miR-23a和ESRP1在直肠癌中的作用

发布时间：2018-05-15 09:11

本文选题：直肠癌 + miR-a　；参考：《中国病理生理杂志》2017年05期

【摘要】：目的:研究miR-23a和上皮剪接调节蛋白1(epithelial splicing regulatory protein 1,ESRP1)在直肠癌组织及细胞系中的表达,以及对体外直肠癌细胞活力和凋亡的作用。方法:采用RT-q PCR分析miR-23a在36例直肠癌组织和癌旁组织中的表达,免疫组化检测ESRP1在直肠癌组织中的表达,分析miR-23a和ESRPl在直肠癌组织中的相关性;利用RT-q PCR检测miR-23a在直肠癌Caco-2和SW480细胞及人正常结肠上皮细胞株NCM460中的表达;合成miR-23a inhibitor和inhibitor阴性对照(inhibitor NC),并将其分别转染至SW480细胞后,通过CCK-8法检测miR-23a inhibitor转染SW480细胞后对细胞活力的影响,流式细胞术检测转染后细胞凋亡率,Transwell小室实验检测细胞侵袭;通过Western blot技术检测SW480细胞中ESRPl蛋白的表达;构建野生型pGL3-ESRP1-3’UTR(wt-pGL3-ESRP1-3’UTR)或突变型pGL3-ESRP1-3’UTR(mut-pGL3-ESRP1-3’UTR)质粒,并分别与miR-23a inhibitor或inhibitor NC共转染至HEK293和SW480细胞中,利用双萤光素酶报告基因检测试剂盒说明检测双萤光素酶活性;将ESRP1 mimic或mimic NC瞬时转染SW480细胞后,CCK-8法和流式细胞术分别检测细胞活力和凋亡;Western blot法检测瞬转ESRP1 mimic后对ESRP1、caspase-3、Smac和XIAP蛋白表达的影响。结果:miR-23a和ESRP1在直肠癌组织的表达较癌旁正常组织分别上调和下调,两者呈明显负相关(P0.01);miR-23a的表达与直肠癌的淋巴结转移和肿瘤浸润深度相关;与NCM460细胞相比较,miR-23a在SW480细胞中的表达量显著上调(P0.01);转染miR-23a inhibitor后,SW480细胞活力较inhibitor NC组显著下降(P0.01);转染miR-23a inhibitor后SW480细胞早期凋亡率明显升高,同时细胞体外侵袭能力受到抑制;萤光素酶报告基因结果表明ESRP1是miR-23a的直接靶基因;转染miR-23a inhibitor至SW480细胞后ESRP1蛋白表达水平明显升高;ESRP1 mimic转染SW480细胞后可抑制细胞活力并诱导细胞凋亡,同时上调caspase-3和Smac的表达,下调XIAP的表达。结论:miR-23a可通过负向调控下游靶基因ESRP1从而影响直肠癌细胞生长和凋亡。
[Abstract]:Aim: to investigate the expression of miR-23a and ESRP1 in rectal cancer tissues and cell lines, and the effects of ESRP1 on the viability and apoptosis of rectal cancer cells in vitro. Methods: RT-q PCR was used to analyze the expression of miR-23a in 36 cases of rectal cancer and adjacent tissues. Immunohistochemical staining was used to detect the expression of ESRP1 in rectal cancer. The correlation between miR-23a and ESRPl in rectal carcinoma was analyzed. RT-q PCR was used to detect the expression of miR-23a in Caco-2 and SW480 cells and normal colonic epithelial cell line NCM460, miR-23a inhibitor and inhibitor negative control inhibitor NCX were synthesized and transfected into SW480 cells, respectively. The effect of miR-23a inhibitor transfection on the viability of SW480 cells was detected by CCK-8, apoptosis rate was detected by flow cytometry and cell invasion was detected by transwell chamber assay, and the expression of ESRPl protein in SW480 cells was detected by Western blot technique. The wild-type pGL3-ESRP1-3 UTR-wt-pGL3-ESRP1-3UTR) or mutant pGL3-ESRP1-3USRP1-3UTR) plasmid were constructed and cotransfected with miR-23a inhibitor or inhibitor NC into HEK293 and SW480 cells. After transient transfection of ESRP1 mimic or mimic NC into SW480 cells, CCK-8 assay and flow cytometry were used to detect the cell viability and apoptosis. Western blot assay was used to detect the effect of ESRP1 mimic on the expression of ESRP1caspase-3 and XIAP protein. Results the expression of miR-23a and ESRP1 were up-regulated and down-regulated in rectal cancer tissues, respectively. There was a significant negative correlation between the expression of MR-23a and lymph node metastasis and depth of tumor invasion. Compared with NCM460 cells, the expression of miR-23a in SW480 cells was significantly up-regulated, and the viability of SW480 cells was significantly lower than that of inhibitor NC group. The early apoptotic rate of SW480 cells was significantly increased after miR-23a inhibitor transfection, and the invasion ability of SW480 cells was inhibited in vitro. The results of luciferase reporter gene showed that ESRP1 was the direct target gene of miR-23a, and the expression of ESRP1 protein increased significantly after transfection of miR-23a inhibitor to SW480 cells. ESRP1 protein expression in SW480 cells was inhibited and apoptosis induced by ESRP1 mimic, and the expression of caspase-3 and Smac was up-regulated. The expression of XIAP was down-regulated. Conclusion the growth and apoptosis of rectal cancer cells can be affected by the negative regulation of downstream target gene ESRP1.
【作者单位】：绍兴第二医院肛肠外科;岳阳市一人民医院胃肠外科;
【基金】：岳阳市2014年第三批科技资助项目
【分类号】：R363;;R735.3+7

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