【摘要】 目的探讨miRNA-1236对结直肠癌细胞增殖的作用及其相关机制。方法通过实时定量PCR检测转染miR-1236后miRNA-1236的表达,使用CCK-8以及克隆形成方法检测转染miRNA-1236后HCT116细胞的活性,ki67方法检测转染miRNA-1236后HCT116细胞增殖能力的变化,生物信息方法筛选miR-1236的潜在靶基因FOXO3a。Luciferase报告检测miRNA-1236与FOXO3a的结合位点,通过实时定量PCR方法检测转染miRNA-1236后FOXO3a基因的变化;通过免疫组化的方法检测结直肠癌患者组织中FOXO3a的表达,通过western blot检测低表达miRNA-1236后HCT116细胞中FOXO3a、PCNA以及Bax蛋白的变化。通过TCGA数据库检测FOXO3a基因在结直肠癌患者癌-癌旁,不同肿瘤阶段与不同性别中的表达。结果本研究发现高表达miRNA-1236后HCT116细胞活性相对于阴性对照组明显增加,而低表达miRNA-1236后活性降低。低表达miRNA-1236后HCT116细胞增殖能力相对于阴性对照组明显降低。生物信息学预测miR-1236与FOXO3a有潜在的结合位点。采用Western blot以及逆转录实时定量PCR验证,过表达miRNA-1236后FOXO3a表达相对于阴性对照组明显降低,而低表达miRNA-1236后,FOXO3a表达明显升高,同时luciferase结果显示,FOXO3a是miRNA-1236的靶基因。并且,FOXO3a在结直肠癌症组织中明显降低。结论 miR-1236通过靶向FOXO3a的表达调节结直肠癌细胞HCT116增殖,同时为miR-1236成为诊断、预防以及治疗结直肠癌的生物学标记物提供理论基础。更多还原

【Abstract】 Objective To investigate the role and mechanism of miRNA-1236 on proliferation in colorectal cancer. Method The expression of miRNA-1236 was detect by quantitative real-time PCR. The cell viability of HCT116 cells after transfected with miRNA-1236 was detected by CCK-8. The proliferation of miR-1236 in HCT116 cell was tested by colony formation assay and Ki67 staining. The potential target gene FOXO3 a of miR-1236 was screened by bioinformatics. Observing the level of FOXO3 a gene with transfection of miRNA-1236 by real-time quantitative PCR detection. Luciferase reports detected the binding site of miRNA-1236 with FOXO3 a; The expression of FOXO3 a in colorectal cancer was detected by immunohistochemistry. Detected the protein level of FOXO3 a, PCNA and Bax in HCT116 cells after transfection with miR-1236 by western blotting. The expression of FOXO3 a in colorectal Cancer-Para cancer patients with different cancer stages and genders was detected by TCGA database. Results The cell viability of HCT116 cells was signi?cantly increased with the miRNA-1236 mimic compared with negative control group, while it was decreased with the miRNA-1236 inhibitor. The proliferation of HCT116 cells was signi?cantly reduced compared with the negative control group by colony formation assay and Ki67 staining.Bioinformatics predicted potential binding sites between miR-1236 and FOXO3 a. Using Western blot and quantitative real-time PCR of reverse transcription to verify that FOXO3 a expression was significantly reduced after overexpression of miRNA-1236 than the negative control group, while FOXO3 a expression was significantly increased after treat with miRNA-1236 inhibitor. Meanwhile, luciferase results showed that FOXO3 a was a direct target gene of miRNA-1236. Simultaneously, FOXO3 a was signi?cantly reduced in colorectal cancer tissue. Conclusion miRNA-1236 regulate the proliferation of colorectal cancer by targeting FOXO3 a, and signi?cantly affects survival of colorectal cancer patients.更多还原