过表达PLAC1基因对子宫颈癌HeLa细胞侵袭和转移能力影响

Effect of overexpression of PLAC1 gene on cell invasion and metastasis in cervical cancer HeLa cells

目的探讨过表达PLAC1基因对子宫颈癌HeLa细胞侵袭和转移能力影响。方法通过慢病毒感染技术构建稳定过表达PLAC1基因的HeLa细胞株,Transwell实验和划痕实验评估细胞的侵袭和迁移能力,蛋白质印迹法检测Slug、Snail、vimentin、p65和基质金属蛋白酶2(MMP2)等肿瘤相关分子表达水平,对癌症基因组图谱(TCGA)和基因表达综合(GEO)数据库中宫颈癌RNA-Seq数据和相关临床资料进行分析,探讨PLAC1表达情况与宫颈癌患者生存预后关系。结果 HeLa^(PLAC1)组细胞迁移数为(179.40±31.86)个,多于HeLa^(Vector)组的(103.2±8.17)个,t=5.968,P<0.001;侵袭数为(170.00±49.10)个,多于HeLa^(Vector)组的(87.40±13.90)个,t=2.352,P=0.047。在细胞划痕实验中,HeLa^(PLAC1)组迁移率为(35.29±1.94)%,高于HeLa^(Vector)组的(21.56±2.44)%,t=8.809,P<0.001。HeLa^(PLAC1)组Slug表达量为5.42±1.24,高于HeLa^(Vector)组的0.42±0.08,t=11.764,P<0.001;HeLa^(PLAC1)组Snail表达量为0.72±0.02,高于HeLa^(Vector)组的0.67±0.03,t=3.107,P=0.038;HeLa^(PLAC1)组p65表达量为0.80±0.03,低于HeLa^(Vector)组的0.97±0.02,t=8.709,P=0.003;HeLa^(PLAC1)组p-p65表达量为0.82±0.05,低于HeLa^(Vector)组的1.00±0.05,t=4.510,P=0.011;HeLa^(PLAC1)组MMP2表达量为0.95±0.03,高于HeLa^(Vector)组的0.85±0.03,t=4.984,P=0.008;HeLa^(PLAC1)组vimentin表达量为3.42±1.28,高于HeLa^(Vector)组0.32±0.07,t=7.962,P<0.001。MS751^(shPLAC1-1)组Slug表达量为0.61±0.11,低于MS751^(shNC)组的0.90±0.10,t=10.887,P<0.001;MS751^(shPLAC1-1)组Snail表达量为0.92±0.03,高于MS751^(shNC)组的0.48±0.02,t=19.730,P<0.001;MS751^(shPLAC1-1)组p65表达量为0.93±0.03,高于MS751^(shNC)组的0.66±0.01,t=13.235,P=0.003;MS751^(shPLAC1-1)组p-p65表达量为0.91±0.02,高于MS751^(shNC)组的0.53±0.02,t=26.856,P<0.001;MS751^(shPLAC1-1)组MMP2表达量为0.79±0.01,低于MS751^(shNC)组的0.88±0.01,t=10.246,P=0.001;MS751^(shPLAC1-1)组vimentin表达量为0.64±0.08,低于MS751^(shNC)组的0.88±0.07,t=8.111,P=0.001;MS751^(shPLAC1-2)组Slug表达量为0.66±0.10,低于MS751^(shNC)组的0.90±0.10,t=5.259,P=0.006;MS751^(shPLAC1-2)组Snail表达量为1.25±0.11,高于MS751^(shNC)组的0.48±0.002,t=12.506,P=0.005;MS751^(shPLAC1-2)组p-p65表达量为0.94±0.01,高于MS751^(shNC)组的0.53±0.02,t=38.408,P<0.001;MS751^(shPLAC1-2)组MMP2表达量为0.76±0.01,低于MS751^(shNC)组的0.88±0.01,t=8.906,P=0.002;MS751^(shPLAC1-2)组vimentin表达量为0.60±0.10,低于MS751^(shNC)组的0.88±0.07,t=7.785,P=0.002。在TCGA-CESA数据中,肿瘤组PLAC1中位表达量为37.63,高表达组和低表达组中位生存时间分别为27.9(0.75531.25)和40.33 (0.17534)个月,差异有统计学意义,χ^(2)=5.160,P=0.015。结论过表达PLAC1基因通过上调Slug和vimentin表达增强HeLa细胞侵袭、迁移能力,且PLAC1高表达是宫颈癌患者不良生存预后的独立危险因素。

