DESI2基因干扰联合PI3K抑制剂对人胰腺癌细胞ASPC-1的影响
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摘要
目的研究人凋亡相关新基因DESI2基因和PI3K抑制剂对人胰腺癌细胞ASPC-1的影响。方法构建DESI2慢病毒干扰载体,并将细胞分为四组:(1)溶剂对照组;(2)PI3K抑制剂组;(3)DESI2干扰+PI3K抑制剂组;(4)DESI2空载+PI3K抑制剂组。采用MTT检测细胞活性,流式检测细胞周期和调亡,Transwell检测细胞侵袭情况,qRT-PCR检测细胞DESI2、Caspase3、AKT、PI3K和mTOR mRNA的表达,Western blotting检测细胞DESI2、Caspase3、AKT、p-AKT、PI3K、p-PI3K、mTOR和p-mTOR蛋白表达情况。结果与溶剂对照组相比,PI3K抑制剂组和DESI2空载+PI3K抑制剂组细胞活性明显下降,G_1期比例升高,S期和G_2期比例降低,凋亡明显升高,细胞侵袭能力明显下降,DESI2和Caspase3的mRNA和蛋白水平表达明显升高,AKT、PI3K和mTOR及其磷酸化的蛋白表达量明显下降。与PI3K抑制剂组相比,DESI2干扰+PI3K抑制剂组细胞活性明显升高,G_1期比例降低,S期比例升高,凋亡明显下降,细胞侵袭能力明显升高,DESI2和Caspase3 mRNA和蛋白水平表达明显下降,AKT、PI3K和mTOR及其磷酸化的蛋白表达量明显升高,研究数据组间比较均达到统计学显著水平(P<0.05)。结论 PI3K抑制剂通过阻断PI3K/AKT/mTOR信号通路,可以抑制人胰腺癌细胞ASPC-1的增殖及侵袭,促进细胞调亡;PI3K/AKT/mTOR信号通路的功能状态能反馈调节上游DESI2基因的表达。
Objective To explore the influence of desumoylating isopeptidase 2(DESI2) gene and PI3 K inhibitors on pancreatic cancer cells line ASPC-1. Methods Construction of DESI2 lentivirus interference vector,and its effect was verified by qRT-PCR and Western blotting. Pancreatic cancer cells line ASPC-1 was divided into four groups:(1)control group;(2)PI3 K inhibitor group;(3)DESI2 interference+PI3 K inhibitor group;(4)DESI2 interference control+PI3 K inhibitor group. Cell activity was measured with MTT, cell cycle and apoptosis were measured with flow cytomertry and cell invasion was assayed by transwell. mRNA expressions of DESI2,Caspase3, AKT, PI3 K and mTOR were quantified with qRT-PCR. Protein expression of DESI2, Caspase3,AKT, p-AKT, PI3 K, p-PI3 K, mTOR and p-mTOR were assessed with Western blotting. Results Compared with the control group, the cell activites in PI3 K inhibitor group and DESI2 interference control+PI3 K inhibitor group were significantly decreased; cell cycle in G_1 phase significantly increased, S and G_2 phase significantly decreased, the apoptosis significantly increased, cell invasion ability significantly decreased, mRNA and protein expression of DESI2 and Caspase3 significantly increased, protein expression of AKT, PI3 K, mTOR and its phosphorylation significantly decreased. Compared with PI3 K inhibitors group, the cell activity in DESI2 interference+PI3 K inhibitor group significantly increased, cell cycle in G_1 phase significantly decreased and S phase significantly increased. The apoptosis significantly decreased,cell invasion ability increased significantly,mRNA and protein expression of DESI2 and Caspase3 significantly decreased, protein expression of AKT, PI3 K,mTOR and its phosphorylation significantly increased(all P<0.05). Conclusion PI3 K inhibitors can block the PI3 K/AKT/mTOR signaling pathway, inhibit the proliferation and invasion of human pancreatic cancer cell ASPC-1 and promote cell apoptosis. And, the functional state of PI3 K/AKT/mTOR signaling pathway is a feedback regulation on expression of upstream DESI2 gene.
Objective To explore the influence of desumoylating isopeptidase 2(DESI2) gene and PI3 K inhibitors on pancreatic cancer cells line ASPC-1. Methods Construction of DESI2 lentivirus interference vector,and its effect was verified by qRT-PCR and Western blotting. Pancreatic cancer cells line ASPC-1 was divided into four groups:(1)control group;(2)PI3 K inhibitor group;(3)DESI2 interference+PI3 K inhibitor group;(4)DESI2 interference control+PI3 K inhibitor group. Cell activity was measured with MTT, cell cycle and apoptosis were measured with flow cytomertry and cell invasion was assayed by transwell. mRNA expressions of DESI2,Caspase3, AKT, PI3 K and mTOR were quantified with qRT-PCR. Protein expression of DESI2, Caspase3,AKT, p-AKT, PI3 K, p-PI3 K, mTOR and p-mTOR were assessed with Western blotting. Results Compared with the control group, the cell activites in PI3 K inhibitor group and DESI2 interference control+PI3 K inhibitor group were significantly decreased; cell cycle in G_1 phase significantly increased, S and G_2 phase significantly decreased, the apoptosis significantly increased, cell invasion ability significantly decreased, mRNA and protein expression of DESI2 and Caspase3 significantly increased, protein expression of AKT, PI3 K, mTOR and its phosphorylation significantly decreased. Compared with PI3 K inhibitors group, the cell activity in DESI2 interference+PI3 K inhibitor group significantly increased, cell cycle in G_1 phase significantly decreased and S phase significantly increased. The apoptosis significantly decreased,cell invasion ability increased significantly,mRNA and protein expression of DESI2 and Caspase3 significantly decreased, protein expression of AKT, PI3 K,mTOR and its phosphorylation significantly increased(all P<0.05). Conclusion PI3 K inhibitors can block the PI3 K/AKT/mTOR signaling pathway, inhibit the proliferation and invasion of human pancreatic cancer cell ASPC-1 and promote cell apoptosis. And, the functional state of PI3 K/AKT/mTOR signaling pathway is a feedback regulation on expression of upstream DESI2 gene.
