COX2抑制剂联合KDM1A基因对肺癌A549细胞凋亡的诱导作用研究
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摘要
目的探讨COX2抑制剂塞来昔布或KDM1A siRNA对肺癌A549细胞增殖凋亡的影响及其机制。方法以LipofectamineTM 2000为载体,将合成的KDM1A siRNA转染人肺腺癌A549细胞,Western blot检测转染后A549细胞中KDM1A的表达。塞来昔布或si-KDM1A处理A549细胞,分为NC组、塞来昔布组、si-KDM1A组、塞来昔布+si-KDM1A组。CCK8法检测细胞活力,流式细胞术检测细胞凋亡率;H2DCFHDA荧光探针检测ROS含量;Western blot检测STAT3、磷酸化STAT3及下游与增殖凋亡相关的基因PCNA和Bax蛋白表达。结果转染si-KDM1A后,A549细胞KDM1A表达明显受到抑制,与空白对照组比较,差异具有统计学意义(P<0.05)。塞来昔布和si-KDM1A均能明显抑制A549细胞活力,诱导细胞凋亡,提高细胞内ROS含量,下调p-STAT3和PCNA表达,上调Bax表达,与NC组比较,差异均具有统计学意义(P<0.05);且二者联合对细胞增殖、凋亡及STAT3信号通路的影响更明显,细胞活力、凋亡率、ROS含量及p-STAT3、PCNA、Bax表达水平与塞来昔布组和si-KDM1A组比较,差异均具有统计学意义(P<0.05)。结论 COX2抑制剂或KDM1A siRNA均可抑制肺癌A549细胞活力,诱导细胞凋亡,二者联用强于单独使用,其机制可能与提高细胞内ROS含量及抑制STAT3信号通路有关。
Objective To investigate the effect of COX2 inhibitor celecoxib or KDM1 A siRNA on the proliferation and apoptosis of A549 lung cancer cells. Methods Lipofectamine TM2000 was used as a carrier. KDM1 A siRNA was transfected into human lung adenocarcinoma A549 cells, and the KDM1 A expression in transfected A549 cells was detected by Western blot. After treated with celecoxib or si-KDM1 A,the A549 cells were divided into NC group, celecoxib group, si-KDM1 A group, celecoxib + si-KDM1 A group. The cell viability was detected by CCK8 assay, and the apoptosis rate was detected by flow cytometry. The content of ROS was detected through the H2 DCFHDA fluorescent probe. The expression of STAT3, p-STAT3, PCNA and Bax proteins were detected by Western blot. Results The expression of KDM1 A in A549 cells after transfected with si-KDM1 A was significantly inhibited, and there was significant difference when compared with the NC group(P<0.05). Celecoxib or si-KDM1 A both could significantly inhibit A549 cell viability, induce apoptosis, increase the content of ROS in cells, down-regulate the expression of p-STAT3 and PCNA and up-regulate the expression of Bax. All differences were statistically significant when compared with NC group(P<0.05). The combination of celecoxib and si-KDM1 A had much greater effects on cell proliferation, apoptosis and STAT3 signaling pathway. The results of cell viability, apoptosis rate, ROS content and expression of p-STAT3,PCNA and Bax were significantly different from those in celecoxib group and si-KDM1 A group(P<0.05). Conclusion COX2 inhibitors or KDM1 A siRNA both inhibit A549 cell viability and induce cell apoptosis. Their combination was stronger than single use. The mechanism may be related to the increase of intracellular ROS content and the inhibition of STAT3 signaling pathway.
Objective To investigate the effect of COX2 inhibitor celecoxib or KDM1 A siRNA on the proliferation and apoptosis of A549 lung cancer cells. Methods Lipofectamine TM2000 was used as a carrier. KDM1 A siRNA was transfected into human lung adenocarcinoma A549 cells, and the KDM1 A expression in transfected A549 cells was detected by Western blot. After treated with celecoxib or si-KDM1 A,the A549 cells were divided into NC group, celecoxib group, si-KDM1 A group, celecoxib + si-KDM1 A group. The cell viability was detected by CCK8 assay, and the apoptosis rate was detected by flow cytometry. The content of ROS was detected through the H2 DCFHDA fluorescent probe. The expression of STAT3, p-STAT3, PCNA and Bax proteins were detected by Western blot. Results The expression of KDM1 A in A549 cells after transfected with si-KDM1 A was significantly inhibited, and there was significant difference when compared with the NC group(P<0.05). Celecoxib or si-KDM1 A both could significantly inhibit A549 cell viability, induce apoptosis, increase the content of ROS in cells, down-regulate the expression of p-STAT3 and PCNA and up-regulate the expression of Bax. All differences were statistically significant when compared with NC group(P<0.05). The combination of celecoxib and si-KDM1 A had much greater effects on cell proliferation, apoptosis and STAT3 signaling pathway. The results of cell viability, apoptosis rate, ROS content and expression of p-STAT3,PCNA and Bax were significantly different from those in celecoxib group and si-KDM1 A group(P<0.05). Conclusion COX2 inhibitors or KDM1 A siRNA both inhibit A549 cell viability and induce cell apoptosis. Their combination was stronger than single use. The mechanism may be related to the increase of intracellular ROS content and the inhibition of STAT3 signaling pathway.
引文
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[15]Ma Y, Zhang J, Zhang Q, et al. Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction[J]. Biochem Biophys Res Commun, 2014, 448(1):8-14. doi:10.1016/j. bbrc.2014.04.007.
[16]Liu X, Pei C, Yan S, et al. NADPH oxidase 1-dependent ROS is crucial for TLR4 signaling to promote tumor metastasis of non-small cell lung cancer[J]. Tumour Biol, 2015,36(3):1493-1502. doi:10.1007/s13277-014-2639-9.
