黄连素与miRNA-29b-3p对结直肠癌细胞增殖和凋亡的影响
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摘要
目的探讨黄连素对结直肠癌HCT-116细胞增殖、凋亡的影响,并探讨其作用机制。方法采用0、25、50、100、150和200μmol/L的黄连素处理HCT-116细胞24、48 h后,实时荧光定量逆转录聚合酶链反应(qRTPCR)检测人正常结肠上皮细胞株NCM460和人结直肠癌细胞株HCT-116、Caco-2细胞中miRNA-29b-3p相对表达量及黄连素干预后HCT-116细胞中miRNA-29b-3p表达水平变化。噻唑蓝(MTT)法检测HCT-116细胞存活情况;流式细胞仪检测150μmol/L黄连素处理48 h后的HCT-116细胞凋亡情况;流式细胞仪检测miRNA-29b-3p过表达和低表达对HCT-116细胞存活和凋亡的影响。结果 qRT-PCR法检测发现,HCT-116细胞中miRNA-29b-3p的相对表达量,低于Caco-2细胞和NCM460细胞,黄连素干预后HCT-116细胞中miRNA-29b-3p的相对表达量明显上调(P﹤0.01)。150μmol/L黄连素处理48 h后,HCT-116细胞存活率明显降低(P﹤0.01),凋亡率明显升高(P﹤0.01)。miRNA-29b-3p过表达可抑制HCT-116细胞的增殖并诱导其凋亡;miRNA-29b-3p低表达可逆转黄连素对HCT-116细胞的抑增殖和促凋亡作用。结论黄连素可能通过促进miRNA-29b-3p表达来抑制结直肠癌HCT-116细胞增殖并诱导其凋亡。
Objective To investigate the effect of berberine on proliferation and apoptosis of colorectal cancer HCT-116 cells and its potential mechanism.Method Cell proliferation of HCT-116 cells after treating with varying berberine concentrations(0 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L,150 μmol/L,and 200 μmol/L) for 24 h or 48 h were detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay.Cell apoptosis of HCT-116 cells after treating with150 μmol/L berberine for 48 h were determined by flow cytometry.The expression of miRNA-29 b-3 p in human normal colonic epithelial cell line NCM460 and human colorectal cancer cell lines HCT-116 and Caco-2 after treating with berberine was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Cell proliferation and apoptosis of HCT-116 after over-expression or inhibition of miRNA-29 b-3 p were detected by flow cytometry,respectively.Result qRT-PCR assay showed that,the relative expression of miRNA-29 b-3 p was down-regulated in HCT-116 cells compared with that in Caco-2 and NCM460 cells,while was up-regulated in HCT-116 cells after treating with berberine(P<0.01).150 μmol/L berberine for 48 h,the survival rate of HCT-116 cells decreased significantly(P<0.01),and the apoptotic rate increased significantly(P<0.01).Over-expression of miRNA-29 b-3 p inhibited the proliferation and promoted apoptosis of HCT-116 cells.Inhibition of miRNA-29 b-3 p expression could reverse the inhibitory effect of berberine on the proliferation and the promotional effect on the apoptosis of HCT-116 cells.Conclusion Berberine may inhibit the proliferation and promote apoptosis of HCT-116 cells by up-regulating the expression of miRNA-29 b-3 p.
Objective To investigate the effect of berberine on proliferation and apoptosis of colorectal cancer HCT-116 cells and its potential mechanism.Method Cell proliferation of HCT-116 cells after treating with varying berberine concentrations(0 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L,150 μmol/L,and 200 μmol/L) for 24 h or 48 h were detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay.Cell apoptosis of HCT-116 cells after treating with150 μmol/L berberine for 48 h were determined by flow cytometry.The expression of miRNA-29 b-3 p in human normal colonic epithelial cell line NCM460 and human colorectal cancer cell lines HCT-116 and Caco-2 after treating with berberine was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Cell proliferation and apoptosis of HCT-116 after over-expression or inhibition of miRNA-29 b-3 p were detected by flow cytometry,respectively.Result qRT-PCR assay showed that,the relative expression of miRNA-29 b-3 p was down-regulated in HCT-116 cells compared with that in Caco-2 and NCM460 cells,while was up-regulated in HCT-116 cells after treating with berberine(P<0.01).150 μmol/L berberine for 48 h,the survival rate of HCT-116 cells decreased significantly(P<0.01),and the apoptotic rate increased significantly(P<0.01).Over-expression of miRNA-29 b-3 p inhibited the proliferation and promoted apoptosis of HCT-116 cells.Inhibition of miRNA-29 b-3 p expression could reverse the inhibitory effect of berberine on the proliferation and the promotional effect on the apoptosis of HCT-116 cells.Conclusion Berberine may inhibit the proliferation and promote apoptosis of HCT-116 cells by up-regulating the expression of miRNA-29 b-3 p.
