment of esophageal cancer.