Objective To explore the effect of expression of PLAC1 gene on the invasion and metastasis of cervical cancer HeLa cells.Methods The HeLa cell line stably overexpressing PLAC1 gene was constructed by lentiviral infection technique.Transwell assay and wound healing assay were used to evaluate the cell invasion and migration ability.Westernblotting was used to detect the expression levels of tumor-related molecules such as Slug,Snail,vimentin,p65 and matrix metalloproteinase 2(MMP2).Clinical information and gene expression data of patients with cervical cancer were obtained from TCGA and GEO database,which were analyzed to investigate the correlation between the expression level of PLAC1 and the survival prognosis of patients with cervical cancer.Results The migration number of HeLa^(PLAC1) group was 179.40±31.86,which was higher than that in the HeLa^(Vector) group(103.2±8.17),t=5.968,P<0.001.The invasion number of HeLa^(PLAC1) group was(170.00±49.10),which was higher than that of HeLa^(Vector) group(87.40±13.90),t=2.352,P=0.047.In the wound healing assay,the mobility of HeLa^(PLAC1) group was(35.29±1.94)%,which was higher than that of HeLa^(Vector) group(21.56±2.44)%,t=8.809,P<0.001.The expression level of Slug in HeLa^(PLAC1) group was 5.42±1.24,which was higher than that of HeLa^(Vector) group(0.42±0.08),t=11.764,P<0.001.Snail level of HeLa^(PLAC1) group was 0.72±0.02,higher than that of HeLa^(Vector) group(0.67±0.03),t=3.107,P=0.038.The expression level of p65 in HeLa^(PLAC1) group was 0.80±0.03,lower than that in HeLa^(Vector) group(0.97±0.02),t=8.709,P=0.003.The expression level of p-p65 in HeLa^(PLAC1) group was(0.82±0.05),which was lower than that of HeLa^(Vector) group(1.00±0.05),t=4.510,P=0.011.The expression level of MMP2 in HeLa^(PLAC1) group was(0.95±0.03),higher than that in HeLa^(Vector) group(0.85±0.03),t=4.984,P=0.008.The expression of vimentin in HeLa^(PLAC1) group was 3.42±1.28,higher than that in HeLa^(Vector) group(0.32±0.07),t=7.962,P<0.001.Moreover,the Slug expression level of MS751^(shPLAC1-1) group was(0.61±0.11),lower than that of MS751^(shNC) group(0.90±0.10),t=10.887,P<0.001.Snail level of MS751^(shPLAC1-1) group was(0.92±0.03),higher than that of MS751^(shNC) group(0.48±0.02),t=19.730,P<0.001.The p65 level of MS751^(shPLAC1-1) group was(0.93±0.03),higher than that of MS751^(shNC) group(0.66±0.01),t=13.235,P=0.003.The p-p65 expression level of MS751^(shPLAC1-1) group was 0.91±0.02,higher than that of MS751^(shNC) group(0.53±0.02),t=26.856,P<0.001.The expression of MMP2 in MS751^(shPLAC1-1) group was 0.79±0.01,lower than that in MS751^(shNC) group(0.88±0.01),t=10.246,P=0.001.vimentin level of MS751^(shPLAC1-1) was 0.64±0.08,lower than that of MS751^(shNC) group(0.88±0.07),t=8.111,P=0.001.Slug level of MS751^(shPLAC1-2) group was 0.66±0.10,lower than that of MS751^(shNC) group(0.90±0.10),t=5.259,P=0.006.Snail-expression level of MS751^(shPLAC1-2) group was 1.25±0.11,higher than that of MS751^(shNC) group(0.48±0.002),t=12.506,P=0.005.The expression of p-p65 in MS751^(shPLAC1-2) group was0.94±0.01,higher than that in MS751^(shNC) group(0.53±0.02),t=38.408,P<0.001.The expression level of MMP2 in MS751^(shPLAC1-2) group was(0.76±0.01),lower than that in MS751^(shNC) group(0.88±0.01),t=8.906,P=0.002.vimentin expression level of MS751^(shPLAC1-2) was 0.60±0.10,lower than that of MS751^(shNC) group(0.88±0.07),t=7.785,P=0.002.In the TCGA-CESA data,the median expression level of PLAC1 was 37.63 in the tumor group,the median survival time in the high expression group was 27.9(0.75-531.25)months,and the median survival time in the low expression group was 40.33(0.17-534)months,with statistical significance(χ^(2)=5.160,P=0.015).Conclusions Overexpression of PLAC1 gene enhances the invasion and migration ability of HeLa cells by up-regulating the expression of Slug and vimentin,and the high expression of PLAC1 is an independent risk factor for the poor survival and prognosis of cervical cancer patients.