HBx上调lncRNA-HEIH对肝癌细胞增殖的影响

Effect of up-regulating lncRNA-HEIH by HBx on proliferation of hepatocellular carcinoma cells

目的探讨HBx上调肝细胞癌(肝癌)中高表达的长链非编码RNA(lncR NA-HEIH)对肝癌细胞增殖的影响。方法标本来源于2015年9月至2016年3月中山大学附属第三医院和中山大学附属肿瘤医院30例HBV相关性肝癌患者手术组织。所有患者均签署知情同意书,符合医学伦理学规定。其中男24例,女6例;年龄20~65岁,中位年龄52岁。采用RT-PCR检测30例患者肝癌组织及癌旁组织中HBx、lncRNA-HEIH m RNA的表达水平。构建稳定表达HBx的肝癌细胞系SMMC-7721-HBx及Bel-7404-HBx(实验细胞),及其对照细胞SMMC-7721-Vector和Bel-7404-Vector。RT-PCR及Western blot检测细胞株lncRNA-HEIH的表达。根据有否转染s i R N A-H E I H,将实验细胞分别分为干扰组和非干扰组。MTT检测两组吸光度值(A_(490))。实验数据比较采用t检验,lncRNA-HEIH与HBx表达相关性检验采用Pearson相关。结果肝癌组织中HBx、lncRNA-HEIH m RNA表达量分别为0.91±0.04、0.93±0.05,明显高于癌旁组织的0.53±0.01、0.35±0.02(t=7.95,10.42;P<0.05)。lncRNA-HEIH m RNA与HBx m RNA的表达水平呈正相关(r=0.840,P<0.05)。实验细胞SMMC-7721-HBx和Bel-7404-HBx中lncRNA-HEIH表达量分别为1.05±0.22、1.20±0.17,明显高于对照细胞的0.45±0.10、0.50±0.12(t=5.69,3.78;P<0.05)。SMMC-7721-HBx和Bel-7404-HBx干扰组48、72、96 h的A_(490)分别为0.26±0.05、0.38±0.04、0.43±0.10和0.27±0.05、0.30±0.12、0.39±0.05,明显低于非干扰组的0.37±0.07、0.59±0.12、0.80±0.10和0.38±0.07、0.62±0.12、0.78±0.11(t=-5.72,-3.47,-3.82和-3.89,-5.57,-8.34;P<0.05)。结论 lncRNA-HEIH高表达可能与HBV相关性肝癌发生有关,HBx基因可通过上调lncRNA-HEIH的表达参与HBx基因促进肝癌细胞增殖。

Objective To investigate the effect of up-regulating long noncoding RNA high expression in hepatocellular carcinoma （lncRNA-HEIH） by HBx on the proliferation of hepatocellular carcinoma （HCC） cells. Methods The samples were collected from 30 patients with hepatitis B virus （HBV）-related HCC who received surgery in the Third Affiliated Hospital of Sun Yat-sen University and Cancer Center of Sun Yat-sen University between September 2015 and March 2016. The informed consents of all patients were obtained and the local ethical committee approval was received. Among 30 patients, 24were males and 6 were females, aged 20-65 years old with a median age of 52 years old. The expression levels ofHBx mRNA and lncRNA-HEIH mRNA in the HCC tissues and paracarcinoma tissues were detected by RT-PCR. HCC cell lines SMMC-7721-HBx and Bel-7404-HBx stably expressing HBx were established as the experimental cells. And the control cells SMMC-7721-Vector and Bel-7404-Vector were also established. The expression of lncRNA-HE1H in the cell lines was detected by RT-PCR and Western blot. According to siRNA-HEIH was transfected or not, the experimental cells were respectively divided into the interference group and the non-interference group. The absorbance values （A490） of two groups were detected by MTT. The experimental data were compared using t test. The relationship between the expression of lncRNA-HEIH and HBx was analyzed using Pearson correlation analysis. Results The expression levels of HBx mRNA and lncRNA-HEIH mRNA in the HCC tissues were respectively 0.91±0.04 and 0.93±0.05, significantly higher than 0.53±0.01 and 0.35±0.02 in the paracarcinoma tissues （t=-7.95, 10.42; P〈0.05）. The expression level of lncRNA-HEIH mRNA was positively correlated with that of HBx mRNA （r=0.840, P〈0.05）. The expression level of lncRNA-HEIH in the SMMC-7721-HBx and Bel-7404-HBx cells was respectively 1.05±0.22 and 1.20±0.17, significantly higher than 0.45±0.10 and 0.50±0.12 in the control cells （t=5.69, 3.78; P〈0.05）. The A490 in the SMMC-7721-HBx and Bel-7404-HBx interference groups was respectively 0.26±0.05, 0.38±0.04, 0.43±0.10 and 0.27±0.05, 0.30±0.12, 0.3±0.05 at 48, 72 and 96 h, significantly lower than 0.37±0.07, 0.59±0.12, 0.80±0.10 and 0.38±0.07, 0.62±0.12, 0.78±0.11 in the non-interference group （t=-5.72, -3.47, -3.82 and -3.89, -5.57, -8.34; P〈0.05）. Conclusions High expression of lncRNA-HEIH is probably correlated with the incidence of HBV-related HCC. HBx gene is involved in promoting the proliferation of HCC cells via up-regulating the expression of lncRNA-HEIH.