下调Girdin基因表达对肺癌细胞增殖凋亡、免疫因子IL-8和TNF-α及顺铂化疗敏感性的研究
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摘要
目的:探讨下调微丝附着梁蛋白(Girdin)基因表达对肺癌细胞增殖凋亡、免疫因子IL-8和TNF-α及顺铂化疗敏感性的影响。方法:以人胚肺成纤维细胞MRC5作为对照细胞,RT-PCR及Western blot分别检测Girdin在PC9、SPC-A-1、H322、H1299、A549肺癌细胞中的表达; SPC-A-1细胞分为空白对照组、阴性对照组、Girdin-siRNA组、顺铂组和Girdin-siRNA+顺铂组,各组细胞培养48 h,Western blot检测Girdin-siRNA转染SPC-A-1细胞的效果; MTT法检测各组细胞活力;流式细胞术检测各组细胞凋亡率; RT-PCR检测IL-8和TNF-α的mRNA表达; Western blot检测增殖相关蛋白细胞增殖核抗原(PCNA)、凋亡相关蛋白含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、Bcl-2相关X蛋白(Bax)及PI3K/AKT信号通路磷脂酰肌醇-3激酶(PI3K)和磷酸化的丝氨酸苏氨酸激酶(p-AKT)的蛋白表达。结果:肺癌细胞中Girdin的mRNA及蛋白表达均明显高于在MRC5细胞表达(P<0. 05); Girdin-siRNA转染SPC-A-1细胞后Girdin的表达显著降低(P<0. 05);与空白对照组比较,Girdin-siRNA组和顺铂组OD值及IL-8、TNF-α、PCNA、PI3K、p-AKT的表达均显著降低,细胞凋亡率及Caspase-3和Bax表达均显著升高(P<0. 05); Girdin-siRNA+顺铂组OD值及IL-8、TNF-α、PCNA、PI3K、p-AKT的表达均显著低于Girdin-siRNA组和顺铂组,细胞凋亡率及Caspase-3和Bax表达均显著高于Girdin-siRNA组和顺铂组(P<0. 05)。结论:抑制肺癌细胞Girdin表达可通过降低癌细胞增殖、诱导细胞凋亡和提高免疫增强乳腺癌顺铂化疗敏感性,对细胞增殖凋亡的机制可能与下调PI3K/AKT信号通路有关。
Objective: To investigate the effect of down-regulation of Girdin gene expression on proliferation,apoptosis,immune factors and cisplatin chemosensitivity of lung cancer cells. Methods: Human embryonic lung fibroblast MRC5 cells as control,RT-PCR and Western blot were used to detect the expression of Girdin in PC9,SPC-A-1,H322,H1299,A549 in lung cancer cells; SPC-A-1 cells were divided into blank control group,negative control group,Girdin-siRNA group,cisplatin group and Girdin-siRNA+cisplatin group,cells were cultured for 48 h,effect of Girdin-siRNA transfected SPC-A-1 cells were detected by Western blot; cell viability were detected by MTT; cell apoptosis was detected by flow cytometry; the expression of IL-8 and TNF-α mRNA were detected by RT-PCR; the expression of PCNA,Caspase-3,PI3 K and p-AKT protein were detected by Western blot. Results: The mRNA and protein expression of Girdin in lung cancer cells were significantly higher than the expression in MRC5 cells( P<0. 05); the expression of Girdin after Girdin-siRNA transfected SPC-A-1 cells was significantly decreased( P<0. 05); compared with the control group,OD value and the expression of IL-8,TNF-α,PCNA,PI3 K,p-AKT in Girdin-siRNA group and cisplatin group were significantly decreased,the apoptosis rate and the expression of Bax and Caspase-3 were significantly increased( P<0. 05); OD value and the expression of IL-8,TNF-α,PCNA,PI3 K,pAKT in Girdin-siRNA+cisplatin group were significantly lower than Girdin-siRNA group and cisplatin group,the apoptosis rate and the expression of Caspase-3 and Bax were significantly higher than Girdin-siRNA group and cisplatin group( P<0. 05). Conclusion: Inhibiting of Girdin expression in lung cancer cells can reduce the proliferation of cancer cells,induce apoptosis and improve immunity to enhance the sensitivity of cisplatin chemotherapy. The mechanism of cell proliferation and apoptosis may be related to the downregulation of PI3 K/AKT signaling pathway.
