hsa-miR-302a-3p靶向VEGFA抑制胃癌细胞增殖的机制研究
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摘要
目的探讨过表达hsa-miR-302a-3p下调血管内皮生长因子A(VEGFA)对胃癌细胞SGC-7901存活、运动和上皮间质转化的调节作用。方法采用细胞转染,将hsa-miR-302a-3p mimic转染于miR组细胞,pcVEGFA转染于VEGFA组细胞,同时将两个基因共转染于miR+VEGFA组。采用RT-PCR和Western blot检测转染效率,生物信息学靶向预测和荧光素实验验证两个基因靶向关系,采用CCK-8法检测各组细胞增殖,Transwell检测各组细胞侵袭能力,划痕实验检测各组细胞迁移能力,显微镜下观察细胞的形态是否发生上皮间质转化(EMT),采用Western blot检测各组细胞存活相关蛋白Ki67和Caspase-3、EMT相关蛋白E-cadherin、Vimentin、N-cadherin和Snail以及VEGFA下游靶基因p-P38、p-MAPKAPK和p-Hsp27蛋白表达水平。结果VEGFA是miR-302a-3p的预测靶位点;与对照组相比,miR组细胞数量、侵袭和迁移率均下降(P<0.05),VEGFA组细胞数量、侵袭和迁移率均升高(P<0.05);与VEGFA组相比,miR+VEGFA组细胞数量、侵袭和迁移率均下降(P<0.05),E-cadherin蛋白表达水平上调(P<0.05),Vimentin、N-cadherin和Snail蛋白表达水平下调(P<0.05),p-P38、p-MAPKAPK和p-Hsp27蛋白表达水平下调(P<0.05)。结论 hsa-miR-302a-3p通过抑制VEGFA的表达,抑制胃癌细胞SGC-7901的增殖、侵袭和迁移。
Objective To explore the role mechanism of hsa-miR-302a-3p overexpression in the inhibition of proliferation of gastric cancer cell SGC-7901 by targeted-regulating vascular endothelial growth factor A(VEGFA).Methods The cell transfection was used to transfect hsa-miR-302a-3p mimic into miR mimic group and transfect pcVEGFAinto VEGFA group,and the two genes were co-transfected into miR+VEGFA group.The transfection efficiency was detected by RT-PCR and Western blot.The bioinformatics targeting prediction and fluorescein assay were used to verify the targeting relationship between the two genes.Cell proliferation was detected by CCK-8test,and Transwell assay was used to detect the invasion ability of each group,and scratch assay was used to detect the migration ability of each group.The morphology changes of epithelial-mesenchymal transition(EMT)in cells were observed under microscope.Western blot was used to detect the protein expression levels of survival-related proteins Ki67 and Caspase-3,EMT-related proteins E-cadherin,Vimentin,N-cadherin and Snail and VEGFA downstream target genes p-P38,p-MAPKAPK and p-Hsp27.Results VEGFA was the predicted target site of miR-302a-3p.Compared with control group,the number of cells,the invasion and migration rates were also reduced(P<0.05)in miR mimic group,and the number of cells was increased(P<0.05)as well as the invasion and migration rates in VEGFA group.Compared with VEGFA group,the number of cells,the invasion and migration rates were also decreased(P<0.05)in miR+VEGFA group.The protein expression level of E-cadherin was up-regulated(P<0.05)while the protein expression levels of Vimentin,N-cadherin and Snail were down-regulated(P<0.05),and the protein expression levels of p-P38,p-MAPKAPK and p-Hsp27 were also down-regulated(P <0.05).Conclusion hsa-miR-302a-3p overexpression can inhibit the proliferation and promote apoptosis of gastric cancer cell SGC-7901 by targeting negative regulation of VEGFAexpression.
Objective To explore the role mechanism of hsa-miR-302a-3p overexpression in the inhibition of proliferation of gastric cancer cell SGC-7901 by targeted-regulating vascular endothelial growth factor A(VEGFA).Methods The cell transfection was used to transfect hsa-miR-302a-3p mimic into miR mimic group and transfect pcVEGFAinto VEGFA group,and the two genes were co-transfected into miR+VEGFA group.The transfection efficiency was detected by RT-PCR and Western blot.The bioinformatics targeting prediction and fluorescein assay were used to verify the targeting relationship between the two genes.Cell proliferation was detected by CCK-8test,and Transwell assay was used to detect the invasion ability of each group,and scratch assay was used to detect the migration ability of each group.The morphology changes of epithelial-mesenchymal transition(EMT)in cells were observed under microscope.Western blot was used to detect the protein expression levels of survival-related proteins Ki67 and Caspase-3,EMT-related proteins E-cadherin,Vimentin,N-cadherin and Snail and VEGFA downstream target genes p-P38,p-MAPKAPK and p-Hsp27.Results VEGFA was the predicted target site of miR-302a-3p.Compared with control group,the number of cells,the invasion and migration rates were also reduced(P<0.05)in miR mimic group,and the number of cells was increased(P<0.05)as well as the invasion and migration rates in VEGFA group.Compared with VEGFA group,the number of cells,the invasion and migration rates were also decreased(P<0.05)in miR+VEGFA group.The protein expression level of E-cadherin was up-regulated(P<0.05)while the protein expression levels of Vimentin,N-cadherin and Snail were down-regulated(P<0.05),and the protein expression levels of p-P38,p-MAPKAPK and p-Hsp27 were also down-regulated(P <0.05).Conclusion hsa-miR-302a-3p overexpression can inhibit the proliferation and promote apoptosis of gastric cancer cell SGC-7901 by targeting negative regulation of VEGFAexpression.
