miRNA-96-5p靶向调控叉头框蛋白Q1的表达抑制胃癌细胞增殖侵袭和上皮间质转化

miRNA-96-5p inhibits the proliferation and migration of gastric cancer cells by targeting FoxQ1

目的研究miRNA-96-5p在胃癌细胞和组织中的表达水平以及对胃癌细胞增殖、侵袭的影响和分子机制。方法收集2015年6月至2017年1月手术切除的胃癌标本53例,采用实时荧光定量PCR法检测胃癌组织和癌旁组织中miRNA-96-5p的表达水平,采用免疫组化法检测叉头框蛋白Q1(FoxQ1)蛋白的表达,分析miRNA-96-5p的表达与胃癌患者临床病理特征和FoxQ1蛋白表达的相关性。采用实时荧光定量PCR法检测miRNA-96-5p在胃癌细胞和正常胃黏膜上皮细胞中的表达。以miRNA-96-5p模拟物转染人胃癌BGC-823细胞,采用细胞计数盒8(CCK-8)法检测BGC-823细胞的增殖,采用Transwell小室法检测BGC-823细胞的侵袭,采用Western blot法检测FoxQ1、E-cadherin和Vimentin蛋白的表达。采用双荧光素酶活性实验验证miRNA-96-5p对FoxQ1的靶向作用。结果胃癌组织中miRNA-96-5p的中位表达水平为1.05,明显低于癌旁组织(3.23,P<0.05)。胃癌组织中FoxQ1蛋白的阳性表达率(71.7%)明显高于癌旁组织(28.3%,P<0.05)。相关性分析显示,FoxQ1蛋白在胃癌组织中的表达与miRNA-96-5p的表达呈负相关(r=-0.613,P=0.006)。miRNA-96-5p在胃癌BGC-823细胞中的表达水平为0.96±0.08,低于正常胃黏膜上皮细胞GES-1(2.84±0.15,P<0.05)。与空白对照组和miRNA模拟物对照组BGC-823细胞比较,miRNA-96-5p模拟物组BGC-823细胞的增殖和侵袭能力均受到明显抑制(均P<0.05),且miRNA-96-5p模拟物组BGC-823细胞中FoxQ1和Vimentin蛋白的表达水平降低,E-cadherin蛋白的表达水平增高。双荧光素酶报告基因检测结果显示,FoxQ1是miRNA-96-5p的靶基因。结论 miRNA-96-5p在胃癌组织和细胞中表达下调,miRNA-96-5p通过调控FoxQ1基因的表达影响胃癌细胞的增殖、侵袭和上皮间质转化。

Objective To investigate the role of microRNA-96-5p in the proliferation and invasion of gastric cancer cells and its molecular mechanism. Methods From June 2015 to January 2017, 53 resected specimens were collected. The transcriptional levels of microRNA-96-5p and forkhead box Q1 (FoxQ1) in gastric cancer tissues and the matched para-cancerous tissues were quantified by quantitative real-time PCR (qRT-PCR). The expression of FoxQ1 protein was also detected by immunohistochemistry (IHC). The relationship between microRNA-96-5p expression and the clinicopathological features of gastric cancer and its correlation with FoxQ1 expression were analyzed. The expressions of miRNA-96-5p in gastric cancer tissue and adjacent normal tissue were detected by qRT-PCR. miRNA-96-5p mimics was transfected to BGC-823 gastric cancer cells. The effects of miRNA-96-5p on cell proliferation and invasion were detected by cell counting kit-8 (CCK-8) assay and Transwell assay, respectively. The protein expressions of FoxQ1, E-cadherin and vimentin were determined by western blot. The relationship between FoxQ1 and miRNA-96-5p expressed in BGC-823 cells was detected by dual-luciferase reporter assay. Results The median expression of miRNA-96-5p in gastric cancer tissue was 1.05, significantly lower than 3.23 of para-cancerous tissues (P<0.05). The positive rate of FoxQ1 expression in gastric cancer tissue was 71.7%, significantly higher than 28.3% of para-cancerous tissues (P<0.05). The expression of FoxQ1 was negatively corelated with the level of miRNA-96-5p (r=-0.613, P=0.006). The expression of miRNA-96-5p in gastric cancer cell BGC-823 was significantly decreased compared with normal gastric epithelial cell (0.96±0.08 vs 2.84±0.15, P<0.05). The results of CCK-8 assay and Transwell assay showed that overexpression of miRNA-96-5p significantly reduced the proliferation and invasion abilities of gastric cancer cells (P<0.05). Overexpression of miRNA-96-5p decreased the protein level of FoxQ1. Moreover, it upregulated the expression of E-cadherin and downregulated the expression of vimentin. The result of dual-luciferase-3′-UTR reporter assay confirmed that miRNA-96-5p binds to the 3′UTR of FoxQ1. Conclusion miRNA-96-5p may suppress the proliferation, migration and epithelial-mesenchymal transition (EMT) of gastric cancer cell by down-regulation of FoxQ1.