miRNA-24通过靶向CARMA3基因调控胃癌AGS细胞的增殖和凋亡
|
摘要
目的:探讨miRNA-24对胃癌AGS细胞增殖和凋亡的影响及其潜在的作用机制。方法:实时定量聚合酶链式反应(q RT-PCR)检测不同胃癌细胞株(AGS、MKN74、HGC27)和胃黏膜上皮GES-1细胞中miRNA-24的表达水平。建立miRNA-24过表达的AGS细胞株,采用CCK-8法检测细胞增殖活力,流式细胞术检测细胞周期和凋亡情况,Western blotting检测细胞周期和凋亡相关cyclin D1、CDK2、Bcl-2、p-IκB-α/IκB-α和p-Rb/Rb蛋白的表达水平;双荧光素酶报告基因分析法预测和验证miRNA-24可能的靶基因。结果:3株胃癌细胞中miRNA-24的表达均低于GES-1 cell。转染miRNA-24 mimic(miR-24组)48 h后AGS细胞增殖活力显著低于miR-NC组[(119.62±12.63)%vs(147.79±11.89)%,P<0.05],并出现周期阻滞,且早期和晚期细胞凋亡率明显较miR-NC组上升[早期凋亡率:(11.32±2.27)%vs(0.57±0.08)%;晚期凋亡率:(15.56±2.27)%vs(0.85±0.16)%,均P<0.05]。转染miRNA-24 mimic后,细胞中cyclin D1、CDK2、Bcl-2及p-Rb的蛋白表达水平均较miR-NC组显著降低,p-IκB-α蛋白的表达水平较miR-NC组显著上升。共转染miRNA-24 mimic和miRNA-24可能作用靶点CARMA3基因过表达质粒的miR-24+pc DNA-CARMA3组AGS细胞CARMA3蛋白表达较miR-24组明显增加(1.74±0.09 vs 1.03±0.06,P<0.05)。miR-24+pc DNA3-CARMA3组AGS细胞48 h增殖活力较miR-24组显著升高[(137.85±15.34)%vs(102.31±11.23)%,P<0.05];而miR-24+pc DNA3-CARMA3组AGS细胞早期凋亡率和晚期凋亡率均较miR-24组显著降低[早期凋亡率:(4.24±0.56)%vs(11.32±2.27)%,P<0.05;晚期凋亡率:(6.38±0.63)%vs(15.56±2.27)%,P<0.05]。CARMA3过表达可部分逆转miRNA-24对胃癌AGS细胞增殖及凋亡的作用。结论:miRNA-24可通过靶向CARMA3基因抑制胃癌细胞的增殖、促进其凋亡。
Objective:To explore effect of miRNA-24 on proliferation and apoptosis of the gastric cancer AGS cell and its potential mechanism. Methods: Expressions of miRNA-24 in gastric cancer AGS, MKN and HGC27 cells as well as gastric mucosal epithelium GES-1 cell were detected by q RT-PCR. The AGS cell line with over-expression of miRNA-24 was constructed. CCK-8, flow cytometry and Western blotting assays were respectively used to measure proliferation vibility of the cells, cell cycle and apoptosis, and expressions of cell cycle and apoptosis-related cyclin D1, CDK2, Bcl-2, p-IκB-α/IκB-α, p-Rb/Rb proteins. Possible target gene of miRNA-24 was forecasted and verified with dual luciferase report gene assay. Results: Expressions of miRNA-24 in the gastric cells was obviously lower than that in the GES-1 cell(P<0.05). Proliferation vibility of the AGS cell at 48 h in the transfected with miRNA-24 mimic(miR-24) group was remarkably lower than that in the control miR-NC group([119.62±12.63]%vs [147.79±11.89]%, P<0.05), an arrest of cell cycle occurred, and its early and later apoptosis rates were evidently more increased than those in the miR-NC group(early apoptosis rate:[11.32+2.27]% vs [0.57±0.08]%; later apoptosis rate:([15.56 ± 2.27]% vs [0.85 ± 0.16]%, all P<0.05). In the miRNA-24 group, expression levels of cyclin D1,CDK2, Bcl2 and p-Rb proteins were all obviously lower than those in the miR-NC group, and expression level of pIκB-α protein was obviously higher than that in the miR-NC group. In co-transfection of miRNA-24 mimic and over-expression plasmid of CARMA3 that may be action target of miRNA-24(miR-24+pc DNA-CARMA3) group,CARMA3 protein expression of the AGS cell was significantly higher than that in the miR-24 group(1.74±0.09 vs1.03±0.06, P<0.05). Proliferation vibility of the AGS cell at 48 h in the miR-24+pc DNA-CARMA3 group was obviously higher than that in the miR-24 group([137.85±15.34]% vs [102.31±11.23]%, P<0.05), however, early and later apoptosis rates of the AGS cell in the miR-24+pc DNA-CARMA3 group were all lower than those in the miR-24 group(early apoptosis rate: [4.24±0.56]% vs [11.32±2.27]%, later apoptosis rate: [6.38+0.63]% vs [15.56±2.27]%,all P<0.05). Effect of miRNA-24 on proliferation of apoptosis of the gastric cancer AGS cell can be partially reversed by over-expression of CARMA3. Conclusion: miRNA-24 could inhibit proliferation of the gastric caner cells and promote their apoptosis through targeting CARMA3.
Objective:To explore effect of miRNA-24 on proliferation and apoptosis of the gastric cancer AGS cell and its potential mechanism. Methods: Expressions of miRNA-24 in gastric cancer AGS, MKN and HGC27 cells as well as gastric mucosal epithelium GES-1 cell were detected by q RT-PCR. The AGS cell line with over-expression of miRNA-24 was constructed. CCK-8, flow cytometry and Western blotting assays were respectively used to measure proliferation vibility of the cells, cell cycle and apoptosis, and expressions of cell cycle and apoptosis-related cyclin D1, CDK2, Bcl-2, p-IκB-α/IκB-α, p-Rb/Rb proteins. Possible target gene of miRNA-24 was forecasted and verified with dual luciferase report gene assay. Results: Expressions of miRNA-24 in the gastric cells was obviously lower than that in the GES-1 cell(P<0.05). Proliferation vibility of the AGS cell at 48 h in the transfected with miRNA-24 mimic(miR-24) group was remarkably lower than that in the control miR-NC group([119.62±12.63]%vs [147.79±11.89]%, P<0.05), an arrest of cell cycle occurred, and its early and later apoptosis rates were evidently more increased than those in the miR-NC group(early apoptosis rate:[11.32+2.27]% vs [0.57±0.08]%; later apoptosis rate:([15.56 ± 2.27]% vs [0.85 ± 0.16]%, all P<0.05). In the miRNA-24 group, expression levels of cyclin D1,CDK2, Bcl2 and p-Rb proteins were all obviously lower than those in the miR-NC group, and expression level of pIκB-α protein was obviously higher than that in the miR-NC group. In co-transfection of miRNA-24 mimic and over-expression plasmid of CARMA3 that may be action target of miRNA-24(miR-24+pc DNA-CARMA3) group,CARMA3 protein expression of the AGS cell was significantly higher than that in the miR-24 group(1.74±0.09 vs1.03±0.06, P<0.05). Proliferation vibility of the AGS cell at 48 h in the miR-24+pc DNA-CARMA3 group was obviously higher than that in the miR-24 group([137.85±15.34]% vs [102.31±11.23]%, P<0.05), however, early and later apoptosis rates of the AGS cell in the miR-24+pc DNA-CARMA3 group were all lower than those in the miR-24 group(early apoptosis rate: [4.24±0.56]% vs [11.32±2.27]%, later apoptosis rate: [6.38+0.63]% vs [15.56±2.27]%,all P<0.05). Effect of miRNA-24 on proliferation of apoptosis of the gastric cancer AGS cell can be partially reversed by over-expression of CARMA3. Conclusion: miRNA-24 could inhibit proliferation of the gastric caner cells and promote their apoptosis through targeting CARMA3.
