泛素特异性肽酶22对肝癌细胞凋亡的影响及机制研究
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摘要
目的探讨泛素特异性肽酶22(USP22)对肝癌细胞凋亡的影响及其可能的分子机制。方法采用RT-PCR和Western blotting分别检测人正常肝Chang liver细胞和人肝癌HepG2细胞中USP22 mRNA及蛋白表达。利用脂质体转染USP22-siRNA靶向沉默USP22基因,并采用RT-PCR和Western blotting检测其沉默效果;流式细胞仪检测USP22基因沉默对HepG2细胞周期和细胞凋亡的影响,Western blotting检测USP22基因沉默对HepG2细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved caspase-9)、细胞周期素D1(Cyclin D1)和细胞周期蛋白依赖性激酶2(CDK2)表达的影响。结果 USP22基因在人肝癌HepG2细胞中表达明显高于人正常肝Chang liver细胞(P<0.05);siRNA干扰HepG2细胞后,与对照组相比,干预组中USP22 mRNA和蛋白的表达均显著降低(P<0.05),而阴性组差异无统计学意义(P>0.05)。靶向沉默USP22基因后,与对照组相比,干扰组中G_0/G_1期细胞所占比例显著升高,S期和G_2/M期细胞所占比例显著下降,细胞凋亡率明显升高,差异均有统计学意义(P<0.05);阴性组与对照组间G_0/G_1期、S期、G_2/M期细胞所占比例和细胞凋亡率相比,差异无统计学意义(P>0.05)。与对照组相比,干预组中Cleaved caspase-3、Cleaved caspase-9、Cyclin D1和CDK2蛋白的相对表达量均显著降低(P<0.05),阴性组与对照组间差异无统计学意义(P>0.05)。结论 USP22基因沉默可诱导肝癌HepG2细胞凋亡,其作用机制可能与周期蛋白Cyclin D1、CDK2及凋亡蛋白Cleaved caspase-3、Cleaved caspase-9的表达下降有关。
Objective To investigate the effect of ubiquitin specific peptidase 22( USP22) on apoptosis of hepatocellular carcinoma cells and its possible molecular mechanism. Methods RT-PCR and Western blotting were used to detect the expression of USP22 mRNA and protein in human normal Chang liver cells and human hepatoma HepG2 cells. The USP22 gene was silenced by transfection of USP22-siRNA with liposome,and the silencing effect was detected by RT-PCR and Western blotting. The effects of USP22 gene silencing on HepG2 cell cycle and apoptosis were investigated by flow cytometry,and Western blotting was used to detect the effect of USP22 gene silencing on the expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins. Results The expression of USP22 gene in human hepatocellular carcinoma HepG2 cells was significantly higher than that in human normal Chang liver cells( P < 0. 05). After siRNA interference with HepG2 cells,the expressions of USP22 mRNA and protein in the intervention group were significantly lower than those in the control group( P < 0. 05),but there was no significant difference in the negative group( P > 0. 05). After the siRNA interfered with HepG2 cells,compared with the control group,the expressions of USP22 mRNA and protein in the intervention group were significantly decreased( P < 0. 05),but the difference in the negative group was not significant( P > 0. 05). After silence of the USP22 gene,compared with the control group,the proportion of G_0/G_1 phase cells was significantly increased,the proportions of S phase and G_2/M phase cells were decreased significantly,and the rate of apoptosis was increased significantly,whose differences were statistically significant( P < 0. 05); but there was no significant difference in the percentage of G_0/G_1 phase,phase S and phase G_2/M cells and apoptosis rate between the negative group and the control group( P > 0. 05). Compared with the control group,the relative expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins in the intervention group were significantly reduced( P < 0. 05),and the difference between the negative group and the control group was not significant( P > 0. 05). Conclusion The gene silencing of USP22 can induce apoptosis of HepG2 cells in hepatocellular carcinoma,and its mechanism may be related to the decline in expression of cell cycle correlated proteins Cyclin D1,CDK2,and apoptotic proteins Cleaved caspase-3 and Cleaved caspase-9.
