Circ0066629促进结直肠癌细胞活力的体外研究

Circ0066629 promotes colorectal cancer cell viability in vitro

目的探讨环状RNA circ0066629促进结直肠癌细胞活力作用及其调控的分子机制。方法 35例结直肠癌5-氟尿嘧啶(5-Fu)及伊立替康耐药和不耐药患者组织来源于南京医科大学附属上海市松江中心医院,不同结直肠癌细胞系来源于美国菌种保藏中心,利用RT-q PCR测定circ0066629在结直肠癌组织及细胞系中的表达水平。Circ0066629过表达质粒、siRNA质粒、miR-219a-2-3p过表达质粒(miR-219a-2-3p mimics)及miR-219a-2-3p干扰质粒(miR-219a-2-3p inhibitor)用于调控circ0066629和miR-219a-2-3p的表达。RT-q PCR用于测定转染效率。随后,应用CCK-8和细胞划痕实验测定LOVO细胞活力。荧光素酶报告基因实验用于评估circ0066629与miR-219a-2-3p以及p53与miR-219a-2-3p的靶向关系。蛋白免疫印迹实验用于测定p53和鼠双微体基因2(MDM2)在LOVO细胞中的蛋白表达水平。结果 circ0066629在结直肠癌5-Fu及伊立替康耐药患者组织中表达下调,但在不同结直肠癌细胞系中表达上调。过表达circ0066629明显促进了5-Fu和伊立替康处理下的LOVO细胞活力。MiR-219a-2-3p被预测为circ0066629的靶基因,并且miR-219a-2-3p过表达对LOVO细胞活力的作用与circ0066629相反。另外,p53是miR219a-2-3p的预测靶向基因,p53特异性抑制剂(PFT-α)明显抑制LOVO细胞活力。结论 circ0066629通过调控miR-219a-2-3p/P53/MDM2轴促进结直肠癌细胞活力。

Objective To explore the role of circ0066629 in colorectal cancer cell viability and reveal its regulatory molecular mechanisms.Methods The tissues of 35 colorectal cancer patients with 5-FU and irinotecan resistance or non resistance were obtained from Shanghai Songjiang Central Hospital of Nanjing Medical University,and different colorectal cancer cell lines were from the American Type Culture Collection(ATCC).RT-qPCR assay was employed circ0066629 expression in above tissues and cell lines.The circ0066629 overexpressed/siRNA vectors and miR-219a-2-3p mimics/miR-219a-2-3p inhibitor vectors were used to regulate circ0066629 and 219a-2-3p expression.Cell transfection efficiency was detected via RT-qPCR.Subsequently,CCK-8 and the scratch assay was adopted to evaluate the vitality ability of LO-VO cells.Luciferase reporter assay was utilized to predicate the targeted correlation between circ0066629 and miR-219a-2-3p or p53 and miR-219a-2-3p.Western blotting assay was implemented to determine the protein levels of p53 and murine double minute 2 (MDM2) in LOVO cells.Results Circ0066629 was significantly down-regulated in 5-Fu and irinotecan resistant colorectal cancer tissues,but up-regulated in diverse colorectal cancer cell lines.Overexpressed circ0066629 accelerated 5-FU and irinotecan-disposed LOVO cell viability.MiR-219a-2-3p was predicated as a direct target gene of circ0066629,and the functions of miR-219a-2-3p overexpression on LOVO cell viability was contrary to circ006629.Additionally,p53 was a predicated target gene of miR-219a-2-3p,and p53 inhibitor (PFT-α) clearly restrained cell viability LOVO cells.Conclusion Circ0066629 promoted colorectal cancer cell viability via regulating miR-219a-2-3p/p53/MDM2 aixs.