引文
[1]CHENG X J,LIN J C,TU S P.Etiology and prevention of gastric Cancer[J].Gastrointest Tumors,2016,3(1):25-36.DOI:10.1159/000443995.
[2]GAO P,TSAI C,YANG Y,et al.Intraoperative radiotherapy in gastric and esophageal cancer:meta-analysis of long-term outcomes and complications[J].Minerva Med,2017,108(1):74-83.DOI:10.23736/S0026-4806.16.04628-0.
[3]HARADA K,MIZRAK KAYA D,SHIMODAIRA Y,et al.Global chemotherapy development for gastric cancer[J].Gastric Cancer,2017,20(Suppl 1):92-101.DOI:10.1007/s10120-016-0655-8.
[4]MERHAUTOVA J,DEMLOVA R,SLABY O.Micro RNA based therapy in animal models of selected gastrointestinal cancers[J/OL].Front Pharmacol,2016,7:329[2017-01-10].http://journal.frontiersin.org/article/10.3389/fphar.2016.00329/full.DOI:10.3389/fphar.2016.00329.
[5]SUN F,WANG J,PAN Q,et al.Characterization of function and regulation of mi R-24-1 and mi R-31[J].Biochem Biophys Res Commun,2009,380(3):660-665.DOI:10.1016/j.bbrc.2009.01.161.
[6]FANG Z,TANG J,BAI Y,et al.Plasma levels of micro RNA-24,micro RNA-320a,and micro RNA-423-5p are potential biomarkers for colorectal carcinoma[J].J Exp Clin Cancer Res,2015,34(2):86-90.DOI:10.1186/s13046-015-0198-6.
[7]XU L,CHEN Z,XUE F,et al.Micro RNA-24 inhibits growth,induces apoptosis,and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein[J/OL].Cancer Cell Int,2015,15:61[2017-01-10].https://cancerci.biomedcentral.com/articles/10.1186/s12935-015-0217-x.DOI:10.1186/s12935-015-0217-x.
[8]ZHANG S,ZHANG C,LIU W,et al.Micro RNA-24 upregulation inhibits proliferation,metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3[J].Int J Oncol,2015,47(4):1351-1360.DOI:10.3892/ijo.2015.3117.
[9]INOGUCHI S,SEKI N,CHIYOMARU T,et al.Tumour-suppressive micro RNA-24-1 inhibits cancer cell proliferation through targeting FOXM1 in bladder cancer[J].FEBS Lett,2014,588(17):3170-3179.DOI:10.1016/j.febslet.2014.06.058.
[10]XIA Z X,LI Z X,ZHANG M,et al.CARMA3 regulates the invasion,migration,and apoptosis of non-small cell lung cancer cells by activating NF-κB and suppressing the P38 MAPK signaling pathway[J].Exp Mol Pathol,2016,100(2):353-360.DOI:10.1016/j.yexmp.2015.
[11]XIE C,HAN Y,FU L,et al.Overexpression of CARMA3 is associated with advanced tumor stage,cell cycle progression,and cisplatin resistance in human epithelial ovarian cancer[J].Tumour Biol,2014,35(8):7957-7964.DOI:10.1007/s13277-014-2070-2.
[12]DU S,JIA L,ZHANG Y,et al.CARMA3 is upregulated in human pancreatic carcinoma,and its depletion inhibits tumor proliferation,migration,and invasion[J].Tumour Biol,2014,35(6):5965-5970.DOI:10.1007/s13277-014-1791-6.
[13]FENG X,MIAO G,HAN Y,et al.CARMA3 is overexpressed in human glioma and promotes cell invasion through MMP9 regulation in A172 cell line[J].Tumour Biol,2014,35(1):149-154.DOI:10.1007/s13277-013-1018-2.
