HOXA末端转录本反义RNA在肝癌组织中的表达及对肝癌HepG2细胞增殖、侵袭与迁移能力的影响

Expression of HOXA terminal transcript antisense RNA in hepatocellular carcinoma tissues and its effect on proliferation, invasion and migration of hepatocellular carcinoma HepG2 cells

目的探讨HOXA末端转录本反义RNA(HOTTIP)在肝癌组织中的表达以及对肝癌HepG2细胞增殖、侵袭与迁移能力的影响。方法收集2012年1月至2018年6月在辽宁省丹东市第一医院经手术切除的肝癌组织及对应的癌旁组织标本各60例,应用实时荧光定量聚合酶链反应(RT-qPCR)检测HOTTIP在肝癌组织及癌旁组织中的表达情况,并分析表达情况与临床病理特征的关系。通过细胞转染技术构建HOTTIP高表达HepG2细胞株为实验组,设空载质粒pcDNA3.1-NC为对照组。CCK-8法检测HOTTIP对HepG2细胞增殖能力的影响,Transwell法检测HOTTIP对HepG2细胞侵袭与迁移能力的影响。结果HOTTIP mRNA在肝癌组织中的表达高于癌旁组织,差异有统计学意义(1.9±0.6比0.9±0.7,t=6.069,P<0.01)。以HOTTIP mRNA表达中位值(1.92)为临界点,将60例肝癌病例分为高表达组(30例)和低表达组(30例)。HOTTIP表达与TNM分期、分化程度及淋巴结转移有关(χ2值分别为10.800、8.076、5.711,均P<0.05),与患者年龄、性别、肿瘤直径、肿瘤数目、有无合并肝炎及甲胎蛋白(AFP)水平无关(均P>0.05)。RT-qPCR结果显示转染过表达HOTTIP肝癌HepG2细胞中HOTTIP mRNA表达增加,与对照组相比差异有统计学意义(63±6比13±9,t=9.129,P<0.01)。CCK-8法实验结果提示HepG2细胞过表达HOTTIP后细胞增殖活力增强(24、36、72 h在490 nm处吸光度分别为1.497±0.017比0.826±0.006、2.002±0.025比1.211±0.020、3.257±0.042比1.772±0.021),差异均有统计学意义(t值分别为5.321、7.349、8.793,均P<0.01)。Transwell侵袭实验中,实验组转入底层膜的细胞数为(101±9)个,多于对照组的(41±11)个,差异有统计学意义(t=6.839,P<0.01)。Transwell迁移实验中,实验组转入底层膜的细胞数为(112±9)个,多于对照组的(53±11)个,差异有统计学意义(t=7.105,P<0.01)。结论HOTTIP在肝癌组织中表达上调,过表达HOTTIP后能够促进肝癌细胞的增殖、侵袭与迁移。

Objective To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P < 0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ2 values were 10.800, 8.076, 5.711, all P < 0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P > 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.