miR-23a和ESRP1在直肠癌中的作用

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。

miR-23a regulates cell growth and apoptosis of rectal cancer via targeting ESRP1 gene

AIM: To investigate the expression of miR-23a and epithelial splicing regulatory protein 1(ESRP1) in rectal cancer tissues and cell lines as well as their effects on rectal cancer cell viability and apoptosis.METHODS: The relative levels of miR-23a in the rectal cancer tissues and cultured cells were assessed by RT-qPCR. The positive expression of ESRP1 in the rectal cancer tissues and non-cancer tissues was detected by immunohistochemical staining. The sequences of miR-23a inhibitor and inhibitor negative control (NC) were synthesized, and transfected into the SW480 cells. The cell viability was measured by CCK-8 assay. The apoptotic rate was analyzed by flow cytometry. The cell invasion was evaluated by Matrigel counting assay. The expression of ESRP1 was determined by Western blot. The wild-type pGL3-ESRP1-3'UTR (wt-pGL3-ESRP1-3'UTR) or mutant pGL3-ESRP1-3'UTR (mut-pGL3-ESRP1-3'UTR) plasmid and miR-23a inhibitor or inhibitor NC were co-transfected into the HEK293 and SW480 cells. The dual luciferase activity was detected according to Promega dual luciferase reporter gene assay kit instructions. The cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry analysis, respectively, after the SW480 cells were transfected with ESRP1 mimic or mimic NC. The expression of ESRP1, caspase-3, Smac and X-linked inhibitor of apoptosis protein (XIAP) in the SW480 cells was detected by Western blot.RESULTS: The expression of miR-23a was significantly up-re-gulated in the rectal cancer tissues and cell lines, while the positive expression of ESRP1 was significantly decreased in the rectal cancer specimens. The miR-23a expression was also closely related to lymphnode metastasis and TNM stages of rectal cancer patients. ESRP1 was inversely correlated with miR-23a in the rectal cancer tissues. After transfection with miR-23a inhibitor in human rectal cancer SW480 cells, the down-regulation of miR-23a induced significant inhibition of cell viability as compared with the cells transfected with inhibitor NC (P<0.01). Furthermore, the apoptotic rate induced by the miR-23a inhibitor transfection was markedly higher than that of control (P<0.01). Luciferase assay showed that ESRP1 was a direct target gene of miR-23a. The cell viability and apoptosis were inhibited and promoted, respectively, after transfection with ESRP1 mimic in the SW480 cells. Promoted expression of ESRP1 significantly up-regulated the levels of caspase-3 and Smac as well as down-regulated the expression of XIAP in the SW480 cells.CONCLUSION: The expression of miR-23a is significantly associated with the growth and apoptosis of human rectal cancer cells by targeting ESRP1. miR-23a may be a potential therapeutic target for the treatment of rectal cancer in the future.