过表达PRRX1通过p53介导的线粒体凋亡途径诱导肝癌SMMC7721细胞凋亡
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摘要
目的:探讨配对相关同源框1蛋白(PRRX1)过表达对肝癌SMMC7721细胞凋亡的影响及其分子机制。方法:分别用慢病毒介导PRRX1过表达载体(pGMLV-PRRX1)、空载质粒(Vector)感染人肝癌SMMC7721细胞,用qPCR和WB实验检测慢病毒感染后细胞中PRRX1 mRNA和蛋白的表达变化,用CCK-8法、Annexin-V FITC/PI染色流式细胞术分别检测PRRX1过表达对SMMC7721细胞增殖、凋亡的影响,用线粒体膜电位检测试剂盒(JC-10染色法)检测细胞线粒体膜电位变化,用caspase活性检测试剂盒(分光光度法)测定细胞中caspase-8和caspase-9酶活性,用WB实验检测细胞中p53、Bcl-2、Bax、Fas、Cleaved-caspase-3以及线粒体和细胞质中细胞色素C(Cty C)蛋白的表达。结果:成功构建PRRX1过表达的SMMC7721细胞株,感染细胞中PRRX1 mRNA和蛋白的表达水平显著升高(均P<0.01)。与对照组和空载组比较,PRRX1过表达组SMMC7721细胞的增殖能力显著下降、细胞凋亡率显著增高、Cleaved-caspase-3剪切水平显著升高、线粒体膜电位显著下降、线粒体中Cty C蛋白表达下调、胞质中Cty C蛋白表达上调以及caspase-9酶活性升高(P<0.05或P<0.01),同时p53和Bax蛋白表达增加而Bcl-2蛋白表达降低(均P<0.05),但Fas蛋白表达及caspase-8酶活性无显著变化(均P>0.05)。结论:PRRX1过表达可诱导肝癌SMMC7721细胞凋亡,其机制可能与p53介导的线粒体凋亡途径被激活有关。
Objective: To investigate the effect of over-expression of paired related homoeobox 1(PRRX1) on apoptosis of hepatocellular carcinoma SMMC7721 cells, and to explore its detailed mechanism. Methods:Lentivirus-mediated PRRX1 over-expression vector(pGMLV-PRRX1) and empty vector(Vector) were respectively infected into SMMC7721 cells, and the mRNA and protein expression levels of PRRX1 in infected cells were detected by qPCR and WB. The effect of PRRX1 over-expression on the cell proliferation and apoptosis of SMMC7721 cells were assessed by CCK-8 assay and Annexin-V FITC/PI double staining flow cytometry assay, respectively. The change of mitochondrial membrane potential of SMMC7721 cells was detected by mitochondrial membrane potential assay kit(JC-10 staining assay). The enzymatic activities of caspase-8 and caspase-9 in infected cells were detected by using caspase activity assay kit(spectrophotometric method). The protein expression levels of p53, Bcl-2, Bax, Fas, Cleaved-caspase-3 and Cty C expressed in mitochondria and cytosol were evaluated by WB. Results:PRRX1 over-expressed SMMC7721 cell line was successfully constructed, and the protein and mRNA expression levels of PRRX1 were significantly increased in lentivirus infected cells(all P<0.01). Compared with control group and vector group, over-expression of PRRX1 significantly inhibited cell proliferation, weakened mitochondrial membrane potential, but increased the rate of apoptosis, elevated the shear level of caspase-3, promoted the release of Cyt C protein from mitochondrial into cytosol and increased the enzymatic activity of caspase-9(all P<0.05 or P<0.01). In addition,over-expression of PRRX1 also promoted the protein expressions of p53 and Bax but inhibited the protein expression of Bcl-2(all P<0.05 or P<0.01); however,it had no significant effect on the expression of Fas protein and the enzymatic activity of caspase-8(all P>0.05). Conclusion:Over-expression of PRRX1 induces apoptosis in hepatocellular carcinoma SMMC7721 cells, which may be related to the activation of p53-mediated mitochondrial apoptotic pathway.