Objective: To investigate the effect of down-regulation of Girdin gene expression on proliferation,apoptosis,immune factors and cisplatin chemosensitivity of lung cancer cells. Methods: Human embryonic lung fibroblast MRC5 cells as control,RT-PCR and Western blot were used to detect the expression of Girdin in PC9,SPC-A-1,H322,H1299,A549 in lung cancer cells; SPC-A-1 cells were divided into blank control group,negative control group,Girdin-siRNA group,cisplatin group and Girdin-siRNA+cisplatin group,cells were cultured for 48 h,effect of Girdin-siRNA transfected SPC-A-1 cells were detected by Western blot; cell viability were detected by MTT; cell apoptosis was detected by flow cytometry; the expression of IL-8 and TNF-α mRNA were detected by RT-PCR; the expression of PCNA,Caspase-3,PI3 K and p-AKT protein were detected by Western blot. Results: The mRNA and protein expression of Girdin in lung cancer cells were significantly higher than the expression in MRC5 cells( P<0. 05); the expression of Girdin after Girdin-siRNA transfected SPC-A-1 cells was significantly decreased( P<0. 05); compared with the control group,OD value and the expression of IL-8,TNF-α,PCNA,PI3 K,p-AKT in Girdin-siRNA group and cisplatin group were significantly decreased,the apoptosis rate and the expression of Bax and Caspase-3 were significantly increased( P<0. 05); OD value and the expression of IL-8,TNF-α,PCNA,PI3 K,pAKT in Girdin-siRNA+cisplatin group were significantly lower than Girdin-siRNA group and cisplatin group,the apoptosis rate and the expression of Caspase-3 and Bax were significantly higher than Girdin-siRNA group and cisplatin group( P<0. 05). Conclusion: Inhibiting of Girdin expression in lung cancer cells can reduce the proliferation of cancer cells,induce apoptosis and improve immunity to enhance the sensitivity of cisplatin chemotherapy. The mechanism of cell proliferation and apoptosis may be related to the downregulation of PI3 K/AKT signaling pathway.
引文
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[2]Chen G,Umelo IA,Lv S,et al.miR-146a inhibits cell growth,cell migration and induces apoptosis in non-small cell lung cancer cells[J].PLo S One,2013,8(3):e60317.
[3]Hsieh MJ,Tsai TL,Hsieh YS,et al.Dioscin-induced autophagy mitigates cell apoptosis through modulation of PI3K/Akt and ERKand JNK signaling pathways in human lung cancer cell lines[J].Archiv Toxicol,2013,87(11):1927-1937.
[4]Gutschner T,Hmmerle M,Eimann M,et al.The noncoding RNAMALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells[J].Cancer Res,2013,73(3):1180-1189.
[5]Wynes MW,Hinz TK,Gao D,et al.FGFR1 mRNA and protein expression,not gene copy number,predict FGFR TKI sensitivity across all lung cancer histologies[J].Clin Cancer Res,2014,20(12):3299-3309.
[6]Ke Y,Bao T,Wu X,et al.Scutellarin suppresses migration and invasion of human hepatocellular carcinoma by inhibiting the STAT3/Girdin/Akt activity[J].Biochem Biophys Res Commun,2017,483(1):509-515.
[7]朱明文,孙欣,林方才.Girdin蛋白与乳腺癌复发转移危险度的关系[J].医学研究生学报,2014,27(1):70-72.Zhu MW,Sun X,Lin FC.Relationship between expression of girdin protein and risk of recurrence and metastasis in breast cancer[J].J Med Postgraduates,2014,27(1):70-72.
[8]Song JY,Jiang P,Li N,et al.Clinical significance of Girdin expression detected by immunohistochemistry in non small cell lung cancer[J].Oncol Lett,2014,7(2):337-341.
[9]吴志豪,刘海斌,郑敏.K-ras对girdin蛋白表达的调控机制及二者在结直肠癌中的表达[J].中国病理生理杂志,2017,33(2):297-301.Wu ZH,Liu HB,Zheng M.Regulatory mechanisms of K-ras to girdin in COS7 cells and expression of K-ras and girdin in colorectal carcinoma[J].Chin J Pathophysiol,2017,33(2):297-301.
[10]林宪仁,何佳,巴一,等.Girdin蛋白在脑胶质瘤组织中的表达及其与临床病理特征和预后的关系[J].天津医科大学学报,2015,21(5):397-400.Lin XR,He J,Ba Y,et al.Expression of Girdin in glioma tissues and its relationship with pathological clinical characteristics and prognosis of the patients[J].J Tianjin Med Univ,2015,21(5):397-400.
[11]高志强,汪俊峰,陈德华,等.Girdin基因在肝癌细胞表达及抑制其表达的生物学特性[J].中华实验外科杂志,2017,34(12):2109-2112.Gao ZQ,Wang JF,Chen DH,et al.Expression of Girdin gene in hepatocellular carcinoma cells and inhibition of its expression on biological characteristics[J].Chin J Exp Surg,2017,34(12):2109-2112.
[12]Cao K,Lu C,Han S,et al.Expression of Girdin in primary hepatocellular carcinoma and its effect on cell proliferation and invasion[J].Inter J Clin Exp Pathol,2015,8(1):551-559.
[13]Li X,An Z,Li P,et al.A prognostic model for lung adenocarcinoma patient survival with a focus on four miRNAs[J].Oncol Lett,2017,14(3):2991-2995.
