环状RNA circKDM4B调控肝癌细胞增殖及侵袭迁移的实验研究

doi:10.3969/j.issn.1009-976X.2021.06.005

摘要/Abstract

摘要： 目的探索环状RNA circKDM4B对肝癌细胞增殖、凋亡和侵袭迁移的影响,明确其在肝癌进展中所发挥的调控作用。方法采用核糖核酸酶R（RNase R）消化实验和荧光原位杂交实验（FISH）验证circKDM4B的环状结构稳定性及其在细胞中的定位;利用小干扰RNA（siRNA）敲减肝癌细胞circKDM4B的表达,并通过qRT-PCR实验验证敲减效率;采用细胞计数方法（CCK-8）实验、EdU增殖实验检测circKDM4B对肝癌细胞增殖的影响,流式细胞技术检测其对肝癌细胞凋亡的影响;同时应用划痕实验、Transwell实验检测circKDM4B对肝癌细胞迁移侵袭能力的影响。结果相较于线性KDM4B mRNA,circKDM4B对RNase R耐受性更加稳定,FISH结果显示circKDM4B主要定位于肝癌细胞质中;在转染了circKDM4B siRNA后,circKDM4B在肝癌细胞表达下调,而亲本基因KDM4B不受影响。CCK-8和EdU增殖实验结果显示,敲减circKDM4B可抑制肝癌细胞的增殖;流式细胞实验显示敲减circKDM4B可明显促进肝癌细胞凋亡;划痕实验和Transwell实验表明敲减circKDM4B后肝癌细胞的侵袭和迁移能力明显减弱。结论 circKDM4B稳定表达于肝癌细胞质中,并具有促进肝癌细胞增殖、侵袭迁移、抑制肝癌细胞凋亡的能力,这提示circKDM4B有望成为新的肝癌治疗靶点。

关键词: 肝细胞癌, 环状RNA, 增殖, 凋亡, 迁移

Abstract: Objective To clarify the role of circKDM4B on the proliferation, apoptosis, invasion and migration of hepatocellular carcinoma （HCC） cells. Methods In vitro stability of circKDM4B was examined by RNase R treatment, and its location in cells was determined by fluorescence in situ technique （FISH）. The expression of circKDM4B was silenced by small interfering RNA （siRNA） in SNU 387 and Huh7 cells, respectively, then verified by qRT-PCR. After that, CCK-8 assay, EdU assay, wound healing assay, transwell assays and flow cytometry were used to detect the effects of circKDM4B silencing on HCC cell proliferation, apoptosis, invasion and migration. Results circKDM4B was stable in vitro and not affected by RNase R. FISH showed that circKDM4B was mainly located in cytoplasm. After transfection with si-circKDM4B, the expression levels of circKDM4B were down-regulated. CCK-8, EdU assay and flow cytometry showed that knock-down of circKDM4B significantly inhibited the proliferation ability of HCC cells, and promoted the apoptosis of HCC cells. While wound healing and transwell assay confirmed that knock-down of circKDM4B inhibited the migration and invasion ability of HCC cells. Conclusion circKDM4B is stably located in the cytoplasm of HCC cells, and promotes the proliferation, migration and invasion while inhibits apoptosis of HCC cells, which suggests its potential role as a novel therapy target for HCC.

Key words: hepatocellular carcinoma, circKDM4B, proliferation, apoptosis, migration

中图分类号:

R735.7

马晓武, 谭文亮, 杨磊, 谢智钦, 王庆斌, 陈亚进, 商昌珍. 环状RNA circKDM4B调控肝癌细胞增殖及侵袭迁移的实验研究[J]. 岭南现代临床外科, 2021, 21(06): 614-619.

MA Xiao-wu, TAN Wen-liang, YANG Lei, XIE Zhi-qin, WANG Qing-bin, CHEN Ya-jin, SHANG Chang-zhen. Experimental study of circKDM4B on the proliferation, migration and invasion of hepatocellular carcinoma cells[J]. Lingnan Modern Clinics In Surgery, 2021, 21(06): 614-619.

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