长链非编码RNA同源盒基因A11反义RNA对肝癌细胞增殖和肝癌相关基因表达的影响

Effects of long non-coding RNA homeobox A11 antisense knockdown on the proliferation and liver cancer-related genes expression of hepatocellular carcinoma cells

目的探究长链非编码RNA(lncRNA)同源盒基因A11反义RNA(HOXA11-AS)对肝细胞癌(HCC)细胞增殖及HCC相关基因表达的影响。方法15例人HCC组织及癌旁组织由西安交通大学第二附属医院生物诊疗中心样本库于2017年6月至2019年11月收集。采用实时定量聚合酶链反应(qPCR)检测HOXA11-AS的表达并用RNA荧光原位杂交进行亚细胞定位;通过转染lncRNA Smart Silencer敲低Bel-7402和Hep3细胞中HOXA11-AS的表达,并采用细胞计数试剂(CCK-8)、平板克隆形成实验、流式细胞术检测细胞的增殖、周期及凋亡情况;利用qPCR芯片检测敲低HOXA11-AS对HCC相关基因表达的影响,使用R语言clusterProfiler包对差异基因进行功能富集分析,并通过rescue实验验证差异基因介导HOXA11-AS调控HCC细胞增殖的作用。分别采用t检验和Turkey检验分析两组或多组间差异。结果HOXA11-AS在HCC组织中的表达明显高于癌旁组织(9.72±5.99比5.53±4.92,t=2.09,P<0.05),在3种HCC细胞系(Bel-7402、HepG2和Hep3B)中的表达显著高于与正常肝细胞系L-O2(1.81±0.33、1.49±0.17、3.53±0.30比1.00±0.02,t=4.20、4.94、14.34,P均<0.05)。RNA荧光原位杂交显示HOXA11-AS在HCC细胞的胞核和胞质均有分布。HOXA11-AS功能缺失实验提示敲低组细胞增殖活性明显低于对照组(Bel-7402:0.95±0.03比1.27±0.07,t=8.60,P<0.01;Hep3B:0.59±0.04比0.74±0.03,t=6.00,P<0.01);敲低组细胞克隆形成数量显著低于对照组(Bel-7402:228.33±25.18比572.33±26.08,t=19.74,P<0.01;Hep3B:112.33±11.50比214.33±14.64,t=9.49,P<0.01);敲低组S期细胞比例显著高于对照组[Bel-7402:(33.13±2.37)%比(18.92±0.49)%,t=10.15,P<0.01;Hep3B:(27.66±0.85)%比(21.88±0.81)%,t=8.48,P<0.01];敲低组凋亡细胞比例显著高于对照组[Bel-7402:(18.50±0.95)%比(15.43±1.24)%,t=3.41,P<0.05;Hep3B:(8.65±0.88)%比(3.28±0.34)%,t=9.82,P<0.01]。qPCR芯片检测显示敲低HOXA11-AS可显著改变46个HCC相关基因的表达,这些基因显著富集于细胞增殖、周期和凋亡等相关生物学过程以及肿瘤坏死因子(TNF)、酪氨酸激酶(JAK)/信号转导与转录激活因子(STAT)、磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)、丝裂原活化蛋白激酶(MAPK)和Ras等肿瘤相关通路。过表达KIT、SPARC、HDAC10可逆转HOXA11-AS敲低对HCC细胞增殖的抑制作用。结论HOXA11-AS在HCC细胞中高表达,可通过影响细胞周期、凋亡以及HCC相关基因的表达促进HCC细胞增殖。

Objective To investigate effects of long non-coding RNA(lncRNA)homeobox A11 antisense(HOXA11-AS)knockdown on the proliferation of hepatocellular carcinoma(HCC)cells and the expression of HCC-related genes.Methods Fifteen cases of HCC and adjacent tissues were collected by the Biobank of National&Local Joint Engineering Research Center of Biodiagnositcs and Biotherapy of the Second Affiliated Hospital of Xi’an Jiaotong University from June 2017 to November 2019.The expression of HOXA11-AS was examined by quantitative polymerase chain reaction(qPCR)and its subcellular distribution of in HCC cells was detected by RNA-Fluorescence in situ hybridization(FISH).HOXA11-AS was knocked down by transfecting the lncRNA Smart Silencer in Bel-7402 and Hep3B cells,then the proliferation ability,cell cycle,and apoptosis of HCC cells were evaluated by cell counting kit(CCK-8),clone formation,and flow cytometry assays.The effect of HOXA11-AS silencing on the expression of HCC-related genes was detected by qPCR array,and functional enrichment analysis were conducted using R package cluster Profiler.The mediation of HCC-related genes in HOXA11-AS regulating HCC cell proliferation were verified by rescue experiments.The t-test and Turkey test were used to analyze the differences between two or more groups,respectively.Results HOXA11-AS was up-regulated in HCC tissues and three HCC cell lines(Bel-7402,HepG2 and Hep3B).RNA-FISH showed that HOXA11-AS was distributed in both the nucleus and cytoplasm of HCC cells.Upon HOXA11-AS knockdown,the proliferative activity and colony formation ability of Bel-7402 and Hep3B cells were significantly suppressed,while the proportion of S-phase and apoptotic cells were significantly increased.The qPCR array disclosed that 46 of HCC-related genes whose expression was dramatically altered when HOXA11-AS silencing,and these genes were significantly enriched in biological processes related to cell proliferation,cycle and apoptosis,as well as several critical tumor-related pathways such as tumor necrosis factor(TNF),janus kinase(JAK)/signal transducer and activators of transcription(STAT),phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt),mitogen-activated protein kinase(MAPK)and Ras.Overexpression of KIT,SPARC and HDAC10 could reverse the inhibitory effect of HOXA11-AS silencing on the proliferation of HCC cells.Conclusion Elevated HOXA11-AS contributes to HCC proliferation through affecting cell cycle,apoptosis as well as the expression of HCC-related genes.