| BackgroundMicroRNAs (miRNAs) are a class of small (19～24nucleotide) noncoding RNAs that mediate post-transcriptional regulation of target genes. The mature miRNAs can combine with mRNA3’UTR of downstream target genes in incomplete matching way, thus interference and repress the mRNA transcription of target genes. In addition, miRNAs can promote mRNA degradation of target genes by the deadenylation of target genes mRNA so that inhibit the expression of target genes. MiRNAs are important in the regulation of various biological and pathological processes, such as cellular proliferation, differentiation, apoptosis, emergency response and immune response, which play an important role in the regulation of tumour cell growth, proliferation, invasion, metastasis and angiogenesis as a novel species of oncogenes or tumour suppressor genes. At present, many miRNAs expression profiles of malignant tumours have been established through the miRNAs chip hybridization method, some of which can be as potential therapeutic targets and tumour markers for clinical prognosis of patients. Of course, more and more research about miRNAs in human esophageal carcinoma have been reported, with the deepening of the study, we have known the mechanism of a large number of miRNAs in esophageal squamous cell carcinoma(ESCC). It has been found that miR-675is up-regulated in human colon cancer, pancreatic cancer, serous endometrial tumours, endometrial carcinosarcomas, and in blood cells of lung cancer patients compared to blood cells of COPD patients. Recently, H19has been shown to be the primary miRNA precursor of miR-675in human, moreover, H19expression has been previously reported to be up-regulated in many cancers including hepatocellular carcinoma, ovarian cancer, bladder cancer, breast cancer, testicular cancer, choriocarcinoma, colorectal cancer, lung cancer, gastric cancer, cervical cancer, esophageal cancer. As H19is overexpressed in ESCC tissues and that is the primary miRNA precursor of miR-675, we speculate that miR-675might be involved in the tumoriogenesis and progression of ESCC induced by H19. The study about miR-675in ESCC has not been reported at home and abroad, so we carried out this research. Because H19can be transcribed to two mature miR-675-5p and miR-675-3p sequences, in this study we used miR-675-5p to study.ObjectiveStudy the expression of miR-675-5p in ESCC tissues and its clinical significance, meanwhile investigate the effects of miR-675-5p on the biological behavior of esophageal cancer cell line and cell pathway, and predict the target gene of miR-675-5p.Methods1. Detected the expression of H19and miR-675-5p in ESCC tissues and cell lines by real-time quantitative PCR(qRT-PCR), meanwhile analysed the relationship between H19and miR-675-5p, especially focused on the analysis of H19, miR-675-5p and clinical pathological characteristics of the patients with ESCC;2. Through infection to Eca109and EC9706cells by lentivirus, miR-675-5p expression was up-regulated and down-regulated, then to observe its effect on the cell biological characteristics, such as proliferation of ESCC cells, cell cycle, apoptosis, migration and invasion and tumorigenicity in nude mice; 3. The luciferase reporter assay was used to assess the target genes of miR-675-5p in Eca109and EC9706cell lines;4. Target gene REPS2mRNA and protein expression levels in the Eca109and EC9706cell lines transfected by LV-miR-675-precursor and LV-miR-675-5p-inhibition were observed by qRT-PCR and Western blotting, at the same time, RAC1and CDC42protein expression levels were also observed in the Eca109and EC9706cell lines by Western blotting;5. Expression levels of REPS2mRNA and protein in ESCC tissues and cell lines were observed by qRT-PCR and Western blotting, and the expression of REPS2protein was studied by immunohistochemical method in120cases of ESCC, moreover, the relationship between the expression level of REPS2protein in ESCC and the clinical pathological characteristics and prognosis of ESCC patients was analyzed.Results1. The expression levels of miR-675-5p and H19mRNA in ESCC tissues were significantly increased than that in adjacent non-cancerous tissues and normal tissues, meanwhile the expression levels of miR-675-5p and H19mRNA in four human ESCC cell lines were also higher than that in normal human esophageal epithelial cell line (HEEpic). Moreover, logarithmic ratio values of miR-675-5p expression in ESCC tissues and cell lines were positively correlated with that of H19mRNA expression in ESCC tissues and cell lines.2. Overexpression of miR-675-5p was significantly associated with primary tumour, TNM stage, lymph node metastasis respectively, and overexpression of H19mRNA was significantly associated with TNM stage and lymph node metastasis respectively. 3. Upregulation of miR-675-5p promoted the growth, proliferation, clone formation, invasion and migration, inhibited apoptosis and reduced cell cycle arrest at G1phase in vitro, and promoted the tumorigenicity graft growth of nude mice in vivo; but downregulation of miR-675-5p inhibited the growth, proliferation, clone formation, invasion and migration, induced apoptosis and increased cell cycle arrest at G1phase in vitro, and suppressed the tumorigenicity graft growth of nude mice in vivo.4. The luciferase reporter assay showed that REPS2was a direct target gene of miR-675-5p.5. Upregulation and downregulation of miR-675-5p did not affect the transcription level of REPS2mRNA, but could lead to the downregulation and upregulation of REPS2protein expression level, and led to the upregulation and downregulation of RAC1and CDC42protein expression levels.6. The expression levels of REPS2mRNA and protein in ESCC tissues were significantly decreased than that in adjacent non-cancerous tissues and normal tissues, meanwhile the expression levels of REPS2mRNA and protein in four human ESCC cell lines were also lower than that in normal HEEpic cell line. Moreover, logarithmic ratio values of miR-675-5p expression in ESCC tissues were negatively correlated with that of REPS2mRNA expression in ESCC tissues.7. The protein expression of REPS2was significantly associated with primary tumour, TNM stage, lymph node metastasis and recurrence respectively. Kaplan-Meier curves showed that down-regulated REPS2protein expression was associated with shorter overall survival and disease-free survival in ESCC patients. When REPS2protein expression and lymph node metastasis status were combined, patients with low REPS2protein expression/lymph node(+) had both poorer overall and disease-free survival than that of others. Cox multivariate regression analysis further revealed that REPS2protein expression was an independent prognostic factor for ESCC patients.Conclusion1. The expression levels of miR-675-5p and H19mRNA in ESCC tissues and cells were up-regulated, meanwhile logarithmic ratio values of miR-675-5p expression in ESCC tissues and cell lines were positively correlated with that of H19mRNA expression in ESCC tissues and cell lines.2. Overexpression of miR-675-5p was significantly associated with primary tumour, TNM stage, lymph node metastasis respectively, and overexpression of H19mRNA was significantly associated with TNM stage and lymph node metastasis respectively.3. MiR-675-5p promoted the growth, proliferation, clone formation, invasion and migration, inhibited apoptosis and reduced cell cycle arrest at G1phase in vitro, and promoted the tumorigenicity graft growth of nude mice in vivo.4. REPS2was a direct target gene of miR-675-5p. The regulation of miR-675-5p to REPS2was a post-transcriptional regulation, and miR-675-5p participated in the regulation of Ras/Ral signal transduction pathway.5. The expression levels of REPS2mRNA and protein in ESCC tissues and cells were down-regulated, meanwhile logarithmic ratio values of miR-675-5p expression in ESCC tissues and cell lines were negatively correlated with that of REPS2mRNA expression in ESCC tissues and cell lines.6. The protein expression of REPS2was significantly associated with primary tumour, TNM stage, lymph node metastasis and recurrence respectively. REPS2protein expression level influenced the survival prognosis of ESCC patients, and REPS2protein expression was an independent prognostic factor for ESCC patients. |
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