Objective To investigate the effect of ubiquitin specific peptidase 22( USP22) on apoptosis of hepatocellular carcinoma cells and its possible molecular mechanism. Methods RT-PCR and Western blotting were used to detect the expression of USP22 mRNA and protein in human normal Chang liver cells and human hepatoma HepG2 cells. The USP22 gene was silenced by transfection of USP22-siRNA with liposome,and the silencing effect was detected by RT-PCR and Western blotting. The effects of USP22 gene silencing on HepG2 cell cycle and apoptosis were investigated by flow cytometry,and Western blotting was used to detect the effect of USP22 gene silencing on the expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins. Results The expression of USP22 gene in human hepatocellular carcinoma HepG2 cells was significantly higher than that in human normal Chang liver cells( P < 0. 05). After siRNA interference with HepG2 cells,the expressions of USP22 mRNA and protein in the intervention group were significantly lower than those in the control group( P < 0. 05),but there was no significant difference in the negative group( P > 0. 05). After the siRNA interfered with HepG2 cells,compared with the control group,the expressions of USP22 mRNA and protein in the intervention group were significantly decreased( P < 0. 05),but the difference in the negative group was not significant( P > 0. 05). After silence of the USP22 gene,compared with the control group,the proportion of G_0/G_1 phase cells was significantly increased,the proportions of S phase and G_2/M phase cells were decreased significantly,and the rate of apoptosis was increased significantly,whose differences were statistically significant( P < 0. 05); but there was no significant difference in the percentage of G_0/G_1 phase,phase S and phase G_2/M cells and apoptosis rate between the negative group and the control group( P > 0. 05). Compared with the control group,the relative expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins in the intervention group were significantly reduced( P < 0. 05),and the difference between the negative group and the control group was not significant( P > 0. 05). Conclusion The gene silencing of USP22 can induce apoptosis of HepG2 cells in hepatocellular carcinoma,and its mechanism may be related to the decline in expression of cell cycle correlated proteins Cyclin D1,CDK2,and apoptotic proteins Cleaved caspase-3 and Cleaved caspase-9.
引文
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[17]陈绍发.CKS1和Cyclin D1在老年肝细胞癌中的表达[J].中国老年学杂志,2013,33(18):4556-4557.DOI:10.3969/j.issn.1005-9202.2013.18.091.
[18]WANG X,LU Y,FENG W,et al.A two kinase-gene signature model using CDK2 and PAK4 expression predicts poor outcome in non-small cell lung cancers[J].Neoplasma,2016,63(2):322-329.DOI:10.4149/220_150817N448.
[19]LI Z H,YU Y,DU C,et al.RNA interference-mediated USP22 gene silencing promotes human brain glioma apoptosis and induces cell cycle arrest[J].Oncol Lett,2013,5(4):1290-1294.DOI:10.3892/ol.2013.1188.
[20]ZHUANG Y J,LIAO Z W,YU H W,et al.ShRNA-mediated silencing of the ubiquitin-specific protease 22 gene restrained cell progression and affected the Akt pathway in nasopharyngeal carcinoma[J].Cancer Biol Ther,2015,16(1):88-96.DOI:10.4161/15384047.2014.987029.
[21]黄婷,邵勇.caspase与妊娠并发症的研究进展[J].中华内分泌外科杂志,2014,8(6):512-514.DOI:10.3760/cma.j.issn.1674-6090.2014.06.021.
[22]SHI J X,WANG Q J,LI H,et al.Silencing of USP22 suppresses high glucose-induced apoptosis,ROS production and inflammation in podocytes[J].Mol Biosyst,2016,12(5):1445-1456.DOI:10.1039/c5mb00722d.
[23]李朝晖,王建,付红,等.siRNA沉默USP22基因对胶质瘤细胞增殖的抑制作用[J].中国微侵袭神经外科杂志,2015,20(9):419-423.DOI:10.11850/j.issn.1009-122X.2015.09.014.LI Z H,WANG J,FU H,et al.siRNA-mediated silencing of the USP22 gene inhibits cell proliferation in human glioma cells[J].Chin J Minim Invasive Neurosurg,2015,20(9):419-423.DOI:10.11850/j.issn.1009-122X.2015.09.014.
[2]BEARD R E,HANTO D W,GAUTAM S,et al.A comparison of surgical outcomes for noncirrhotic and cirrhotic hepatocellular carcinoma patients in a Western institution[J].Surgery,2013,154(3):545-555.DOI:10.1016/j.surg.2013.02.019.
[3]DONGIOVANNI P,ROMEO S,VALENTI L.Hepatocellular carcinoma in nonalcoholic fatty liver:role of environmental and genetic factors[J].World J Gastroenterol,2014,20(36):12945-12955.DOI:10.3748/wjg.v20.i36.12945.
[4]柏雪丽,胡素香,欧阳玲.USP22与肿瘤关系的研究进展[J].现代肿瘤医学,2014,22(2):460-463.DOI:10.3969/j.issn.1672-4992.2014.02.70.BAI X L,HU S X,OUYANG L.The research progression of the relationship between USP22 and tumors[J].Modern Oncology,2014,22(2):460-463.DOI:10.3969/j.issn.1672-4992.2014.02.70.
[5]WANG H,LI Y P,CHEN J H,et al.Prognostic significance of USP22as an oncogene in papillary thyroid carcinoma[J].Tumour Biol,2013,34(3):1635-1639.DOI:10.1007/s13277-013-0696-0.
[6]胡晶,李国强,金然,等.USP22和PTEN在非小细胞肺癌中的表达及临床意义[J].哈尔滨医科大学学报,2015,49(1):45-51.HU J,LI G Q,JIN R,et al.Expressions of USP22 and PTEN in nonsmall cell lung cancer and their clinical significances[J].Journal of Harbin Medical University,2015,49(1):45-51.
[7]MA Y,FU H L,WANG Z,et al.USP22 maintains gastric cancer stem cell stemness and promotes gastric cancer progression by stabilizing BMI1 protein[J].Oncotarget,2017,8(20):33329-33342.DOI:10.18632/oncotarget.16445.