引文
[1]饶慕圣,曹胜华,王学成.直肠癌诊断的研究进展综述[J].基层医学论坛,2018,22(20):2870-2872.
[2]韦金磊,张森.结直肠癌的临床治疗进展[J].中国临床新医学,2018,11(2):202-208.
[3]Cicero AF,Baggioni A.Berberine and its role in Chronic disease[J].Adv Exp Med Biol,2016,928:27-45.
[4]Qadir SA,Kwon MC,Han JG,et al.Effect of different extraction protocols on anticancer and antioxidant activities of Berberis koreana bark extracts[J].J Biosci Bioeng,2009,107(3):331-338.
[5]Shukla S,Rizvi F,Raisuddin S,et al.FoxO proteins'nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberinetreated HepG2 cells[J].Free Radic Biol Med,2014,76(2):185-199.
[6]Qi HW,Xin LY,Xu X,et al.Epithelial-to-mesenchymal transition markers to predict response of berberine in suppressing lung cancer invasion and metastasis[J].J Transl Med,2014,12(1):22.
[7]Park KS,Kim JB,Bae J,et al.Berberine inhibited the growth of thyroid cancer cell lines 8505C and TPC1[J].Yonsei Med J,2012,53(2):346-351.
[8]Li XK,Motwani M,Tong W,et al.Huanglian,a Chinese herbal extract,inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CD2 kinase activity in human cancer cells[J].Mol Pharmacol,2000,58(6):1287-1293.
[9]彭洪,林中超,彭明沙,等.黄连素抑制直肠癌SW480细胞增殖的实验研究[J].西部医学,2018,30(2):182-186;191.
[10]Ambros V,Lee RC,Lavanway A,et al.MicroRNAs and other tiny endogenous RNAs in C.elegans[J].Curr Biol,2003,13(10):807-818.
[11]马肖,袁伟,唐万燕,等.miR-29b在结直肠癌组织中的表达及其临床意义[J].中华肿瘤杂志,2016,38(6):430-433.
[12]Casey SC,Amedei A,Aquilano K,et al.Cancer prevention and therapy through the modulation of the tumor microenvironment[J].Semin Cancer Biol,2015,35(Suppl):S199-S223.
[13]Tang J,Feng Y,Tsao S,et al.Berberine and coptidis rhizoma as novel antineoplastic agents:a review of traditional use and biomedical investigations[J].J Ethnopharmacol,2009,126(1):5-17.
[14]Iizuka N,Miyamoto K,Okita K,et al.Inhibitory effect of coptidis rhizoma and berberine on the proliferation of human esophageal cancer cell lines[J].Cancer Lett,2000,148(1):19-25.
[15]Park SH,Sung JH,Kim EJ,et al.Berberine induces apoptosis via ROS generation in PANC-1 and MIA-PaCa2 pancreatic cell lines[J].Braz J Med Biol Res,2015,48(2):111-119.
[16]Yip NK,Ho WS.Berberine induces apoptosis via the mitochondrial pathway in liver cancer cells[J].Oncol Rep,2013,30(3):1107-1112.
[17]Esquela-Kerscher A,Slack FJ.Oncomirs-microRNAs with a role in cancer[J].Nat Rev Cancer,2006,6(4):259-269.
[18]Amodio N,Leotta M,Bellizzi D,et al.DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma[J].Oncotarget,2012,3(10):1246-1258.
[19]Wang Y,Zhang X,Li H,et al.The role of miRNA-29 family in cancer[J].Eur J Cell Biol,2013,92(3):123-128.
[20]Liu H,Wang B,Lin J,et al.microRNA-29b:an emerging player in human cancer[J].Asian Pac J Cancer Prev,2014,15(21):9059-9064.
[21]Steele R,Mott JL,Ray RB.MBP-1 upregulates miR-29b that represses Mcl-1,collagens,and matrix-metalloproteinase-2 in prostate cancer cells[J].Genes Cancer,2015,1(4):381-387.
[22]Kong Y,Zou S,Yang F,et al.RUNX3-mediated up-regulation of miR-29b suppresses the proliferation and migration of gastric cancer cells by targeting KDM2A[J].Cancer Lett,2016,381(1):138-148.
[23]Lv M,Zhong Z,Huang M,et al.lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by miR-29b-3p as competing endogenous RNA[J].Biochim Biophys Acta Mol Cell Res,2017,1864(10):1887-1899.
