miR-4695-5p在结直肠癌患者血清中的表达及其对结直肠癌细胞增殖和侵袭的作用机制研究

Expression of miR-4695-5p in the serum of colorectal cancer patients and the mechanism of its effect on the proliferation and invasion of colorectal cancer cells

目的探讨微小RNA(microRNA)-4695-5p在结直肠癌患者血清中的表达及其对结直肠癌CACO-2细胞增殖与侵袭的影响。方法选取2018年3月—2021年11月郑州大学附属洛阳市中心医院胃肠外科收治的结直肠癌患者血清标本43例,以43例门诊体检健康者的血清为对照。采用实时定量聚合酶链式反应(qRT-PCR)检测结直肠癌患者血清和体检健康者血清中的相对表达量。将miR-4695-5p过表达质粒或阴性对照质粒转染CACO-2细胞,即miR-4695-5p组和对照组,qRT-PCR验证转染效率。CCK8法及Transwell实验分别检测过表达miR-4695-5p对CACO-2细胞增殖及侵袭能力的影响。双荧光素酶报告基因实验验证miR-4695-5p与Ras相关的C3肉毒素底物1(RAC1)的靶向结合关系。qRT-PCR和Western blotting分别检测过表达miR-4695-5p对RAC1基因及Wnt/β-Catenin信号通路蛋白表达的影响。采用SPSS 28.0软件进行统计分析。正态分布的计量资料以均数±标准差(±s)表示,组间比较采用t检验,多组间比较采用方差分析。结果结直肠癌患者血清中miR-4695-5p表达水平明显低于体检健康者(P<0.01)。对照组和miR-4695-5p组中miR-4695-5p相对表达量分别为1.09±0.65和8.83±2.03,miR-4695-5p组CACO-2细胞中miR-4695-5p表达水平是对照组的8.10倍,过表达miR-4695-5p的CACO-2细胞构建成功(P<0.01)。与对照组比较,miR-4695-5p组CACO-2细胞的增殖能力明显下降(P<0.05),CACO-2细胞的侵袭能力明显降低(P<0.01)。生物信息学工具和双荧光素酶报告基因实验证实miR-4695-5p能够与RAC1靶向结合(P<0.01)。与对照组相比,miR-4695-5p组RAC1基因表达明显下降(P<0.01),Wnt/β-Catenin信号通路蛋白Wnt3a、β-catenin、c-MYC表达显著下降。结论miR-4695-5p在结直肠癌患者血清中呈低表达状态,miR-4695-5p通过靶向抑制RAC1基因表达下调Wnt/β-Catenin信号通路活性,从而抑制结直肠癌CACO-2细胞的增殖与侵袭。

Objective To explore the expression of microRNA-4695-5p in the serum of colorectal cancer patients and its effect on the proliferation and invasion of colorectal cancer CACO-2 cells.Methods A total of 43 serum samples of colorectal cancer patients who were admitted to the Department of Gastrointestinal Surgery,Luoyang Central Hospital Affiliated to Zhengzhou University from March 2018 to November 2021 were selected,and serum samples of 43 healthy people who underwent outpatient physical examination were used as controls.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression levels in the serum of colorectal cancer patients and those of healthy individuals.The miR-4695-5p overexpression plasmid or the negative control plasmid were transfected into CACO-2 cells,namely the miR-4695-5p group and the control group,and the transfection efficiency was verified by qRT-PCR.CCK8 method and Transwell experiment were used to detect the effect of overexpression of miR-4695-5p on the proliferation and invasion of CACO-2 cells.The dual luciferase reporter gene experiment was used to verify the targeted binding relationship between miR-4695-5p and Ras-related C3 botulinum toxin substrate 1(RAC1).qRT-PCR and Western blot were used to detect the effect of overexpression of miR-4695-5p on the expression of RAC1 gene and Wnt/β-Catenin signaling pathway protein.The software of SPSS28.0 was used to conduct data analysis.The measurement data of normal distribution were espressed by Mean±SD.The t-test was used to compare the means between two groups,and the one-way analysis of variance was used to compare the means of multiple groups.Results The expression level of miR-4695-5p in the serum of patients with colorectal cancer was significantly lower than that of healthy individuals(P<0.01).The relative expression levels of miR-4695-5p in the control group and miR-4695-5p group were 1.09±0.65 and 8.83±2.03,respectively.The expression level of miR-4695-5p in CACO-2 cells in the miR-4695-5p group was 8.10 times that of the control group,and CACO-2 cells overexpressing miR-4695-5p were successfully constructed(P<0.01).Compared with the control group,the proliferation ability of CACO-2 cells in the miR-4695-5p group was significantly reduced(P<0.05),and the invasion ability of CACO-2 cells was significantly reduced(P<0.01).Bioinformatics tools and dual luciferase reporter gene experiments confirmed that miR-4695-5p can target and bind RAC1(P<0.01).Compared with the control group,the expression of RAC1 gene in the miR-4695-5p group was significantly decreased(P<0.01),and the expression of Wnt/β-Catenin signaling pathway proteins Wnt3a,β-catenin,and c-MYC decreased significantly.Conclusions miR-4695-5p is lowly expressed in the serum of colorectal cancer patients.miR-4695-5p inhibits the proliferation and invasion of colorectal cancer CACO-2 cells by targeting the inhibition of RAC1 gene expression and down-regulating the activity of the Wnt/β-Catenin signaling pathway.