LINC00665靶向miR-379-5p对肝癌细胞生物行为的影响及其机制研究

Effect of LINC00665 on hepatocellular carcinoma cell proliferation,apoptosis,migration and invasion by targeting miR-379-5p

目的研究LINC00665对肝癌细胞增殖、迁移、侵袭和凋亡的影响和潜在的分子机制。方法选取2013年6月至2018年6月于济南市第三人民医院病理检查确诊为肝癌且经手术切除126例患者的肝癌组织及癌旁组织(距离肝癌组织边缘>2 cm),其中男86例,女40例,年龄25.0~72.0(48.2±9.9)岁。qRT-PCR检测126例肝癌组织和癌旁组织中LINC00665的表达,CCK8法测定肝癌HCC9204细胞存活率,流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告系统验证LINC00665与miR-379-5p的调控关系。结果与癌旁组织相比,肝癌组织组中的LINC00665表达量升高[(1.00±0.10)比(1.82±0.18)],差异具有统计学意义(P<0.05)。LINC00665含量降低后[(1.01±0.10)比(0.53±0.05)],HCC9204细胞中细胞周期蛋白D1(CyclinD1)、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、基质金属蛋白酶-2(MMP-2)表达量降低[(0.74±0.07)比(0.13±0.01);(0.81±0.08)比(0.35±0.03);(0.69±0.07)比(0.22±0.02)],p21、Bax和E-钙黏蛋白(E-cadherin)表达升高[(0.20±0.02)比(0.66±0.06);(0.26±0.03)比(0.78±0.08);(0.17±0.02)比(0.72±0.07)],细胞存活率降低[(100.13±10.14)%比(46.22±4.73)%],细胞凋亡率上升[(8.42±0.91)%比(23.34±2.37)%],迁移和侵袭细胞数均下降[(109±11)个比(53±5)个;(101±10)个比(47±5)个],差异均具有统计学意义(均P<0.05)。过表达miR-379-5p组共转染野生型WT-LINC00665报告载体的细胞中萤火虫荧光素酶相对活性显著降低[(1.03±0.10)比(0.24±0.02)],转染突变型MUT-LINC00665的细胞中荧光素酶相对活性差异不具有统计学意义(P>0.05);LINC00665过表达组(pcDNA3.1-LINC00665)的miR-379-5p含量下降[(1.01±0.10)比(0.37±0.04)];LINC00665抑制组(si-LINC00665)的miR-379-5p含量上升[(0.98±0.10)比(1.66±0.17)],差异均具有统计学意义(P<0.05);降低LINC00665含量同时降低miR-379-5p含量,细胞存活率升高[(46.53±4.72)%比(82.26±8.34)%],细胞凋亡率降低[(23.51±2.44)%比(12.07±1.21)],迁移细胞数和侵袭细胞数均升高[(54±6)个比(92±9)个;(48±5)个比(88±9)个],差异均具有统计学意义(均P<0.05)。结论在肝细胞癌HCC9204中,LINC00665靶向调控miR-379-5p的表达,进而调控肝癌细胞HCC9204增殖、迁移、侵袭和凋亡,是肝癌的潜在分子靶点。

Objective To investigate the effect and mechanism of LINC00665 on hepatocellular carcinoma cell proliferation,migration,invasion and apoptosis.Methods From June 2013 to June 2018,126 liver cancer tissue and adjacent tissue(more than 2 cm from the edge of liver cancer tissue)specimens were collected in the Third People's Hospital of Jinan,86 male and 40 female were included,aged 25.0-72.0(48.2±9.9)years.The expression level of LINC00665 in 126 liver cancer tissues and adjacent tissues were detected by qRT-PCR.The survival rate of hepatocellular carcinoma HCC9204 cells was determined by CCK8 assay.The apoptosis of HCC9204 cells was detected by flow cytometry,cell migration and invasion were detected by Transwell assay.And the dual-luciferase reporter assay system was implemented to investigate the correlations between LINC00665 and miR-379-5p.Results Compared with the adjacent tissues group,the expression level of LINC00665 in liver cancer tissues group was increased(1.00±0.10 vs.1.82±0.18),with statistically significant difference(P<0.05).When LINC00665 was inhibited[(1.01±0.10)vs.(0.53±0.05)],the expression of Cyclin D1,B-cell lymphoma-2(Bcl-2)and matrix metalloproteinase-2(MMP-2)in HCC9204 cells were decreased as well[(0.74±0.07)vs.(0.13±0.01);(0.81±0.08)vs.(0.35±0.03);(0.69±0.07)vs.(0.22±0.02)],but the level of p21,Bax and E-cadherin were increased[(0.20±0.02)vs.(0.66±0.06);(0.26±0.03)vs.(0.78±0.08);(0.17±0.02)vs.(0.72±0.07)],cell survival rate was decreased[(100.13±10.14)%vs.(46.22±4.73)%],apoptosis rate was increased[(8.42±0.91)%vs.(23.34±2.37)%],and the number of migrating and invading cells were decreased[(109±11)vs.(53±5);(101±10)vs.(47±5)],the differences were statistically significant(all P<0.05).In miR-379-5p overexpression group,the relative activity of firefly luciferase in cells which were co-transfected with wild-type WT-LINC00665 report vector was significantly decreased[(1.03±0.10)ratio(0.24±0.02)],and the relative activity of luciferase in cells which were transfected with mutant MUT-LINC00665 was not decreased significantly(P>0.05);The level of miR-379-5p in the LINC00665 overexpression group(pcDNA3.1-LINC00665)was decreased[(1.01±0.10)vs(0.37±0.04)];but was increased in the LINC00665 inhibition group(si-LINC00665)[(0.98±0.10)vs(1.66±0.17)],with statistically significant differences(P<0.05);Decreasing the content of LINC00665 and miR-379-5p,The cell survival rate was increased[(46.53±4.72)%vs.(82.26±8.34)%],the apoptosis rate was decreased(23.51±2.44)%vs.(12.07±1.21)%],and the number of migrating cells and invasive cells were increased[(54±6)vs.(92±9);(48±5)vs.(88±9)]when LINC00665 and miR-379-5p were inhibited the difference were statistically significant(all P<0.05).Conclusions In hepatocellular carcinoma HCC9204,LINC00665 targets the regulation of miR-379-5p expression,thereby regulating the proliferation,migration,invasion,and apoptosis of hepatocellular carcinoma HCC9204,which is a potential molecular target for liver cancer.