[8]TANG B,TANG F,LI B,et al.High USP22 expression indicates poor prognosis in hepatocellular carcinoma[J].Oncotarget,2015,6(14):12654-12667.DOI:10.18632/oncotarget.3705.
[9]王技军,贺岩.USP22在恶性肿瘤中的研究进展[J].实用肿瘤学杂志,2016,30(6):551-554.DOI:10.11904/j.issn.1002-3070.2016.06.015.WANG J J,HE Y.The research progresses in USP22 in malignant tumors[J].Journal of Practical Oncology,2016,30(6):551-554.DOI:10.11904/j.issn.1002-3070.2016.06.015.
[10]ZHOU D,LIU P,SUN D W,et al.USP22 down-regulation facilitates human retinoblastoma cell aging and apoptosis via inhibiting TERT/P53 pathway[J].Eur Rev Med Pharmacol Sci,2017,21(12):2785-2792.
[11]JI M,SHI H,XIE Y,et al.Ubiquitin specific protease 22 promotes cell proliferation and tumor growth of epithelial ovarian cancer through synergy with transforming growth factorβ1[J].Oncol Rep,2015,33(1):133-140.DOI:10.3892/or.2014.3580.
[12]TANG B,LIANG X,TANG F,et al.Expression of USP22 and Survivin is an indicator of malignant behavior in hepatocellular carcinoma[J].Int J Oncol,2015,47(6):2208-2216.DOI:10.3892/ijo.2015.3214.
[13]IHLE M A,HUSS S,JESKE W,et al.Expression of cell cycle regulators and frequency of TP53 mutations in high risk gastrointestinal stromal tumors prior to adjuvant imatinib treatment[J].PLo S One,2018,13(2):e0193048.DOI:10.1371/journal.pone.0193048.
[14]王轶,徐桂芳,张斌,等.TGF-β1通过cyclin D3对肝细胞癌增殖能力的双重调控作用机制[J].胃肠病学和肝病学杂志,2015,24(9):1053-1056.DOI:10.3969/j.issn.1006-5709.2015.09.006.WANG Y,XU G F,ZHAGN B,et al.The double regulation mechanisms of TGF-β1 in the proliferation of hepatocellular carcinoma by cyclin D3[J].Chin J Gastroenterol Hepatol,2015,24(9):1053-1056.DOI:10.3969/j.issn.1006-5709.2015.09.006.
[15]CASIMIRO M C,VELASCO-VELAZQUEZ M,AGUIRRE-ALVARADO C,et al.Overview of cyclins D1 function in cancer and the CDK inhibitor landscape:past and present[J].Expert Opin Investig Drugs,2014,23(3):295-304.DOI:10.1517/13543784.2014.867017.
[16]PEURALA E,KOIVUNEN P,HAAPASAARI K M,et al.The prognostic significance and value of cyclin D1,CDK4 and p16 in human breast cancer[J].Breast Cancer Res,2013,15(1):R5.DOI:10.1186/bcr3376.
[17]陈绍发.CKS1和Cyclin D1在老年肝细胞癌中的表达[J].中国老年学杂志,2013,33(18):4556-4557.DOI:10.3969/j.issn.1005-9202.2013.18.091.
[18]WANG X,LU Y,FENG W,et al.A two kinase-gene signature model using CDK2 and PAK4 expression predicts poor outcome in non-small cell lung cancers[J].Neoplasma,2016,63(2):322-329.DOI:10.4149/220_150817N448.
[19]LI Z H,YU Y,DU C,et al.RNA interference-mediated USP22 gene silencing promotes human brain glioma apoptosis and induces cell cycle arrest[J].Oncol Lett,2013,5(4):1290-1294.DOI:10.3892/ol.2013.1188.
[20]ZHUANG Y J,LIAO Z W,YU H W,et al.ShRNA-mediated silencing of the ubiquitin-specific protease 22 gene restrained cell progression and affected the Akt pathway in nasopharyngeal carcinoma[J].Cancer Biol Ther,2015,16(1):88-96.DOI:10.4161/15384047.2014.987029.
[21]黄婷,邵勇.caspase与妊娠并发症的研究进展[J].中华内分泌外科杂志,2014,8(6):512-514.DOI:10.3760/cma.j.issn.1674-6090.2014.06.021.
[22]SHI J X,WANG Q J,LI H,et al.Silencing of USP22 suppresses high glucose-induced apoptosis,ROS production and inflammation in podocytes[J].Mol Biosyst,2016,12(5):1445-1456.DOI:10.1039/c5mb00722d.
[23]李朝晖,王建,付红,等.siRNA沉默USP22基因对胶质瘤细胞增殖的抑制作用[J].中国微侵袭神经外科杂志,2015,20(9):419-423.DOI:10.11850/j.issn.1009-122X.2015.09.014.LI Z H,WANG J,FU H,et al.siRNA-mediated silencing of the USP22 gene inhibits cell proliferation in human glioma cells[J].Chin J Minim Invasive Neurosurg,2015,20(9):419-423.DOI:10.11850/j.issn.1009-122X.2015.09.014.