[14]MARTIN D,GALISTEO R,GUTKIND J S.CXCL8/IL8 stimulates vascular endothelial growth factor(VEGF)expression and the autocrine activation of VEGFR2 in endothelial cells by activating NF-kappa B through the CBM(Carma3/Bcl10/Malt1)complex[J].J Biol Chem,2009,284(10):6038-6042.DOI:10.1074/jbc.C800207200.
[15]LIN S C,LIU C J,LIN J A,et al.mi R-24 up-regulation in oral carcinoma:positive association from clinical and in vitro analysis[J].Oral Oncol,2010,46(3):204-208.DOI:10.1016/j.oraloncology.2009.12.005.
[16]LYNCH S M,MCKENNA M M,WALSH C P,et al.mi R-24 regulates CDKN1B/p27 expression in prostate cancer[J].Prostate,2016,76(7):637-648.DOI:10.1002/pros.23156.
[17]ZHAO G,LIU L,ZHAO T,et al.Upregulation of mi R-24 promotes cell proliferation by targeting NAIF1 in non-small cell lung cancer[J].Tumour Biol,2015,36(5):3693-3701.DOI:10.1007/s13277-014-3008-4.
[18]DUAN Y,HU L,LIU B,et al.Tumor suppressor mi R-24 restrains gastric cancer progression by downregulating Reg IV[J].Mol Cancer,2014,13:127.DOI:10.1186/1476-4598-13-127
[19]SONG L,YANG J,DUAN P,et al.Micro RNA-24 inhibits osteosarcoma cell proliferation both in vitro and in vivo by targeting LPAATβ[J].Arch Biochem Biophys,2013,535(2):128-135.DOI:10.1016/j.abb.2013.04.001.
[20]MOHAMMADI YEGANEH S,VASEI M,TAVAKOLI R,et al.The effect of mi R-340 over-expression on cell-cycle-related genes in triple-negative breast cancercells[J/OL].Eur J Cancer Care(Engl),2016[2017-01-10],http://onlinelibrary.wiley.com/doi/10.1111/ecc.DOI:10.1111/ecc.12496.
[2]GAO P,TSAI C,YANG Y,et al.Intraoperative radiotherapy in gastric and esophageal cancer:meta-analysis of long-term outcomes and complications[J].Minerva Med,2017,108(1):74-83.DOI:10.23736/S0026-4806.16.04628-0.
[3]HARADA K,MIZRAK KAYA D,SHIMODAIRA Y,et al.Global chemotherapy development for gastric cancer[J].Gastric Cancer,2017,20(Suppl 1):92-101.DOI:10.1007/s10120-016-0655-8.
[4]MERHAUTOVA J,DEMLOVA R,SLABY O.Micro RNA based therapy in animal models of selected gastrointestinal cancers[J/OL].Front Pharmacol,2016,7:329[2017-01-10].http://journal.frontiersin.org/article/10.3389/fphar.2016.00329/full.DOI:10.3389/fphar.2016.00329.
[5]SUN F,WANG J,PAN Q,et al.Characterization of function and regulation of mi R-24-1 and mi R-31[J].Biochem Biophys Res Commun,2009,380(3):660-665.DOI:10.1016/j.bbrc.2009.01.161.
[6]FANG Z,TANG J,BAI Y,et al.Plasma levels of micro RNA-24,micro RNA-320a,and micro RNA-423-5p are potential biomarkers for colorectal carcinoma[J].J Exp Clin Cancer Res,2015,34(2):86-90.DOI:10.1186/s13046-015-0198-6.
[7]XU L,CHEN Z,XUE F,et al.Micro RNA-24 inhibits growth,induces apoptosis,and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein[J/OL].Cancer Cell Int,2015,15:61[2017-01-10].https://cancerci.biomedcentral.com/articles/10.1186/s12935-015-0217-x.DOI:10.1186/s12935-015-0217-x.
