分泌蛋白CRLF1表达对结直肠癌细胞增殖和细胞克隆能力的影响

Effect of CRLF1 expression on proliferation and clonal ability of colorectal cancer cells

目的:深入研究细胞因子受体样因子1(CRLF1)表达对结直肠癌细胞增殖和细胞克隆能力的影响。方法:酶联免疫吸附法(ELISA)检测血清中CRLF1表达水平,提取新鲜肿瘤及癌旁组织样本RNA,测定CRLF1的相对表达量。培养人结直肠癌细胞株HCT116和SW620,合成si-NC和si-CRLF1小干扰RNA,对HCT116和SW620细胞进行转染,使用实时聚合酶链反应(real-time PCR)及ELISA法验证转染效率,CCK8实验检测转染后结直肠癌细胞增殖能力的变化,克隆形成实验检测细胞克隆形成的能力,流式细胞术检测细胞凋亡水平。Western blot检测AKT信号通路及变化。结果:结直肠癌患者血清CRLF1水平显著高于健康捐献者血清。结直肠癌组织中CRLF1 mRNAG表达水平显著高于癌旁组织。分别在两种细胞系中通过转染si-CRLF1以实现敲低CRLF1表达。敲低CRLF1表达后结直肠癌细胞HCT116和SW620的细胞增殖能力及细胞克隆形成能力明显减弱(P<0.05),细胞凋亡率显著升高。敲低CRLF1表达后p-AKT表达水平显著降低。结论:CRLF1表达通过调控AKT信号通路调控结直肠癌进展,该蛋白可能成为诊断结直肠癌的生物标志物以及治疗结直肠癌的潜在靶点。

Objective:To investigate the effect of cytokine receptor like factor l(CRLF1)expression on the proliferation and clonal ability in colorectal cancer cells.Methods:Enzyme linked immunosorbent assay(ELISA)was used to detect the CRLF1 level in serum of colorectal cancer patients and healthy donors.RNA was extracted from fresh cancer tissues and tissues adjacent to cancer,the relative mRNA expression of CRLF1 was determined.Human colorectal cancer cell lines HCT116 and SW620 were cultured.si-NC and si-CRLF1 siRNA were synthesized and transfected into HCT116 and SW620 cells.After verifying the transfection efficiency by real-time PCR and ELISA,CCK8 assay was performed to detect the proliferation ability of colorectal cancer cells.Clone formation assay was also used to detect the ability of clone formation,flow cytometry was used to detect the apoptosis status in colorectal cancer cells after transfection.AKT signaling pathway activity was detected by Western blot.Results:The serum CRLF1 level in colorectal cancer patients was significantly higher than that in healthy donors.Consistently,the expression level of CRLF1 mRNA in colorectal cancer tissue was significantly higher than that in tissue adjacent to cancer.In HCT116 and SW620 cell lines,the expression of CRLF1 was significantly downregulated by si-CRLF1,which significantly reducing the proliferation and colon formation(P<0.05),and inducing apoptosis in both cell lines.In addition,downregulation of CRLF1 significantly reduced the expression of p-AKT.Conclusion:CRLF1 expression regulates the progression of colorectal cancer cells though AKT signaling pathway.CRLF1 may become a biomarker for the diagnosis and a potential target for colorectal cancer.