[14]杨朝阳,王鑫,史炯,等.Girdin表达与非小细胞肺癌侵袭转移及预后的关系[J].现代生物医学进展,2016,16(19):3642-3645.Yang ZY,Wang X,Shi J,et al.Relationship between Girdin expression and invasion and metastasis of non small cell lung cancer and its prognosis[J].Progr Modern Biomed,2016,16(19):3642-3645.
[15]Wu YL,Zhou C,Hu CP,et al.Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations(LUX-Lung 6):an open-label,randomised phase 3 trial[J].Lancet Oncol,2014,15(2):213-222.
[16]He C,Lu K,Liu D,et al.Nanoscale metal-organic frameworks for the co-delivery of cisplatin and pooled siRNAs to enhance therapeutic efficacy in drug-resistant ovarian cancer cells[J].J Am Chem Soc,2014,136(14):5181-5184.
[17]Sato T,Arai E,Kohno T,et al.Epigenetic clustering of lung adenocarcinomas based on DNA methylation profiles in adjacent lung tissue:Its correlation with smoking history and chronic obstructive pulmonary disease[J].Inter J Cancer,2014,135(2):319-334.
[18]Ansari J,Shackelford RE,El-Osta H.Epigenetics in non-small cell lung cancer:from basics to therapeutics[J].Trans Lung Cancer Res,2016,5(2):155-171.
[19]伏勇,洪振强,李楠,等.片仔癀对人骨肉瘤MG63细胞增殖及PI3K/Akt信号通路的作用研究[J].中华中医药杂志,2016,31(11):4675-4677.Fu Y,Hong ZQ,Li N,et al.Effect of Pien Tze Huang on proliferation and PI3K/Akt signaling pathway of human osteosarcoma MG63 cells[J].China J Tradi Chin Med Phar,2016,31(11):4675-4677.
[20]Riquelme E,Behrens C,Lin HY,et al.Modulation of EZH2 expression by MEK-ERK or PI3K-AKT signaling in lung cancer is dictated by different KRAS oncogene mutations[J].Cancer Res,2016,76(3):675-685.
[21]Gil E MC.Targeting the PI3K/AKT/m TOR pathway in estrogen receptor-positive breast cancer[J].Cancer Treat Rev,2014,40(7):862-871.
[22]Vander Broek R,Mohan S,Eytan DF,et al.The PI3K/Akt/m TOR axis in head and neck cancer:functions,aberrations,crosstalk,and therapies[J].Oral Dis,2015,21(7):815-825.
[23]Danielsen SA,Eide PW,Nesbakken A,et al.Portrait of the PI3K/AKT pathway in colorectal cancer[J].Biochimi Biophys Acta,2015,1855(1):104-121.
[24]Wu XS,Bao TH,Ke Y,et al.Hint1 suppresses migration and invasion of hepatocellular carcinoma cells in vitro by modulating girdin activity[J].Tumor Biol,2016,37(11):14711-14719.
[25]Yamamura Y,Asai N,Enomoto A,et al.Akt-Girdin signaling in cancer-associated fibroblasts contributes to tumor progression[J].Cancer Res,2015,75(5):813-823.
[26]王敏,王芸,孙亮,等.Girdin蛋白在胆管癌组织中的表达及意义[J].肝胆外科杂志,2013,21(5):379-381.Wang M,Wang Y,Sun L,et al.Clinical significance of expression of Girdin in cholangiocarcinoma[J].J Hepat Sur,2013,21(5):379-381.
[27]Wang C,Lin J,Li L,et al.Expression and clinical significance of girdin in gastric cancer[J].Mol Clin Oncol,2014,2(3):425-428.
[28]Jiang P,Cui SP,Ren YL,et al.Girdin correlated with autophagy in invasive ductal breast carcinomas[J].Tumori,2013,99(4):530-534.
[29]Shibata T,Matsuo Y,Shamoto T,et al.Girdin,a regulator of cell motility,is a potential prognostic marker for esophageal squamous cell carcinoma[J].Oncol Rep,2013,29(6):2127-2132.
[30]李培栋,王新军,单峤,等.RNA干扰Fascin1表达抑制胶质瘤细胞U87MG的侵袭,转移能力[J].中国免疫学杂志,2015,31(6):764-768.Li PD,Wang XJ,Shan Q,et al.Reversal effect of RNA interference targeting Fascin1 on migration and invasion of glioma cell line U87 MG[J].Chin J Immunol,2015,31(6):764-768.
[31]Li-Byarlay H,Li Y,Stroud H,et al.RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee[J].Proc Natl Acad Sci U S A,2013,110(31):12750-12755.
[32]Shalem O,Sanjana NE,Hartenian E,et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science,2014,343(6166):84-87.
[33]Zhu Z,Sun H,Ma G,et al.Bufalin induces lung cancer cell apoptosis via the inhibition of PI3K/Akt pathway[J].Int J Mol Sci,2012,13(2):2025-2035.
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