引文
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[17]Xu RL,He W,Tang J,et al.Primate-specific miRNA-637 inhibited tumorigenesis in human pancreatic ductal adenocarcinoma cells by suppressing Akt1 expression[J].Exp Cell Res,2018,363(2):310-314.
[18]Shi X,Ran L,Liu Y,et al.Knockdown of hnRNP A2/B1 inhibits cell proliferation,invasion and cell cycle triggering apoptosis in cervical cancer via PI3K/AKT signaling pathway[J].Oncol Rep,2018,39(3):939-950.
[2]Satodahlman M,Wirth K,Yamamoto M.Role of gene therapy in pancreatic cancer-a review[J].Cancers,2018,10(4).pii:E103.
[3]Nath S,Mandal C,Chatterjee U,et al.Association of cytosolic sialidase Neu2 with plasma membrane enhances Fas-mediated apoptosis by impairing PI3K-Akt/mTOR-mediated pathway in pancreatic cancer cells[J].Cell Death Dis,2018,9(2):210.
[4]Chao L,Yan HY,Yang J,et al.Combination of DESI2 and IP10 gene therapy significantly improves therapeutic efficacy against murine carcinoma[J].Oncotarget,2017,8(34):56281-56295.
[5]李丹,赵丹懿,戴朝霞,等.DESI2基因在肝细胞癌中的表达及其对HepG2细胞凋亡的影响[J].大连医科大学学报,2014,35(6):530-532,546.
[6]Qi X,Song X,Liu P,et al.Antitumor effects of PLGAnanoparticles encapsμLating the human PNAS-4 gene combined with cisplatin in ovarian cancer[J].Oncol Rep,2011,26(3):703-710.
[7]Shen CC,Cui XY,He Y,et al.High phosphorylation status of AKT/mTOR signal in DESI2-reduced pancreatic ductal adenocarcinoma[J].Oncol Res,2015,21(2):267-272.
[8]Quirin KA,Kwon JJ,Alioufi A,et al.Safety and efficacy of AAV retrograde pancreatic ductal gene delivery in normal and pancreatic cancer mice[J].Mol Ther Methods Clin Dev,2017,8:8-20.
[9]Strausberg RL,Feingold EA,Grouse LH,et al.Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences[J].PNAS,2002,99(26):16899.
[10]Yan F,Ruan X,Yang H,et al.Identification,characterization,and effects of Xenopus laevis PNAS-4 gene on embryonic development[J].J Biomed Biotechnol,2010,J Biomed Biotechnol,2010,2010:134764.
[11]Mirnezami R,Veselkov K,Strittmatter N,et al.Novel data processing and image co-registration algorithm for regionspecific lipid profiling in colorectal cancer tissue using DE-SI imaging mass spectrometry[J].J Clin Onco,2013,31(15):101-106.
[12]Lin C,Yan H,Yang J,et al.Combination of DESI2 and IP10 gene therapy significantly improves therapeutic efficacy against murine carcinoma[J].Oncotarget,2017,8(34):56281-56295.
[13]Sharma N,Nanta R,Sharma J,et al.PI3K/AKT/mTOR and sonic hedgehog pathways cooperate together to inhibit human pancreatic cancer stem cell characteristics and tumor growth[J].Oncotarget,2015,6(31):32039-32060.
[14]Manna SK,Aggarwal BB.Potentiation of the effect of erlotinib by genistein in pancreatic cancer:the role of Akt and nuclear factor-kappaB[J].Cancer Res,2006,66(21):10553.
[15]Xiao MB,Li T,Ji YF,et al.S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway[J].Oncol Lett,2018,15(1):175-182.
[16]Yang L,Yang G,Ding Y,et al.Inhibition of PI3K/AKT Signaling Pathway Radiosensitizes Pancreatic Cancer Cells with ARID1A Deficiencyin Vitro[J].J Cancer,2018,9(5):890-900.
[17]Xu RL,He W,Tang J,et al.Primate-specific miRNA-637 inhibited tumorigenesis in human pancreatic ductal adenocarcinoma cells by suppressing Akt1 expression[J].Exp Cell Res,2018,363(2):310-314.
[18]Shi X,Ran L,Liu Y,et al.Knockdown of hnRNP A2/B1 inhibits cell proliferation,invasion and cell cycle triggering apoptosis in cervical cancer via PI3K/AKT signaling pathway[J].Oncol Rep,2018,39(3):939-950.