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.%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-qPCR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-qPCR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(P<0.01);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(P<0.01);转染miR-23a inhibitor后,SW480细胞活力较inhibitor NC组显著下降(P<0.01);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.
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