肿瘤蛋白翻译控制反义RNA1靶向调控miR-30c-5p对肝癌细胞放射敏感性和增殖迁移及侵袭的影响

Effects of tumor protein translation control antisense RNA1 on radiosensitivity,proliferation,migration and invasion of hepatocellular carcinoma cells by targeting miR-30c-5p

目的探讨肿瘤蛋白翻译控制反义RNA1(TPT1-AS1)靶向miR-30c-5p对肝癌细胞放射敏感性、细胞增殖、迁移和侵袭的影响。方法34例肝癌组织及癌旁正常组织来源于2016年3月至2018年3月就诊于山西省人民医院的肝癌患者。将肝癌HepG2细胞分为si-NC组(转染si-NC)、si-TPT1-AS1组(转染si-TPT1-AS1)、pcDNA3.1组(转染pcDNA3.1)、pcDNA3.1-TPT1-AS1组(转染pcDNA3.1-TPT1-AS1)、si-TPT1-AS1+anti-miR-NC组(转染si-TPT1-AS1和anti-miR-NC)、si-TPT1-AS1+anti-miR-30c-5p组(转染si-TPT1-AS1+anti-miR-30c-5p)。采用实时荧光定量聚合酶链反应检测癌旁正常组织和肝癌组织中TPT1-AS1和miR-30c-5p的基因表达,克隆形成实验检测细胞放射敏感性,MTT实验检测细胞增殖活力,流式细胞数检测细胞周期分布,Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告基因实验验证TPT1-AS1和miR-30c-5p的靶向关系,Western blot检测增殖、迁移和侵袭相关蛋白的表达。结果肝癌组织中TPT1-AS1和miR-30c-5p的表达水平为0.84±0.08和0.13±0.01,与癌旁正常组织(分别为0.31±0.03和0.50±0.05)比较,差异均有统计学意义(均P<0.05)。采用2、4、6、8 Gy照射细胞时,si-TPT1-AS1组细胞存活分数分别为0.280±0.040、0.069±0.011、0.020±0.003和0.005±0.001,均低于si-NC组(分别为0.648±0.070、0.348±0.080、0.130±0.020和0.060±0.009,均P<0.05),si-TPT1-AS1组细胞放射增敏比为1.672。si-TPT1-AS1组细胞迁移数、侵袭数分别为(50.00±4.36)个和(44.00±4.03)个,低于si-NC组[分别为(109.00±8.68)个和(94.00±7.49)个,均P<0.05]。培养24、48和72 h,si-TPT1-AS1组细胞吸光度(A)值分别为0.28±0.03、0.43±0.04和0.68±0.07,低于si-NC组(分别为0.46±0.04、0.87±0.08和1.35±0.13,均P<0.05)。si-TPT1-AS1组细胞周期素D1(Cyclin D1)、p21、E-cadherin和基质金属蛋白酶2(MMP-2)蛋白表达水平分别为0.25±0.02、0.65±0.06、0.68±0.07和0.27±0.03,与si-NC组(分别为0.88±0.08、0.17±0.02、0.14±0.01和0.89±0.09)比较,差异均有统计学意义(均P<0.05)。si-TPT1-AS1组细胞S期和G2期细胞比例分别为(17.82±1.03)%和(34.15±2.29)%,与si-NC组[(35.14±2.61)%和(16.84±1.21)%]比较,差异均有统计学意义(均P<0.05)。WT-TPT1-AS1+miR-30c-5p组细胞荧光素酶活性为0.26±0.02,低于WT-TPT1-AS1+miR-NC组(0.92±0.09,P<0.05)。照射2、4、6、8 Gy,si-TPT1-AS1+anti-miR-30c-5p组细胞存活分数分别为0.450±0.081、0.200+0.045、0.070±0.010、0.026±0.004,高于si-TPT1-AS1+anti-miR-NC组(分别为0.285±0.043、0.075±0.014、0.028±0.004、0.006±0.001,均P<0.05)。si-TPT1-AS1+anti-miR-30c-5p组细胞放射增敏比为0.694。si-TPT1-AS1+anti-miR-30c-5p组细胞迁移数、侵袭数分别为(79.00±6.65)个和(68.00±6.33)个,高于si-TPT1-AS1+anti-miR-NC组[分别为(52.00±4.41)个和(46.00±4.06)个,均P<0.05]。培养24、48和72 h,si-TPT1-AS1+anti-miR-30c-5p组细胞A值分别为0.37±0.03、0.64±0.06和0.96±0.09,高于si-TPT1-AS1+anti-miR-NC组[分别为0.26±0.03、0.41±0.04和0.65±0.06,均P<0.05]。si-TPT1-AS1+anti-miR-30c-5p组Cyclin D1、p21、E-cadherin和MMP-2蛋白的表达水平分别为0.57±0.06、0.43±0.04、0.43±0.04和0.64±0.06,与si-TPT1-AS1+anti-miR-NC组(分别为0.24±0.02、0.66±0.06、0.65±0.06和0.28±0.03)比较,差异均有统计学意义(均P<0.05)。结论TPT1-AS1在肝癌组织中表达上调,TPT1-AS1可能通过靶向下调miR-30c-5p的表达,降低肝癌细胞的放射敏感性,促进肝癌细胞的增殖、迁移和侵袭。