Objective: To investigate the effect of over-expression of paired related homoeobox 1(PRRX1) on apoptosis of hepatocellular carcinoma SMMC7721 cells, and to explore its detailed mechanism. Methods:Lentivirus-mediated PRRX1 over-expression vector(pGMLV-PRRX1) and empty vector(Vector) were respectively infected into SMMC7721 cells, and the mRNA and protein expression levels of PRRX1 in infected cells were detected by qPCR and WB. The effect of PRRX1 over-expression on the cell proliferation and apoptosis of SMMC7721 cells were assessed by CCK-8 assay and Annexin-V FITC/PI double staining flow cytometry assay, respectively. The change of mitochondrial membrane potential of SMMC7721 cells was detected by mitochondrial membrane potential assay kit(JC-10 staining assay). The enzymatic activities of caspase-8 and caspase-9 in infected cells were detected by using caspase activity assay kit(spectrophotometric method). The protein expression levels of p53, Bcl-2, Bax, Fas, Cleaved-caspase-3 and Cty C expressed in mitochondria and cytosol were evaluated by WB. Results:PRRX1 over-expressed SMMC7721 cell line was successfully constructed, and the protein and mRNA expression levels of PRRX1 were significantly increased in lentivirus infected cells(all P<0.01). Compared with control group and vector group, over-expression of PRRX1 significantly inhibited cell proliferation, weakened mitochondrial membrane potential, but increased the rate of apoptosis, elevated the shear level of caspase-3, promoted the release of Cyt C protein from mitochondrial into cytosol and increased the enzymatic activity of caspase-9(all P<0.05 or P<0.01). In addition,over-expression of PRRX1 also promoted the protein expressions of p53 and Bax but inhibited the protein expression of Bcl-2(all P<0.05 or P<0.01); however,it had no significant effect on the expression of Fas protein and the enzymatic activity of caspase-8(all P>0.05). Conclusion:Over-expression of PRRX1 induces apoptosis in hepatocellular carcinoma SMMC7721 cells, which may be related to the activation of p53-mediated mitochondrial apoptotic pathway.
引文
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[16]LANE D,LEVINE A.p53 Research:the past thirty years and the next thirty years[J].Cold Spring Harb Perspect Biol,2010,2(12):a000893.DOI:10.1101/cshperspect.a000893.
[17]ZHANG L,MA L,YAN T,et al.Activated mitochondrial apoptosis in hESCs after dissociation involving the PKA/p-p53/Bax signaling pathway[J].Exp Cell Res,2018,369(2):226-233.DOI:10.1016/j.yexcr.2018.05.024.
[18]MARTINOU J C,YOULE R J.Mitochondria in apoptosis:Bcl-2family members and mitochondrial dynamics[J/OL].Dev Cell,2011,21(1):92-101[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156409/.DOI:10.1016/j.devcel.2011.06.017.
[19]GAHL R F,DWIVEDI P,TJANDRA N.Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis[J/OL].Cell Death Dis,2016,7(10):e2424[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133987/.DOI:10.1038/cddis.2016.320.
[20]陈嘉羽,蒿艳蓉,陈甲信,等.集落刺激因子1受体介导Bax和Bcl-2表达对人鼻咽癌6-10B细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2017,24(12):1386-1390.DOI:10.3872/j.issn.1007-385x.2017.12.008.
[2]ALLEMANI C,MATSUDA T,DI CARLO V,et al.Global surveillance of trends in cancer survival 2000-14(CONCORD-3):analysis of individual records for 37 513 025 patients diagnosed with one of18 cancers from 322 population-based registries in 71 countries[J/OL].Lancet,2018,391(10125):1023-1075[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879496/.DOI:10.1016/S0140-6736(17)33326-3.
[3]OCA?A O H,CóRCOLES R,FABRA A,et al.Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1[J].Cancer Cell,2012,22(6):709-724.DOI:10.1016/j.ccr.2012.10.012.
[4]TAKANO S,REICHERT M,BAKIR B,et al.Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization[J/OL].Genes Dev,2016,30(2):233-247[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719312/.DOI:10.1101/gad.263327.115.
[5]GUO J,FU Z,WEI J,et al.PRRX1 promotes epithelial-mesenchymal transition through the Wnt/β-catenin pathway in gastric cancer[J].Med Oncol,2015,32(1):393-345.DOI:10.1007/s12032-014-0393-x.