[8]ZHANG S,ZHANG C,LIU W,et al.Micro RNA-24 upregulation inhibits proliferation,metastasis and induces apoptosis in bladder cancer cells by targeting CARMA3[J].Int J Oncol,2015,47(4):1351-1360.DOI:10.3892/ijo.2015.3117.
[9]INOGUCHI S,SEKI N,CHIYOMARU T,et al.Tumour-suppressive micro RNA-24-1 inhibits cancer cell proliferation through targeting FOXM1 in bladder cancer[J].FEBS Lett,2014,588(17):3170-3179.DOI:10.1016/j.febslet.2014.06.058.
[10]XIA Z X,LI Z X,ZHANG M,et al.CARMA3 regulates the invasion,migration,and apoptosis of non-small cell lung cancer cells by activating NF-κB and suppressing the P38 MAPK signaling pathway[J].Exp Mol Pathol,2016,100(2):353-360.DOI:10.1016/j.yexmp.2015.
[11]XIE C,HAN Y,FU L,et al.Overexpression of CARMA3 is associated with advanced tumor stage,cell cycle progression,and cisplatin resistance in human epithelial ovarian cancer[J].Tumour Biol,2014,35(8):7957-7964.DOI:10.1007/s13277-014-2070-2.
[12]DU S,JIA L,ZHANG Y,et al.CARMA3 is upregulated in human pancreatic carcinoma,and its depletion inhibits tumor proliferation,migration,and invasion[J].Tumour Biol,2014,35(6):5965-5970.DOI:10.1007/s13277-014-1791-6.
[13]FENG X,MIAO G,HAN Y,et al.CARMA3 is overexpressed in human glioma and promotes cell invasion through MMP9 regulation in A172 cell line[J].Tumour Biol,2014,35(1):149-154.DOI:10.1007/s13277-013-1018-2.
[14]MARTIN D,GALISTEO R,GUTKIND J S.CXCL8/IL8 stimulates vascular endothelial growth factor(VEGF)expression and the autocrine activation of VEGFR2 in endothelial cells by activating NF-kappa B through the CBM(Carma3/Bcl10/Malt1)complex[J].J Biol Chem,2009,284(10):6038-6042.DOI:10.1074/jbc.C800207200.
[15]LIN S C,LIU C J,LIN J A,et al.mi R-24 up-regulation in oral carcinoma:positive association from clinical and in vitro analysis[J].Oral Oncol,2010,46(3):204-208.DOI:10.1016/j.oraloncology.2009.12.005.
[16]LYNCH S M,MCKENNA M M,WALSH C P,et al.mi R-24 regulates CDKN1B/p27 expression in prostate cancer[J].Prostate,2016,76(7):637-648.DOI:10.1002/pros.23156.
[17]ZHAO G,LIU L,ZHAO T,et al.Upregulation of mi R-24 promotes cell proliferation by targeting NAIF1 in non-small cell lung cancer[J].Tumour Biol,2015,36(5):3693-3701.DOI:10.1007/s13277-014-3008-4.
[18]DUAN Y,HU L,LIU B,et al.Tumor suppressor mi R-24 restrains gastric cancer progression by downregulating Reg IV[J].Mol Cancer,2014,13:127.DOI:10.1186/1476-4598-13-127
[19]SONG L,YANG J,DUAN P,et al.Micro RNA-24 inhibits osteosarcoma cell proliferation both in vitro and in vivo by targeting LPAATβ[J].Arch Biochem Biophys,2013,535(2):128-135.DOI:10.1016/j.abb.2013.04.001.
[20]MOHAMMADI YEGANEH S,VASEI M,TAVAKOLI R,et al.The effect of mi R-340 over-expression on cell-cycle-related genes in triple-negative breast cancercells[J/OL].Eur J Cancer Care(Engl),2016[2017-01-10],http://onlinelibrary.wiley.com/doi/10.1111/ecc.DOI:10.1111/ecc.12496.