[24]Shin J,Shim HG,Hwang T,et al.Restoration of miR-29b exerts anti-cancer effects on glioblastoma[J].Cancer Cell Int,2017,17(1):104.
[2]韦金磊,张森.结直肠癌的临床治疗进展[J].中国临床新医学,2018,11(2):202-208.
[3]Cicero AF,Baggioni A.Berberine and its role in Chronic disease[J].Adv Exp Med Biol,2016,928:27-45.
[4]Qadir SA,Kwon MC,Han JG,et al.Effect of different extraction protocols on anticancer and antioxidant activities of Berberis koreana bark extracts[J].J Biosci Bioeng,2009,107(3):331-338.
[5]Shukla S,Rizvi F,Raisuddin S,et al.FoxO proteins'nuclear retention and BH3-only protein Bim induction evoke mitochondrial dysfunction-mediated apoptosis in berberinetreated HepG2 cells[J].Free Radic Biol Med,2014,76(2):185-199.
[6]Qi HW,Xin LY,Xu X,et al.Epithelial-to-mesenchymal transition markers to predict response of berberine in suppressing lung cancer invasion and metastasis[J].J Transl Med,2014,12(1):22.
[7]Park KS,Kim JB,Bae J,et al.Berberine inhibited the growth of thyroid cancer cell lines 8505C and TPC1[J].Yonsei Med J,2012,53(2):346-351.
[8]Li XK,Motwani M,Tong W,et al.Huanglian,a Chinese herbal extract,inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CD2 kinase activity in human cancer cells[J].Mol Pharmacol,2000,58(6):1287-1293.
[9]彭洪,林中超,彭明沙,等.黄连素抑制直肠癌SW480细胞增殖的实验研究[J].西部医学,2018,30(2):182-186;191.
[10]Ambros V,Lee RC,Lavanway A,et al.MicroRNAs and other tiny endogenous RNAs in C.elegans[J].Curr Biol,2003,13(10):807-818.
[11]马肖,袁伟,唐万燕,等.miR-29b在结直肠癌组织中的表达及其临床意义[J].中华肿瘤杂志,2016,38(6):430-433.
[12]Casey SC,Amedei A,Aquilano K,et al.Cancer prevention and therapy through the modulation of the tumor microenvironment[J].Semin Cancer Biol,2015,35(Suppl):S199-S223.
[13]Tang J,Feng Y,Tsao S,et al.Berberine and coptidis rhizoma as novel antineoplastic agents:a review of traditional use and biomedical investigations[J].J Ethnopharmacol,2009,126(1):5-17.
[14]Iizuka N,Miyamoto K,Okita K,et al.Inhibitory effect of coptidis rhizoma and berberine on the proliferation of human esophageal cancer cell lines[J].Cancer Lett,2000,148(1):19-25.
[15]Park SH,Sung JH,Kim EJ,et al.Berberine induces apoptosis via ROS generation in PANC-1 and MIA-PaCa2 pancreatic cell lines[J].Braz J Med Biol Res,2015,48(2):111-119.
[16]Yip NK,Ho WS.Berberine induces apoptosis via the mitochondrial pathway in liver cancer cells[J].Oncol Rep,2013,30(3):1107-1112.
[17]Esquela-Kerscher A,Slack FJ.Oncomirs-microRNAs with a role in cancer[J].Nat Rev Cancer,2006,6(4):259-269.
[18]Amodio N,Leotta M,Bellizzi D,et al.DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma[J].Oncotarget,2012,3(10):1246-1258.
[19]Wang Y,Zhang X,Li H,et al.The role of miRNA-29 family in cancer[J].Eur J Cell Biol,2013,92(3):123-128.
[20]Liu H,Wang B,Lin J,et al.microRNA-29b:an emerging player in human cancer[J].Asian Pac J Cancer Prev,2014,15(21):9059-9064.
[21]Steele R,Mott JL,Ray RB.MBP-1 upregulates miR-29b that represses Mcl-1,collagens,and matrix-metalloproteinase-2 in prostate cancer cells[J].Genes Cancer,2015,1(4):381-387.
[22]Kong Y,Zou S,Yang F,et al.RUNX3-mediated up-regulation of miR-29b suppresses the proliferation and migration of gastric cancer cells by targeting KDM2A[J].Cancer Lett,2016,381(1):138-148.
[23]Lv M,Zhong Z,Huang M,et al.lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by miR-29b-3p as competing endogenous RNA[J].Biochim Biophys Acta Mol Cell Res,2017,1864(10):1887-1899.
[24]Shin J,Shim HG,Hwang T,et al.Restoration of miR-29b exerts anti-cancer effects on glioblastoma[J].Cancer Cell Int,2017,17(1):104.