环状RNA circ0000601通过miR-31-5p调控NUMB抑制结直肠癌细胞增殖

Circular RNA circ0000601 suppresses colorectal cancer cell proliferation through miR-31-5p and its target NUMB

目的本研究探讨circ0000601对结直肠癌细胞增殖的影响及其可能的作用机制。方法微阵列扫描结直肠癌组织和癌旁正常组织差异性表达的circRNA.PCR检测circ0000601在结直肠癌组织、癌旁正常组织及结直肠癌细胞株中的相对表达。在结直肠癌细胞中转染circ0000601 mimic上调circ0000601的表达,CCK-8检测结直肠癌细胞株增殖能力,通过生物信息学软件分析circ0000601的下游miRNA,双荧光报告基因法进行验证。转染miR-31-5p inhibitor抑制miR-31-5p的表达,CCK-8检测结直肠癌细胞株增殖能力。通过生物信息学数据库miRBase预测miR-31-5p的靶基因,通过荧光素酶报告基因法证实NUMB是miR-31-5p的靶基因。转染miR-31-5p mimic过表达miR-31-5p,检测结直肠癌细胞中NUMB蛋白表达水平。过表达circ0000601检测NUMB mRNA的表达。结果通过t检验及Kruskal-Wallis检验分析数据。circ0000601在结直肠癌组织中的表达水平低于配对正常组织,过表达circ0000601可抑制直肠癌细胞增殖。circ0000601可与miR-31-5p靶向结合。通过miR-31-5p inhbitor下调miR-31-5p的表达可显著抑制结直肠癌细胞的增殖。使用miRBase预测miR-31-5p的靶基因,荧光素酶报告基因法证实NUMB是miR-31-5p的靶基因。过表达miR-31-5p后NUMB mRNA和蛋白的表达水平均降低。过表达circ0000601后NUMB mRNA表达水平增高。结论circ0000601可通过miR-31-5p调控NUMB抑制结直肠癌细胞增殖,circ0000601可作为结直肠癌患者的潜在治疗靶点。

Objective To investigate the effect of circ0000601 on the proliferation of colorectal cancer cell and its possible mechanism. Methods High-throughput circRNA chip was used to screen circRNA gene between colorectal cancer tissues and paired normal tissues. PCR was used to detect the relative expression of circ0000601 in colorectal cancer tissues, paired normal tissues and colorectal cancer cell lines. circ0000601 expression was upregulated through transfecting with circ0000601 mimic and cell proliferation was analyzed by CCK-8 assay. Bioinformatics prediction software was used to analyze circ0000601 target miRNA and luciferasw reporter system was used to verificate circ0000601 target miRNA. The expression of miR-31-5p was down-regulated through transfection of miR-31-5p inhibitor and cell proliferation was analyzed by CCK-8 assay. miRBase was used to predict the target gene of miR-31-5p and Luciferase reporter assay was used to confirm that NUMB was a direct target of miR-31-5p. The expression of miR-31-5p and NUMB protein expression in colorectal cancer cells was detected by Western blotting. circ0000601 expression was upregulated in colorectal cancer cells and NUMB mRNA expression in colorectal cancer cells were detected. Results T-test and Kruskal-Wallis test were used in data analysis. The expression of circ0000601 was lower in colorectal cancer tissues than paired normal tissues and overexpression of circ0000601 reduced colorectal cancer cell proliferation. miR-31-5p was identified and validated to be a target miRNA of circ0000601. The knockdown of miR-31-5p suppressed the proliferation of colorectal cancer cells. Luciferase reporter assay confirmed that NUMB was a direct target of miR-31-5p. The protein level of NUMB was decreased by miR-31-5p Mimic. Also up-regulation of circ0000601 could increase the expression of NUMB mRNA. Conclusion circ0000601 can inhibit colorectal cancer cell proliferation by circ0000601/miR-31-5p/NUMB pathway, therefore, circ0000601 acts as a promising therapeutic target for colorectal cancer patients.