摘要
HMG盒转录因子1(HMG box transcription factor 1,HBP1)作为一个肿瘤抑制因子,具有双重转录因子活性,通过激活或抑制细胞生长相关基因(p16,p21或DNMT1)的表达,抑制肿瘤细胞的增殖。磷脂酶C-γ1(phospholipase C-γ1,PLC-γ1)是磷脂酶C家族的成员,在许多肿瘤细胞PLC-γ1表达升高,与肿瘤细胞的增殖和迁移密切相关。本研究通过生物信息学预测发现PLC-γ1可能是HBP1的下游靶分子;在宫颈癌细胞HeLa中分别过表达和敲低HBP1后,荧光定量PCR及Western印迹检测证明,过表达HBP1,PLC-γ1 mRNA水平下调至49%,蛋白质相对表达量下调至48%(P<0.01);反之,敲低HBP1,PLC-γ1 mRNA水平上调2倍,蛋白质相对表达量上调2倍(P<0.01);双荧光素酶报告基因结果证实,HBP1转录抑制PLC-γ1启动子活性;染色质免疫沉淀结果证实,细胞内HBP1与PLC-γ1启动子结合;细胞划痕实验48 h结果显示,HBP1组划痕面积改变率低于对照组约19%(P<0.05),“HBP1+PLC-γ1”组与HBP1组相比划痕面积改变率增加约17%(P<0.05);Transwell迁移和侵袭实验发现,HBP1组与对照组相比穿过膜的细胞数量分别减少约90和68(P<0.01),“HBP1+PLC-γ1”组与HBP1组相比穿过膜的细胞数量分别增加约89和47(P<0.01),划痕和Transwell实验结果表明,HBP1下调PLC-γ1表达抑制了宫颈癌细胞的迁移和侵袭能力。上述所有结果表明,PLC-γ1是HBP1下游的靶基因,HBP1可以直接结合PLC-γ1基因的启动子,在转录水平抑制PLC-γ1基因的表达,从而抑制宫颈癌细胞的迁移和侵袭。该研究初步阐明了HBP1调控PLC-γ1的分子机制及其在宫颈癌细胞迁移中的作用,为确定宫颈癌治疗的有效分子靶点提供了实验依据。
As a tumor suppressor,HMG box transcription factor 1(HBP1)has dual activity of transcription factor,which inhibits the proliferation of tumor cells by activating or inhibiting the expression of cell growth-related genes(p16,p21,or DNMT1).Phospholipase C-γ1(PLC-γ1)is a member of phospholipase C family.The expression of PLC-γ1 is increased in many tumor cells,which is closely related to the proliferation and migration of tumor cells.In this study,bioinformatics prediction revealed that PLC-γ1 might be the downstream target molecule of HBP1.After overexpression and knockdown of HBP1 in cervical cancer HeLa cells,the expression of PLC-γ1 mRNA and protein decreased to 49% and 48% respectively(P<0.01).On the contrary,when HBP1 was knocked down,PLC-γ1 mRNA level and protein relative expression level were up-regulated by 2 folds(P<0.01).The results of double luciferase reporter gene confirmed that HBP1 transcription inhibited the activity of PLC-γ1 promoter.The binding of HBP1 with PLC-γ1 promoter was confirmed by chromatin immunoprecipitation assay.The results of cell scratch test showed that the change rate of scratch area in HBP1 group was 19% lower than that of control group(P<0.05),and that of"HBP1+PLC-γ1"group was 17% higher than that of HBP1 group(P<0.05).Transwell migration and invasion assays showed that compared with the control group,there was a decrease of about 90 and 68 cells passing through the membrane in HBP1 group(P<0.01),and an increase of 89 and 47 cells passing through the membrane in"HBP1+PLC-γ1"group(P<0.01)respectively.Scratch test and transwell test showed that the down-regulation of PLC-γ1 expression by HBP1 inhibited the migration and invasion of cervical cancer cells.All these results indicate that PLC-γ1 is a target gene downstream of HBP1.HBP1 can directly bind to the promoter of PLC-γ1 gene and inhibit the expression of PLC-γ1 gene at the transcriptional level,thus inhibiting the migration and invasion of cervical cancer cells.This study preliminarily elucidates the molecular
作者
汪军梅
刘宇娟
薛俊慧
曹正意
蒋卫
李慧
张晓伟
WANG Jun-Mei;LIU Yu-Juan;XUN Jun-Hui;CAO Zheng-Yi;JIANG Wei;LI Hui;ZHANG Xiao-Wei(Department of Biochemistry and Biophysics,Peking University Health Science Center,Beijing 100191,China;Department of Biochemistry,Changzhi Medical College,Changzhi 046000,Shanxi,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2020年第12期1455-1463,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金面上项目(No.81874141,81672717,82073068)
北京市自然科学基金面上项目(No.5162013)资助。
关键词
HMG盒转录因子1
磷脂酶C-Γ1
宫颈癌细胞
HMG box transcription factor 1(HBP1)
phospholipase C-γ1(PLC-γ1)
cervical cancer cells
