LETM1在肝癌中的表达及其对SMMC-7721细胞增殖和凋亡的影响

Expression of LETM1 in hepatocellular carcinoma and its effect on proliferation and apoptosis in hepatocellular carcinoma cell line SMMC-7721

目的探讨亮氨酸拉链EF-hand结构域跨膜蛋白1(leucine bnzipper/EF-hand-containing transmembrane protein1,LETM1)在肝癌组织中的表达水平及通过RNAi技术靶向LETM1基因对肝癌细胞株SMMC-7721的增殖及凋亡的影响。方法采用免疫组织化学方法、Western blot检测重庆医科大学附属第一医院肝胆外科2012年9月至2013年4月共60例肝癌组织及其癌旁肝组织中LETM1蛋白表达情况。Western blot检测人肝癌细胞株SMMC-7721和Hep G2及人正常肝细胞株LO2中的LETM1蛋白表达情况。将LETM1 siRNA经Lipofectamine 2000TM脂质体转染SMMC-7721(实验组为si-LETM1、对照组为si-NC),实时荧光定量PCR及Western blot分别检测LETM1转染后肝癌SMMC-7721细胞株中LETM1 mRNA及蛋白的表达。CKK-8及流式细胞术检测LETM1低表达对肝癌细胞增殖及细胞凋亡的影响。结果 1免疫组织化学结果显示,LETM1蛋白在肝癌组织中的阳性表达率为73.33%(44/60),明显高于癌旁组织中的表达率28.33%(17/60,P<0.01);Western blot检测结果显示,LETM1蛋白在肝癌组织中的表达相对量(2.335±0.221)较癌旁组织(1.386±0.081)明显增高(P<0.01)。2LETM1蛋白表达与肝癌肿瘤直径及门静脉侵犯有关(P<0.05),而与肝癌患者性别、年龄、甲胎蛋白、HBs Ag、Edmondson分级及TNM分期无关(P>0.05)。3LETM1蛋白在SMMC-7721及Hep G2中的表达相对量[(分别为(1.447±0.149)、(1.190±0.113)]均明显高于LO2[(0.918±0.027),P<0.05]。4si-LETM1组与si-NC组和空白组比较,si-LETM1组细胞的增殖受到抑制(P<0.01),尤以72 h及96 h最为显著;si-LETM1早期细胞凋亡数(14.6±2.3)%与si-NC早期细胞的凋亡数(6.3±0.7)%比较,差异有统计学意义(P=0.04)。结论 LETM1蛋白在肝癌组织的表达显著高于癌旁组织;基因沉默LETM1后的肝癌细胞其增殖能力明显减弱,凋亡能力明显增强。

Objective To analyze the expression of leucine zipper/EF-hand-containing transmembrane protein 1 （ LETM1 ） in the human hepatocellular carcinoma （HCC） and to determine the effect of RNA interference targeted to silence LETMI gene on the proliferation and apoptosis of HCC cell line SMMC- 7721. Methods Immunohistochemical assay and Western blotting were used to analyze the expression level of LETM1 protein in HCC tissues and paraneoplastic tissues from 60 HCC patients who admitted in our department from September 2012 to April 2013. The relationship between LETM1 expression and clinical pathological parameters was studied. Western blotting was also used to analyze the expression of LETM1 protein in SMMC-7721, HepG2 and normal liver LO2 cells. RNA interference targeted to silence LETM gene was transfected into the SMMC-7721 cells with Lipofectamine 2000 （representing the control group si-NC and the experimental group si-LETM1 ）. Quantitative real-time PCR （qRT-PCR） and Western blotting were used to detect the mRNA and protein levels of LETM1 in the silenced ceils. The proliferation and apoptosis of the transfected cells were assessed by CCK-8 assay and flow cytometry. Results indicated that LETM1 protein was expressed in 73.33% HCC tissues（44/60）, Immunohistochemical assay significantly higher than that of paraneoplastic tissues （28.33%, 17/60, P 〈 0.01 ）. The results showed that the expression at protein level was also higher in the HCC tissues than the paraneoplastic tissues （2. 335 ± 0. 220 vs 1. 386± 0. 080, P 〈0.01 ）. The expression of LETM1 protein was correlated with tumor size and portal vein invasion （P 〈 0. 05）, but not with gender, age, alpha fetoprotein, HBsAg, Edmondson grade and TNM stage （P 〉0.05）. The expression level of LETM1 protein was significantly higher in the SMMC-7721 （ 1. 447 ±0. 149 ） and HepG2 ceils （ 1. 190 ± 0.113 ） than the LO2 cells （0.918 ± 0. 027, P 〈 0.05 ）. Compared with si-NC group and blank group, the cell proliferation was inhibited significantly in si-LETM1 treated cells （P 〈 0.01 ）, especially at 72 and 96 h. The early apoptosis of si-LETM1 transfected cells had a significant increase when compared with si-NC treated cells [ （ 14.6 ± 2.3 ） % vs （6.3± 0.7） %, P = 0.04 ]. Gonclusion The protein expression of LETM1 is significantly higher in HCC tissues than the paraneoplastic tissues. RNAi of LETM1 may inhibit cells proliferation and induce apoptosis in the SMMC-7721 cells.