| Esophageal cancer is the eighth most common malignant cancer in the world, and characterized by high incidence and mortality rate. Esophageal cancer consists of two histologic types: esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma(EA). ESCC is the most predominant subtype in China. Most cases of esophageal cancer are not diagnosed until the disease is at an advanced stage, and the overall five-year survival rate is only 10-20%. The mortality of esophageal cancer is high and the incidence rate of this neoplasm is increasing, Thus, understanding the mechanism of esophageal cancer development is essential for improving disease diagnosis, treatment and prevention.microRNAs(mi RNAs) encode a class of small, short, non-coding RNA which regulate gene expression by targeting specific regions in the 3’UTR of the mRNA. This binding site results in a decrease in the protein levels of target genes either by mRNA degradation or mRNA translation inhibition. Numerous studies have shown that mi RNAs are involved in a lot of biological and pathologic processes including cell proliferation, apoptosis, differentiation and metabolism. mi RNAs play important roles in the initiation and development of cancer and many mi RNAs have been shown to function as oncogenes or tumor suppressors. They are arising as highly tissue-specific biomarkers with potential clinical application for defining cancer types.Recent datas have provided evidence that miRNAs also involve in ESCC initiation and development. Several mi RNAs have been found participating in ESCC cell growth and invasion, such as mi R-21, mi R-10 b, miR-92, mi R-375. Our group previously found that CpG island methylation status and polymorphisms in 3’UTR binding region of mi RNA target genes could affect miRNA function in ESCC tissues. Here, combining miRNA microarray with literature study, we chose mi R-330-3p and mi R-26 b for subsequent study. Thus, the present project is aimed to investigate the effects of mi R-330-3p and miR-26 b on ESCC carcinogenesis and identified a novel upstream and downstream pathway of mi RNA regulation mechanisms.The major results are presented below:1. The study on function and regulation mechanism of mi R-330-3p in esophageal squamous cell carcinoma.1.1 The expression level and biological function of mi R-330-3p in ESCC.To validate microarray data, mi R-330-3p levels were detected using SYBR-Green q RT-PCR in ESCC cell lines and 35 pairs of ESCC tissues. Results showed that mi R-330-3p expressions were significantly higher in ESCC tissues than that in the adjacent normal tissues(P<0.005). Compared with the immortalized human esophageal epithelial cell HET-1A, mi R-330-3p expressions were upregulated in two ESCC cell lines(P<0.05).CCK8 cell viability assays, Ed U assay, flow cytometry analyses, transwell assay and subcutaneous tumor experiments were used to detect the biological function of miR-330-3p. CCK8 assay indicated mi R-330-3p could promote ESCC cell growth in vitro. Also in nude mice, tumors with overexpressed mi R-330-3p emerged a significantly faster growth rate than the control. Ed U assay indicated mi R-330-3p had the ability to stimulate ESCC cell proliferation. FACS studies showed that ESCC cells with high mi R-330-3p levels had a lower proportion of G0/G1 cells. Overexpression and knockdown experiments showed that mi R-330-3p levels were positively related to CDK6 and cyclin A levels and negatively associated with p21Waf1/Cip1 and p27Kip1 levels. In addition, we found that mi R-330-3p could protect ESCC cells against cisplatin-induced cell apoptosis. Except for impact on cell growth, we also investigated whether miR-330-3p could affect ESCC cell motility and found that ectopic expression of mi R-330-3p promoted migration and invasion in ESCC cells. Taken together, our study first demonstrated that mi R-330-3p played an oncogenic role in ESCC initiation and progression through promoting cell cycle, cell survival, migration and invasion.1.2 Mi R-330-3p targets PDCD4 via binding to its 3’UTRIn silico analysis(Target Scan, mi Randa and Pic Tar), we found that the 3’UTR of PDCD4 mRNA contained a highly conserved binding site from position 433 to 439 for mi R-330-3p. To determine if mi R-330-3p could regulate PDCD4 expression in esophageal cancer cells, we detected PDCD4 m RNA and protein level by RT-PCR and western blot. In EC109 cells, the protein and mRNA expression of PDCD4 were significantly suppressed by mi R-330-3p overexpression. Conversely, the expression level of PDCD4 was markedly excited by miR-330-3p inhibition in KYSE150 cells. To further determine the relationship between PDCD4 and mi R-330-3p, we cloned the wild-type and mutant 3’UTR of PDCD4 mRNA that included the binding site of miR-330-3p into pMIR-REPORT vector, named pmiR-PDCD4-WT and pmiR-PDCD4-MUT respectively. And then these constructs were co-transfected with mi R-330-3p mimics or mimics control into EC109 cells. Compared with the NC, the relative luciferase activities were decreased in cells cotransfected with pmiR-PDCD4-WT vectors and mi R-330-3p mimics. However, when the binding site was mutated, this inhibition was attenuated. Collectively, these data showed that miR-330-3p could regulate PDCD4 expression via direct targeting its 3’UTR. We next tested the expression of PDCD4 in ESCC and normal esophageal tissues. As indicated in Figure 6A, 6B, the expression of PDCD4 protein and m RNA were both down-regulated in ESCC tissues. In addition, the Pearson correlation analysis showed that PDCD4 mRNA expression was inversely