小檗碱调控TLR4/NF-κB信号通路抑制宫颈癌Hela细胞增殖研究
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摘要
目的探讨TLR4-NF-κb信号通路在小檗碱发挥抑制HeLa细胞增殖中的作用。方法分别给予5、10和40μg/mL 3种不同剂量干预Hela细胞,并通过特异性siRNA干扰TLR4表达。通过RT-qPCR和Western法检测TLR4、p65和Bcl-2的mRNA和蛋白表达改变,通过MTT法、Transwell小室和TUNEL评价细胞生长、迁移和凋亡情况。结果软件设计+RT-qPCR确定转染抑制效率最高的siRNA-002进行后续实验。小檗碱呈浓度依赖性抑制Hela细胞增殖和迁移,促进细胞凋亡,抑制p65、Bcl-2和TLR4的mRNA和蛋白表达。加用siRNA-002后,小檗碱的上述作用进一步增强。结论小檗碱可调控TLR4-NF-κB信号通路下调Bcl-2表达,显著抑制宫颈癌Hela细胞的增殖和迁移,促进其凋亡,为小檗碱治疗宫颈癌提供新的理论依据。
Objective To evaluate the effects of berberine on the proliferation of Hela cells through TLR4-NF-κB pathway. Methods Three different dose of berberine(5, 10 and 40 μg/mL) and special TLR4-siRNA were used to intervene Hela cells. mRNA and protein of TLR4, p65 and Bcl-2 were evaluated by RT-qPCR and Western blot, respectively. The changes of proliferation, migration and apoptosis of Hela cells were estimated by MTT, Transwell chamber and TUN EL, respectively. Results SiRNA-002 was selected as the strongest silence siRNA by RT-qPCR. The berberine could inhibit the proliferation and migration, promote the apoptosis, down-regulate the mRNA and protein of p65, Bcl-2 and TLR4 of Hela cells, which showed a dose-dependent effect. The effects of berberine above-mentioned were enhanced by siRNA-002. Conclusion Berberine may inhibit Hela cells' proliferation and migration, promote apoptosis by the down-regulation of Bcl-2 through TLR4-NF-κB pathway.
Objective To evaluate the effects of berberine on the proliferation of Hela cells through TLR4-NF-κB pathway. Methods Three different dose of berberine(5, 10 and 40 μg/mL) and special TLR4-siRNA were used to intervene Hela cells. mRNA and protein of TLR4, p65 and Bcl-2 were evaluated by RT-qPCR and Western blot, respectively. The changes of proliferation, migration and apoptosis of Hela cells were estimated by MTT, Transwell chamber and TUN EL, respectively. Results SiRNA-002 was selected as the strongest silence siRNA by RT-qPCR. The berberine could inhibit the proliferation and migration, promote the apoptosis, down-regulate the mRNA and protein of p65, Bcl-2 and TLR4 of Hela cells, which showed a dose-dependent effect. The effects of berberine above-mentioned were enhanced by siRNA-002. Conclusion Berberine may inhibit Hela cells' proliferation and migration, promote apoptosis by the down-regulation of Bcl-2 through TLR4-NF-κB pathway.
引文
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[13]DU Q,WANG Y,CHAO L,et al.Chemopreventive activity of GEN-27,a genistein derivative,in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis[J].Oncotarget,2016,7(14):17870-17884.
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[16]KIM E M,JUNG C H,KIM J,et al.The p53/p21 complex regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins[J].Cancer Res,2017,77(11):3092-3100.
[17]RAHMAN M A,WANG P,ZHAO Z,et al.Systemic delivery of siRNA by DNA Nanoparticles targeting Bcl2 in cancer cell growth[J].Angew Chem Int Ed Engl,2017,129(50):16023.
[2]LI H L,WU H,ZHANG B B,et al.MAPK pathways are involved in the inhibitory effect of berberine hydrochloride on gastric cancer MGC 803 cell proliferation and IL-8 secretion in vitro and in vivo[J].Mol Med Rep,2016,14(2):1430.
[3]WANG H Y,YU H Z,HUANG S M,et al.p53,Bcl-2 and cox-2 are involved in berberine hydrochloride-induced apoptosis of HeLa229 cells[J].Mol Med Rep,2016,14(4):3855-3862.
[4]RAGHAV D,ASHRAF S M,MOHAN L,et al.Berberine induces toxicity in HeLa cells through perturbation of microtubule polymerization by binding to tubulin at a unique site[J].Biochemistry,2017,56(20):334-337.
[5]BOWTELL D,CHENEVIX-TRENCH G,GREEN A,et al.MyD88 and TLR4 expression in epithelial ovarian cancer[J].Mayo Clin Proc,2018,93(3):307-320.
[6]RASHIDIAN A,MUHAMMADNEJAD A,Dehpour A R,et al.Atorvastatin attenuates TNBS-induced rat colitis:the involvement of the TLR4/NF-κB signaling pathway[J].Inflammopharmacology,2016,24(2-3):109-118.
[7]徐小艳,徐宪伟,张果,等.核因子κB和CD147在肺腺癌中的表达及意义[J].实用医学杂志,2017,33(4):583-585.
[8]XU C,LI H,YIN M,et al.Osteopontin is involved in TLR4pathway contributing to ovarian cancer cell proliferation and metastasis[J].Oncotarget,2017,8(58):98394-98404.
[9]MUKHERJEE S,KARMKAR S,BABU S P S.TLR2 and TLR4 mediated host immune responses in major infectious diseases:a review[J].Braz J Infect Dis,2016,20(2):193.
[10]PANG Y N,LIANG Y W,FENG T S,et al.Transportation of berberine into HepG2,HeLa and SY5Y cells:a correlation to its anti-cancer effect[J].Plos One,2014,9(11):e112937.
[11]TONG N,ZHANG J,CHEN Y,et al.Berberine sensitizes mutliple human cancer cells to the anticancer effects of doxorubicin in vitro[J].Oncology Letters,2012,3(6):1263-1267.
[12]HONG-DUCK U.Bcl-2 family proteins as regulators of cancer cell invasion and metastasis:a review focusing on mitochondrial respiration and reactive oxygen species[J].Oncotarget,2016,7(5):5193-5203.
[13]DU Q,WANG Y,CHAO L,et al.Chemopreventive activity of GEN-27,a genistein derivative,in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis[J].Oncotarget,2016,7(14):17870-17884.
[14]UM H D.Bcl-2 family proteins as regulators of cancer cell invasion and metastasis:a review focusing on mitochondrial respiration and reactive oxygen species[J].Oncotarget,2016,7(5):5193-5203.
[15]DELBRIDGE A R D,GRABOW S,STRASSER A,et al.Thirty years of BCL-2:translating cell death discoveries into novel cancer therapies[J].Nat Rev Cancer,2016,16(2):99.
[16]KIM E M,JUNG C H,KIM J,et al.The p53/p21 complex regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins[J].Cancer Res,2017,77(11):3092-3100.
[17]RAHMAN M A,WANG P,ZHAO Z,et al.Systemic delivery of siRNA by DNA Nanoparticles targeting Bcl2 in cancer cell growth[J].Angew Chem Int Ed Engl,2017,129(50):16023.