三结构域蛋白21对结肠癌生物学行为的影响

Effect of TRIM21 on the biological behavior of colon cancer

目的目前三结构域蛋白21(TRIM21)在结肠癌中的临床意义及生物功能仍不明确。文中旨在运用慢病毒构建结肠癌HCT116细胞TRIM 21基因过表达及低表达细胞株,观察TRIM21对结肠癌生物学行为的影响,采用RNA测序以揭示TRIM21调控结肠癌发生发展的下游分子的影响。方法收集2019年10月至2020年5月南京医科大学附属逸夫医院20例结肠癌患者的手术切除标本,分别取癌组织及癌旁肠壁组织,进行石蜡切片免疫组化染色。Western blot检测人正常结肠上皮细胞NCM460及人结肠癌细胞HCT116、HT29、SW620的TRIM21表达,分析结肠癌细胞系的TRIM21蛋白表达水平;通过慢病毒载体分别构建TRIM21稳定过表达、稳定低表达的HCT116细胞株模型,将结肠癌细胞分为对照组(过表达空载)、过表达组(转染TRIM21过表达慢病毒)、空载组(低表达空载对照)、低表达组(转染TRIM21低表达慢病毒),Western blot检测构建的模型;磺酰罗丹明B(SRB)实验、克隆形成实验检测24、48、72、96 h细胞增殖,划痕实验检测0、24、48 h的细胞迁移;RNA测序从转录组水平揭示TRIM21参与调控的下游分子,并进行功能分析。结果结肠癌患者癌组织中TRIM21免疫组化评分[(2.700±0.250)分]明显高于癌旁组织[(2.500±0.346)分],差异有统计学意义(P<0.05)。HCT116、HT29、SW620细胞TRIM21蛋白相对表达量(1.443±0.108、2.413±0.526、1.453±0.154)较NCM460细胞(1.000±0.000)明显升高(P<0.01)。SRB细胞增殖实验结果显示,在48 h、72 h和96 h,过表达组的HCT116结肠癌细胞生长能力(0.382±0.013,1.083±0.176,1.432±0.051)较对照组(0.292±0.039,0.856±0.065,1.186±0.101)明显增强(P<0.05)。48、72、96 h,低表达组HCT116结肠癌细胞生长能力(0.321±0.008,0.725±0.039,1.035±0.071)较空载组(0.392±0.026,0.857±0.055,1.275±0.027)明显降低(P<0.05)。24、48 h,过表达组结肠癌细胞的迁移能力[(43.756±3.987)%、(58.009±7.370)%]较对照组[(27.926±4.312)%、(43.075±2.888)%]明显增强(P<0.01),低表达组细胞的迁移能力[(19.376±5.107)%、(31.506±5.085)%]较空载组[(29.648±4.838)%、(42.731±3.715)%]明显减弱(P<0.01)。结论干扰TRIM 21基因表达可抑制结肠癌HCT116细胞的增殖和迁移,为结肠癌靶向治疗提供了一个新的方向。

Objective Until now,it is still unclear that the clinical significance and biological function of TRIM21 in colon cancer.Therefore,it is of great clinical and theoretical significance to fully understand the expression and function of TRIM21 in colon cancer.The purpose of this study was to investigate the effect of TRIM21 in colon cancer.Lentivirus was used to construct colon cancer HCT116 cells with TRIM21 gene overexpression and low expression.RNA sequencing revealed the downstream molecules that TRIM21 regulated the development of colon cancer.Methods 20 colon cancer specimens were collected from Sir Runrun Hospital of Nanjing Medical University from October 2019 to May 2010.The cancerous tissues and its adjacent tissues were subject to HE staining and immunohistochemical analysis.Western blot was used to detect the expression of TRIM21 in human colon epithelial cells NCM460 and human colon cancer cells HCT116,HT29 and SW620,in order to analyze the endogenic expression of TRIM21.Colon cancer cells were divided into NC group(control group to overexpression),TRIM21 OE group(overexpression lentivirus group),shNC group(control group to low-expression),and TRIM21 KD group(low-expression lentivirus group).The result of Western blot confirmed the success of this studying model.In vitro experiments,cell proliferation was detected by SRB assay and clone formation assay,and cell migration was detected by wound healing assay.RNA sequencing revealed the involvement of TRIM21 in the regulation of downstream molecules at the transcriptome level and functional analysis was performed.Results The relative expression of TRIM21 protein in colon cancer tissues(2.700±0.250)was significantly higher than that in adjacent tissues(2.500±0.346).The relative expression of TRIM21 protein in HCT116,HT29 and SW620 cells(1.443±0.108,2.413±0.526,1.453±0.154)was significantly higher than that in NCM460 cells(1.000±0.000,P<0.01).The data from in vitro experiments showed that the proliferation rate and migration ability of TRIM21 OE group were significantly higher than those of NC group(P<0.01).On the contrary,the proliferation rate and migration ability of TRIM21 KD group were significantly lower than that of shNC group(P<0.01).RNA sequencing revealed that TRIM21 regulated a variety of biological functions.Conclusion Interference with TRIM21 can inhibit the proliferation and migration of colon cancer HCT116 cells,providing a new direction for targeted therapy of colon cancer.