correlated with mi R-330-3p expression(r=-0.5421, p=0.0008). These results suggested that PDCD4 might be a direct downstream target for mi R-330-3p in ESCC cells.1.3 Effect of PDCD4 loss-of-function on cell growth and invasion in ESCC cell lines.To determine the effects of PDCD4 on ESCC cells, we performed loss-of-function study using PDCD4-specific siRNA. CCK8 and Ed U assay revealed that inhibition of PDCD4 promoted cell growth and proliferation in ESCC cells. Furthermore, Flow cytometric analysis showed that knockdown of PDCD4 could promote G1/S transition and inhibit cisplatin-induced apoptosis in both two ESCC cell lines. In addition, transwell assay indicated that knockdown of PDCD4 promoted migration and invasion of EC109 cells. Therefore, down-regulation of PDCD4 played a facilitative role on ESCC cell proliferation and invasion. These results are similar to those obtained with ove r-expression of mi R-330-3p, which indicated that mi R-330-3p might play an oncogenic role in the development of ESCC partially via downregulating the expression of PDCD4.2. The study on the function and regulation mechanism of mi R-26 b in esophageal squamous cell carcinoma.2.1 The expression level of mi R-26 b in ESCC cells and tissuesTo validate microarray data, mi R-26 b levels were further detected using SYBR-Green q RT-PCR in ESCC cell lines and ESCC tissues. Results showed that mi R-26 b expressions were significantly lower in ESCC tissues than that in the adjacent normal tissues(P<0.05). Compared with the immortalized human esophageal epithelial cell HET-1A, miR-26 b expressions were downregulated in three ESCC cell lines(P<0.05).2.2 The biological function of mi R-26 b in ESCC cells.CCK8 cell viability assays, Ed U assay and flow cytometry analyses were used to detect the biological function of miR-26 b. CCK8 assay indicated mi R-26 b could inhibit ESCC cell growth. Ed U assay indicated mi R-26 b had the ability to inhibit ESCC cell proliferation. FACS studies showed that ESCC cells with high miR-26 b levels induced G1 phase arrest. In addition, we found that miR-26 b inhibitor could protect ESCC cells against cisplatin-induced cell apoptosis. Taken together, our study demonstrated that miR-26 b might function as a tumor suppressor in ESCC initiation and progression through inhibiting cell proliferation.2.3 MYCBP may be a downstream target gene which involved in cell growth inhibition induced by mi R-26 b.According to bioinformatics, we found that the 3’UTR of MYCBP contained a highly conserved binding site for mi R-26 b. To determine if mi R-26 b could regulate MYCBP expression in esophageal cancer cells, we detected MYCBP mRNA by RT-PCR after transfected with miR-26 b mimics. Results showed that the mRNA expression of MYCBP was significantly suppressed by miR-26 b. To further determine the relationship between MYCBP and mi R-26 b, we cloned the wild-type 3’UTR of MYCBP into pMIR-REPORT vector, then the construct were co-transfected with miR-26 b mimics or mimics control into EC109 cells. Compared with the NC, luciferase activities were decreased in cells cotransfected with pmi R-MYCBP-WT vectors and mi R-26 b mimics. Moreover, we found that down-regulation of MYCBP played a similar role on ESCC cell growth and proliferation with over-expression of miR-26 b, which indicated that mi R-26 b might play an tumor suppression role in the development of ESCC partially via downregulating MYCBP expression.3. NF-κB regulates miRNAs contributing to ESCC cells growth.3.1 There is a conserved NF-κB binding site in the promoter of miR-330-3p and mi R-26 b.According to the UCSC website provides Sno/mi RNA data combined with Yale TFBS, CpG Island and TFBS conserved data, we found that there was a conserved NF-κB binding site in the promoter region of mi R-330-3p and miR-26 b. This suggested that NF-κB p65 can bind to the regulatory region of these two mi RNAs.3.2 The expression of mi R-330-3p and mi R-26 b are changed by NF-κB p65.To investigate whether these two mi RNAs were regulated by NF-κB, we overexpressed the NF-κB p65 by transient transfecting with p65 expression vector and negative control vector(pcDNA3.1) in EC109 cells. The result showed that enhanced the p65 level, the expression of mi R-330 reduced by half, and inversely, the expression of miR-26 b were increased.3.3 NF-κB p65 can bind regulatory region of miR-330-3p and mi R-26 b.Next, we studied the regulation relationship between NF-κB and miRNAs. Chromatin Immunoprecipitation(Ch IP) assay results showed that the enrichment of NF-κB p65 and p50 subunit were higher than the control IgG in the promoter region of two mi RNAs.3.4 The transcriptional activity of mi R-330-3p by NF-κB p65.In addition, the luciferase activity of cotransfected p65 and 330 P vector into EC109 was reduced compared with the control, suggesting NF-κB was significantly inhibited mi R-330-3p transcriptional activity. Moreover, mutated or deleted the κB site resulted in a rebounded of luciferase activity. These observations indicated that the NF-κB binding site is active and required for the suppression of mi R-330-3p transcriptional activity.In conclusion, we firstly investigate the expression and biological function of mi R-330-3p and miR-26 b in esophageal squamous cell carcinoma, and then study the regulatory mechanism in upstream and downstream of the mi RNAs. The pathway of NF-κB—mi RNAs—targets is established during ESCC development, and this may be a new evidence for ESCC diagnosis and treatment. |
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