二甲双胍对人胚肾HEK293T细胞增殖、凋亡和Cyclin D1表达的影响
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摘要
目的研究二甲双胍对人胚肾细胞系HEK293T增殖的抑制和诱导凋亡的作用,并初步探讨其可能的机制。方法采用0mmol/L、5mmol/L、10mmol/L、20mmol/L二甲双胍浓度梯度处理HEK293T细胞,各组分别处理24、48、72h后,通过MTT实验分析细胞增殖变化;在二甲双胍浓度梯度处理48h后,利用Annexin V-FITC/PI染色检测细胞凋亡;采用实时定量PCR检测Cyclin D1基因mRNA的表达变化。结果二甲双胍处理后,所有处理组在24、48、72h的细胞存活率均显著下降(P<0.05),但并未发现有时间依赖和浓度依赖效应;经流式细胞仪检测,二甲双胍干预48h之后,5mmol/L、10mmol/L和20mmol/L浓度处理组HEK293T细胞凋亡率均明显高于对照组(P<0.05);0mmol/L、5mmol/L、10mmol/L、20mmol/L二甲双胍分别处理HEK293T细胞48h后,实时定量PCR结果显示,10mmol/L和20mmol/L处理组的Cyclin D1表达量出现明显降低(P<0.05),但5mmol/L处理组未发现有明显变化(P>0.05)。结论二甲双胍对HEK293T细胞增殖有明显抑制作用,而对HEK293T细胞凋亡有明显的促进作用,这可能是通过抑制细胞内Cyclin D1表达来实现的。
Objective To investigate the effects of metformin onhuman embryonic kidney cells HEK293 T and its possible mechanism. Methods The HEK293 T cells were treated with 0 mmol/L,5 mmol/L,10 mmol/L and 20 mmol/L metformin treatment,respectively,for 24,48 and 72 h and performed MTT to analyze the cell proliferation;after 24 h treatment using different concentration of metformin,the cells were stained using Annexin V-FITC/PI to detect cell apoptosis;the cells were treated by different concentration of metformin for 48 h and analyzed by real-time PCR to detect mRNA expression of Cyclin D1. Results After metformin treatment,cell proliferations were significantly decreased in all the treatment groups at 24,48 and 72 h(P<0.05),however,no timedependent and dose-dependent were found;Using the flow cytometry,we found that the 5 mmol/L,10 mmol/L and 20 mmol/L metformingroups had higher apoptosis rates compared to the 0 mmol/L after 48 h treatment;after 48 h treatment of metformin on HEK293 T cells,the real-time PCR results showed that10 mmol/L and 20 mmol/L groups had significant lower Cyclin D1 expression compared to control(P<0.05),however,no significantdifference could be found between 5 mmol/L and control(P>0.05). Conclusion Metformin inhibited HEK293 T cell proliferation and induced HEK293 T cell apoptosis which may achieve via suppressing the Cyclin D1 expression.
Objective To investigate the effects of metformin onhuman embryonic kidney cells HEK293 T and its possible mechanism. Methods The HEK293 T cells were treated with 0 mmol/L,5 mmol/L,10 mmol/L and 20 mmol/L metformin treatment,respectively,for 24,48 and 72 h and performed MTT to analyze the cell proliferation;after 24 h treatment using different concentration of metformin,the cells were stained using Annexin V-FITC/PI to detect cell apoptosis;the cells were treated by different concentration of metformin for 48 h and analyzed by real-time PCR to detect mRNA expression of Cyclin D1. Results After metformin treatment,cell proliferations were significantly decreased in all the treatment groups at 24,48 and 72 h(P<0.05),however,no timedependent and dose-dependent were found;Using the flow cytometry,we found that the 5 mmol/L,10 mmol/L and 20 mmol/L metformingroups had higher apoptosis rates compared to the 0 mmol/L after 48 h treatment;after 48 h treatment of metformin on HEK293 T cells,the real-time PCR results showed that10 mmol/L and 20 mmol/L groups had significant lower Cyclin D1 expression compared to control(P<0.05),however,no significantdifference could be found between 5 mmol/L and control(P>0.05). Conclusion Metformin inhibited HEK293 T cell proliferation and induced HEK293 T cell apoptosis which may achieve via suppressing the Cyclin D1 expression.
引文
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[4]Morales DR1,Morris AD.Metformin in cancer treatment and prevention[J].Annu Rev Med,2015,66:17-29.
[5]Dreyer JH,Hauck F,Barros MHM,Niedobitek G.pRb and CyclinD1 Complement p16 as Immunohistochemical Surrogate Markers of HPV Infection in Head and Neck Cancer[J].Appl Immunohistochem Mol Morphol,2017,25(5):366-373.
[6]Yin H,Sheng Z,Zhang X,et al.Overexpression of SOX18 promotes prostate cancer progression via the regulation of TCF1,c-Myc,cyclin D1 and MMP-7[J].Oncol Rep,2017,37(2):1045-1051.
[7]Mills CC,Kolb EA,Sampson VB.Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer[J].Cancer Res,2017,77(23):6489-6498.
[8]Liao S1,Xiao S2,Chen H3,Zhang M1,Chen Z4.The receptor for activated protein kinase C promotes cell growth,invasion and migration in cervical cancer[J].Int J Oncol,2017,51(5):1497-1507.
[9]El-Gendi S1,Abu-Sheasha G2.Ki-67 and Cell Cycle Regulators p53,p63 and cyclinD1 as Prognostic Markers for Recurrence/Progression of Bladder Urothelial Carcinoma[J].Pathol Oncol Res,2018,24(2):309-322.
[10]Livak KJ,Schmittgen TD.Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCTMethod[J].Methods,2001,25(4):402-408.
[11]Courtois S1,Durán RV2,Giraud J1,SifréE1,Izotte J3,Mégraud F4,Lehours P4,Varon C1,Bessède E5.Metformin targets gastric cancer stem cells[J].Eur J Cancer,2017,84:193-201.
[12]胡著云,戈阳华,周琼,等.虎杖提取白藜芦醇对PC-3细胞株的体外实验研究[J].江西医药,2018,53(9):1010-1012.
[13]Kurimoto R,Iwasawa S,Ebata T,et al.Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation[J].Int J Oncol,2016,48(5):1825-36.
[14]Griss T,Vincent EE,Egnatchik R,et al.Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis[J].PLOS Biology,2015,13(12):e1002309.
[15]Costa D,Gigoni A,Würth R,et al.Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA[J].Cancer Cell International,2014,14(1):59.
[16]Sarfstein R,Friedman Y,Attias-Geva Z,et al.Metformin Downregulates the Insulin/IGF-I Signaling Pathway and Inhibits Different Uterine Serous Carcinoma(USC)Cells Proliferation and Migration in p53-Dependent or-Independent Manners[J].PLOSONE,2013,8(4):e61537.
[17]杨文泰.胃蛋白酶原与胃部疾病相关性的研究进展[J].江西医药,2018,53(4):398-400.
[18]Zhao Y,Sun H,Feng M,et al.Metformin is associated with reduced cell proliferation in human endometrial cancer by inbibiting PI3K/AKT/mTOR signaling[J].Gynecological Endocrinology,2018,34(5):428-432.
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