Objective To investigate the effects of tumor protein translation control antisense RNA1(TPT1-AS1)on the radiosensitivity,cell proliferation,migration and invasion of hepatocellular carcinoma cells by targeting microRNA-30c-5p(miR-30c-5p).Methods Thirty-four cases of liver cancer tissues and adjacent normal tissues were derived from liver cancer patients who were admitted to Shanxi Provincial People′s Hospital from March 2016 to March 2018.Liver cancer HepG2 cell was transfected with negative control siRNA(si-NC group),si-TPT1-AS1(si-TPT1-AS1 group),pcDNA3.1(pcDNA3.1 group),pcDNA3.1-TPT1-AS1(pcDNA3.1-TPT1-AS1 group),si-TPT1-AS1 and anti-miR-NC(si-TPT1-AS1+anti-miR-NC group),si-TPT1-AS1 and anti-miR-30c-5p(si-TPT1-AS1+anti-miR-30c-5p group),respectively.Real-time quantitative reverse transcription polymerase chain reaction(qPCR)was used to detect the transcription levels of TPT1-AS1 and miR-30c-5p in normal tissues adjacent to cancer and liver cancer tissues,the clone formation test was used to test the radiosensitivity of HepG2 cells,and the Methyl Thiazolyl Tetrazolium(MTT)test was used to test the proliferation of HepG2 cells.Cell cycle distribution was detected by flow cytometry,Transwell array was used to detect the migration and invasion ability of HepG2 cells,dual luciferase reporter array was used to verify the targeting relationship of TPT1-AS1 and miR-30c-5p,western blot was used to detect the expressions of proliferation,migration and invasion-related proteins.Results The expression levels of TPT1-AS1 and miR-30c-5p in liver cancer tissues were 0.84±0.08 and 0.13±0.01,statistically different from 0.31±0.03 and 0.50±0.05 in normal tissues adjacent to cancer(P<0.05).When the cells were treated with 2,4,6,8 Gy irradiation,the cell survival scores of the si-TPT1-AS1 group were 0.280±0.040,0.069±0.011,0.020±0.003 and 0.005±0.001,respectively,lower than 0.648±0.070,0.348±0.080,0.130±0.020 and 0.060±0.009 of the si-NC group(P<0.05),the radiosensitization ratio of the si-TPT1-AS1 group was 1.672.The number of cell migration and invasion in the si-TPT1-AS1 group were(50.00±4.36)and(44.00±4.03),respectively,which were lower than(109.00±8.68)and(94.00±7.49)in the si-NC group(P<0.05),the cell absorbance(A)values at 24,48 and 72 hours were 0.28±0.03,0.43±0.04 and 0.68±0.07,respectively,lower than 0.46±0.04,0.87±0.08 and 1.35±0.13 of the si-NC group(P<0.05),the protein expression levels of Cyclin D1,p21,E-cadherin and MMP-2 were 0.25±0.02,0.65±0.06,0.68±0.07 and 0.27±0.03,respectively,statistically different from 0.88±0.08,0.17±0.02,0.14±0.01 and 0.89±0.09 of si-NC group(P<0.05),the proportions of S phase and G2 phase in the si-TPT1-AS1 group were(17.82±1.03)%and(34.15±2.29)%,respectively,significantly different from(35.14±2.61)%and(16.84±1.21)%in the si-NC group(P<0.05).The luciferase activity of cells in the WT-TPT1-AS1+miR-30c-5p group was 0.26±0.02,lower than 0.92±0.09 in the WT-TPT1-AS1+miR-NC group(P<0.05).The cell survival scores in the si-TPT1-AS1+anti-miR-30c-5p group were 0.450±0.081,0.200+0.045,0.070±0.010,0.026±0.004 after treatment with 2,4,6,8 Gy irradiation,higher than 0.285±0.043,0.075±0.014,0.028±0.004,0.006±0.001 of si-TPT1-AS1+anti-miR-NC group(P<0.05).The radiosensitization ratio of the si-TPT1-AS1+anti-miR-30c-5p group was 0.694.The number of migration and invasion in the si-TPT1-AS1+anti-miR-30c-5p group were 79.00±6.65 and 68.00±6.33,higher than(52.00±4.41)and(46.00±4.06)of si-TPT1-AS1+anti-miR-NC Group(P<0.05),the A values at 24,48 and 72 hours were 0.37±0.03,0.64±0.06 and 0.96±0.09,respectively,higher than 0.26±0.03,0.41±0.04,and 0.65±0.06 of si-TPT1-AS1+anti-miR-NC group(P<0.05),the expression levels of Cyclin D1,p21,E-cadherin and MMP-2 protein were 0.57±0.06,0.43±0.04,0.43±0.04 and 0.64±0.06,statistically different from 0.24±0.02,0.66±0.06,0.65±0.06 and 0.28±0.03 of the si-TPT1-AS1+anti-miR-NC group(P<0.05).Conclusions The expression of TPT1-AS1 up-regulates in the liver cancer tissues.TPT1-AS1 may down-regulate miR-30c-5p expression,reduce the radiosensitivity of liver cancer cells,and promote the proliferation,migration and invasion of liver cancer cells.