引文
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[17]XU M,ZHOU H,ZHANG C,et al.ADAM17promotes epithelial-mesenchymal transition via TGF-β/Smad pathway in gastric carcinoma cells.Int J Oncol,2016,49(6):2520-2528.
[18]MASUI T,OTA I,YOOK JI,et al.Snail-induced epithelial-mesenchymal transition promotes cancer stem celllike phenotype in head and neck cancer cells.Int J Oncol,2014,44(3):693-699.
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[20]ZHANG Z,RUI W,WANG ZC,et al.Anti-proliferation and anti-metastasis effect of barbaloin in non-small cell lung cancer via inactivating p38MAPK/Cdc25B/Hsp27pathway.Oncol Rep,2017,38(2):1172-1180.
[21]SAWADA J,LI F,KOMATSU M.R-Ras inhibits VEGF-Induced p38MAPK activation and HSP27phosphorylation in endothelial cells.J Vasc Res,2015,52(5):347-359.
[2]SCHWARZENBACH H,NISHIDA N,CALIN GA,et al.Clinical relevance of circulating cell-free microRNAs in cancer.Nat Rev Clin Oncol,2014,11(3):145-156.
[3]NOGUCHI S,YASUI Y,IWASAKI J,et al.Replacement treatment with microRNA-143and-145induces synergistic inhibition of the growth of human bladder cancer cells by regulating PI3K/Akt and MAPK signaling pathways.Cancer Lett,2013,328(2):353-361.
[4]HU S,WILSON KD,GHOSH Z,et al.MicroRNA-302increases reprogramming efficiency via repression of NR2F2.Stem Cells,2013,31(2):259-268.
[5]QIN C,ZHA W,FAN R,et al.MicroRNA-302ainhibits cell proliferation and invasion,and induces cell apoptosis in hepatocellular carcinoma by directly targeting VEGFA.Mol Med Rep,2017,16(5):6360-6367.
[6]GOEL HL,PURSELL B,SHULTZ LD,et al.P-Rex1promotes resistance to VEGF/VEGFR-targeted therapy in prostate cancer.Cell Rep,2016,14(9):2193-2208.
[7]MACEDO F,LADEIRA K,LONGATTOFILHO A,et al.Gastric cancer and angiogenesis:is VEGF a useful biomarker to assess progression and remission?J Gastric Cancer,2017,17(1):1-10.
[8]PENG S,GAO D,GAO C,et al.MicroRNAs regulate signaling pathways in osteogenic differentiation of mesenchymal stem cells(Review).Mol Med Rep,2016,14(1):623-629.
[9]HIBINO S,SAITO Y,MURAMATSU T,et al.Tu1900MiR-1246and Mir-302are novel targets of epigenetic therapy with dznep and SAHA in human cancer cells.Gastroenterology,2013,144(5):S876-S876.
[10]MA G,LI Q,DAI W,et al.Prognostic implications of miR-302a/b/c/d in human gastric cancer.Pathol Oncol Res,2017,23(4):899-905.
[11]WANG X,CHEN X,FANG J,et al.Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis,and silencing of both is effective to inhibit cancer growth.Int J Exp Pathol,2013,6(4):586-597.
[12]TAMMA R,ANNESE T,RUGGIERI S,et al.VEGFAand VEGFR2RNA scope determination in gastric cancer.JMol Histol,2018,49(4):429-435.
[13]BURGESSER MV,RIVA V,OJEDA SM,et al.Expression of VEGF-A,HIF-1A,CD34and Ki67in clear cell renal cell carcinomas and their relationship with conventional prognostic markers.Rev Fac Cien Med Univ Nac Cordoba,2014,71(1):7-15.
[14]JAVAD B,TAYEBE R,ADELEH D,et al.Induction of apoptosis by green synthesized gold nanoparticles through activation of caspase-3and 9in human cervical cancer cells.Avicenna J Med Biotechnol,2016,8(2):75-83.
[15]MARKOSYAN N,RECH A,VONDERHEIDE RH,et al.Abstract 469:mPGES1 deletion and the mechanisms of tumor growth suppression.Cancer Res,2015,75(15Supplement):469-469.
[16]LAMOUILLE S,XU J,DERYNCK R.Molecular mechanisms of epithelial-mesenchymal transition.Nat Rev Mol Cell Biol,2014,15(3):178-196.
[17]XU M,ZHOU H,ZHANG C,et al.ADAM17promotes epithelial-mesenchymal transition via TGF-β/Smad pathway in gastric carcinoma cells.Int J Oncol,2016,49(6):2520-2528.
[18]MASUI T,OTA I,YOOK JI,et al.Snail-induced epithelial-mesenchymal transition promotes cancer stem celllike phenotype in head and neck cancer cells.Int J Oncol,2014,44(3):693-699.
[19]DESAI P,YANG J,TIAN B,et al.Mixed-effects model of epithelial-mesenchymal transition reveals rewiring of signaling networks.Cell Signal,2015,27(7):1413-1425.
[20]ZHANG Z,RUI W,WANG ZC,et al.Anti-proliferation and anti-metastasis effect of barbaloin in non-small cell lung cancer via inactivating p38MAPK/Cdc25B/Hsp27pathway.Oncol Rep,2017,38(2):1172-1180.
[21]SAWADA J,LI F,KOMATSU M.R-Ras inhibits VEGF-Induced p38MAPK activation and HSP27phosphorylation in endothelial cells.J Vasc Res,2015,52(5):347-359.
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