[6]HIRATA H,SUGIMACHI K,TAKAHASHI Y,et al.Downregulation of PRRX1 confers cancer stem cell-like properties and predicts poor prognosis in hepatocellular carcinoma[J].Ann Surg Oncol,2015,22(Suppl 3):S1402-S1409.DOI:10.1245/s10434-014-4242-0.
[7]ZHU H,SUN G,DONG J,et al.The role of PRRX1 in the apoptosis of A549 cells induced by cisplatin[J/OL].Am J Transl Res,2017,9(2):396-402[2019-01-25].http://www.ncbi.nlm.nih.gov/pmc/journals/990/.
[8]ZHANG Y,ZHENG L,HUANG J,et al.miR-124 radiosensitizes human colorectal cancer cells by targeting PRRX1[J/OL].PLoS One,2014,9(4):e93917[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976353/.DOI:10.1371/journal.pone.0093917.
[9]NORRIS R A,SCOTT K K,MOORE C S,et al.Human PRRX1 and PRRX2 genes:cloning,expression,genomic localization,and exclusion as disease genes for Nager syndrome[J].Mamm Genome,2000,11(11):1000-1005.
[10]HIGUCHI M,KATO T,CHEN M,et al.Temporospatial gene expression of Prx1 and Prx2 is involved in morphogenesis of cranial placode-derived tissues through epithelio-mesenchymal interaction during rat embryogenesis[J].Cell Tissue Res,2013,353(1):27-40.DOI:10.1007/s00441-013-1632-8.
[11]DU B,CAWTHORN W P,SU A,et al.The transcription factor paired-related homeobox 1(Prrx1)inhibits adipogenesis by activating transforming growth factor-β(TGF-β)signaling[J].J Biol Chem,2013,288(54):3036-3047.DOI:10.1074/jbc.m112.440370.
[12]FAN M,SHEN J,LIU H,et al.Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma[J].Oncol Rep,2017,38(2):1083-1090.DOI:10.3892/or.2017.5785.
[13]GOLDAR S,KHANIANI M S,DERAKHSHAN S M,et al.Molecular mechanisms of apoptosis and roles in cancer development and treatment[J].Asian Pacific J Cancer Prevention,2015,16(6):2129-2144.DOI:10.7314/apjcp.2015.16.6.2129.
[14]BURKE P J.Mitochondria,bioenergetics and apoptosis in cancer[J].Trends Cancer,2017,3(12):857-870.DOI:10.1016/j.trecan.2017.10.006.
[15]OLA M S,NAWAZ M,AHSAN H.Role of Bcl-2 family proteins and caspases in the regulation of apoptosis[J].Mol Cell Biochem,2011,351(1/2):41-58.DOI:10.1007/s11010-010-0709-x.
[16]LANE D,LEVINE A.p53 Research:the past thirty years and the next thirty years[J].Cold Spring Harb Perspect Biol,2010,2(12):a000893.DOI:10.1101/cshperspect.a000893.
[17]ZHANG L,MA L,YAN T,et al.Activated mitochondrial apoptosis in hESCs after dissociation involving the PKA/p-p53/Bax signaling pathway[J].Exp Cell Res,2018,369(2):226-233.DOI:10.1016/j.yexcr.2018.05.024.
[18]MARTINOU J C,YOULE R J.Mitochondria in apoptosis:Bcl-2family members and mitochondrial dynamics[J/OL].Dev Cell,2011,21(1):92-101[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156409/.DOI:10.1016/j.devcel.2011.06.017.
[19]GAHL R F,DWIVEDI P,TJANDRA N.Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis[J/OL].Cell Death Dis,2016,7(10):e2424[2019-01-25].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133987/.DOI:10.1038/cddis.2016.320.
[20]陈嘉羽,蒿艳蓉,陈甲信,等.集落刺激因子1受体介导Bax和Bcl-2表达对人鼻咽癌6-10B细胞凋亡的影响[J].中国肿瘤生物治疗杂志,2017,24(12):1386-1390.DOI:10.3872/j.issn.1007-385x.2017.12.008.