多组学筛选及验证lncRNA CCAT1通过调节miR-148b-3p和FGF9促进结直肠癌发生及发展机制研究

Multiomics screening and verification of lncRNA CCAT1 promotes the occurrence and development of colorectal cancer by regulating miR-148b-3p and FGF9

目的通过生物信息学及分子生物学探讨lncRNA CCAT1对结直肠癌(colorectal cancer,CRC)细胞增殖的影响,并进一步探讨其机制。方法应用R软件和Bioconductor软件包对9例CRC样本和9例正常样本进行差异表达基因(DEGs)分析,筛选差异表达lncRNA和mRNA。进一步选择30例CRC组织及匹配的癌旁组织(2016年1月至2019年12月从新疆医科大学第一附属医院CRC外科手术中获得的标本),然后通过实时荧光定量PCR(qRT-PCR)检测CRC组织及CRC细胞中CCAT1、miR-148b-3p、FGF9的表达,采用蛋白免疫印迹试验(Western blotting)检测CRC细胞中FGF9的表达情况。细胞转染后,用CCK-8法检测细胞活力,流式细胞术检测细胞周期和凋亡。采用Transwell迁移实验检测各处理组细胞迁移情况。通过双荧光素酶报告基因检测验证CCAT1与miR-148b-3p、miR-148b-3p与FGF9的靶向关系。结果与癌旁组织及人正常结直肠上皮细胞相比,CCAT1在CRC组织和细胞系中高表达且差异有统计学意义(P<0.05)。进一步分析发现,高表达CCAT1的CRC患者与低表达CCAT1的患者相比,肿瘤较大,TNM分期较晚,总生存率较低。生物学功能测定结果表明,CCAT1具有促进细胞增殖的作用。我们还发现miR-148b-3p可以作为CCAT1在CRC细胞中的作用靶点,miR-148b-3p的过表达可以有效抑制由CCAT1敲低诱导的CRC进程。此外,我们发现FGF9作为miR-148b-3p的靶点在CRC中发挥致癌作用。结论CCAT1可通过调控miR-148b-3p及其靶蛋白FGF9促进CRC细胞增殖,这将为CRC的预防及治疗提供一个有效的治疗靶点及理论依据。

Objective To explore the effect of lncRNA CCAT1 on the proliferation of colorectal cancer(CRC)cells through bioinformatics and molecular biology methods,and to further explore its mechanism.Methods R software and Bioconductor software package were used to analyze differentially expressed genes(DEGs)in 9 CRC samples and 9 normal samples,and screen differentially expressed lncRNA and mRNA.30 cases of CRC tissues and matched paracancerous tissues were further selected as the specimens obtained from the CRC surgery of the First Affiliated Hospital of Xinjiang Medical University from Jan.2016 to Dec.2019,and then used real-time fluorescent quantitative PCR(qRT-PCR)to detect the expression of CCAT1,miR-148b-3p,and FGF9 in CRC tissues and CRC cells.Western blotting was used to detect the expression of FGF9 in CRC cells.After cell transfection,CCK-8 was used to detect cell viability,and flow cytometry was used to detect cell cycle and apoptosis.Transwell migration experiment was used to detect cell migration in each treatment group.Through dual luciferase reporter gene detection,the target relationship between CCAT1 and miR-148b-3p,miR-148b-3p and FGF9 was verified.Results Compared with adjacent tissues and human normal colorectal epithelial cells,CCAT1 was highly expressed in CRC tissues and cell lines and the difference was statistically significant(P<0.05).Further analysis found that CRC patients with high expression of CCAT1 had larger tumor diameter,later TNM staging,and poorer overall survival rate than patients with low expression of CCAT1.The functional test results showed that CCAT1 had the effect of promoting cell proliferation.We also found that miR-148b-3p could be used as the target of CCAT1 in CRC cells,and the overexpression of miR-148b-3p could inhibit the CRC process induced by CCAT1 knockdown.In addition,we found that FGF9,as a target of miR-148b-3p,played a carcinogenic effect in CRC.Conclusion CCAT1 can promote CRC cell proliferation by regulating miR-148b-3p and its target protein FGF9,which will provide an effective therapeutic target and theoretical basis for the prevention and treatment of CRC.