[17]张艳,代洪波,陈姗姗,等.雷帕霉素联合紫杉醇对子宫内膜癌STAT3表达的影响[J].现代生物医学进展, 2016,16(20):3898-3901. doi:10.13241/j. cnki. pmb.2016.20.026.
[18]Jiang G, Huang C, Li J, et al. Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145[J]. Mol Cancer Ther,2017, 16(5):924-935. doi:10.1158/1535-7163. MCT-16-0631.
[19]Nan J, Du Y, Chen X, et al. TPCA-1 is a direct dual inhibitor of STAT3 and NF-κB and regresses mutant EGFR-associated human non-small cell lung cancers[J]. Mol Cancer Ther, 2014,13(3):617-629. doi:10.1158/1535-7163. MCT-13-0464.
[20]Wang Y, Chen T, Huang H, et al. miR-363-3p inhibits tumor growth by targeting PCNA in lung adenocarcinoma[J]. Oncotarget, 2017, 8(12):20133-20144. doi:10.18632/oncotarget.15448.
[21]Song S, Jacobson KN, McDermott KM, et al. ATP promotes cell survival via regulation of cytosolic[Ca2+] and Bcl-2/Bax ratio in lung cancer cells[J]. Am J Physiol Cell Physiol,2016, 310(2):C99-C114. doi:10.1152/ajpcell.00092.2015.
[2]Chen S, Li P, Li J, Wang Y, et al. MiR-144 inhibits proliferation and induces apoptosis and autophagy in lung cancer cells by targeting TIGAR[J]. Cell Physiol Biochem, 2015,35(3):997-1007. doi:10.1159/000369755.
[3]Mattsson JS, Bergman B, Grinberg M, et al. Prognostic impact of COX-2 in non-small cell lung cancer:a comprehensive compartment-specific evaluation of tumor and stromal cell expression[J]. Cancer Lett, 2015, 356(2 Pt B):837-845. doi:10.1016/j. canlet.2014.10.032.
[4]Simonsson M, Bj?rner S, Markkula A, et al. The prognostic impact of COX-2 expression in breast cancer depends on oral contraceptive history, preoperative NSAID use, and tumor size[J].Int J Cancer, 2017, 140(1):163-175. doi:10.1002/ijc.30432.
[5]Emami J, Pourmashhadi A, Sadeghi H, et al. Formulation and optimization of celecoxib-loaded PLGA nanoparticles by the Taguchi design and their in vitro cytotoxicity for lung cancer therapy[J]. Pharm Dev Technol, 2014, 19:1-10. doi:10.3109/10837450.2014.920360.
[6]Kong L, Zhang P, Li W, et al. KDM1A promotes tumor cell invasion by silencing TIMP3 in non-small cell lung cancer cells[J]. Oncotarget, 2016, 7(19):27959-27974. doi:10.18632/oncotarget.8563.
[7]Lv T, Yuan D, Miao X, et al. Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer[J]. PLoS One, 2012, 7(4):e35065. doi:10.1371/journal. pone.0035065.
[8]Echizen K, Hirose O, Maeda Y, et al. Inflammation in gastric cancer:Interplay of the COX-2/prostaglandin E2 and Toll-like receptor/MyD88 pathways[J]. Cancer Sci, 2016,107(4):391-397. doi:10.1111/cas.12901.
[9]Kim J, Hong SW, Kim S, et al. Cyclooxygenase-2 expression is induced by celecoxib treatment in lung cancer cells and is transferred to neighbor cells via exosomes[J]. Int J Oncol, 2018, 52(2):613-620. doi:10.3892/ijo.2017.4227.
[10]Hattar K, Franz K, Ludwig M, et al. Interactions between neutrophils and non-small cell lung cancer cells:enhancement of tumor proliferation and inflammatory mediator synthesis[J]. Cancer Immunol Immunother, 2014, 63(12):1297-1306. doi:10.1007/s00262-014-1606-z.
[11]Yu M, Yang H, Wu K, et al. Novel pyrazoline derivatives as bi-inhibitor of COX-2 and B-Raf in treating cervical carcinoma[J]. Bioorg Med Chem, 2014, 22(15):4109-4118. doi:10.1016/j. bmc.2014.05.059.
[12]Mason MD, Clarke NW, James ND, et al. Adding Celecoxib With or Without Zoledronic Acid for Hormone-Na?ve Prostate Cancer:Long-Term Survival Results From an Adaptive, Multiarm, Multistage, Platform, Randomized Controlled Trial[J]. J Clin Oncol,2017, 35(14):1530-1541. doi:10.1200/JCO.2016.69.0677.
[13]Goossens S, Peirs S, Van Loocke W, et al. Oncogenic ZEB2activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia[J]. Blood, 2017,129(8):981-990. doi:10.1182/blood-2016-06-721191.
[14]徐丽伟. KDM1A对胃癌侵袭转移能力的影响及调节机制的研究[D].长沙:中南大学, 2015.
[15]Ma Y, Zhang J, Zhang Q, et al. Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction[J]. Biochem Biophys Res Commun, 2014, 448(1):8-14. doi:10.1016/j. bbrc.2014.04.007.
[16]Liu X, Pei C, Yan S, et al. NADPH oxidase 1-dependent ROS is crucial for TLR4 signaling to promote tumor metastasis of non-small cell lung cancer[J]. Tumour Biol, 2015,36(3):1493-1502. doi:10.1007/s13277-014-2639-9.
[17]张艳,代洪波,陈姗姗,等.雷帕霉素联合紫杉醇对子宫内膜癌STAT3表达的影响[J].现代生物医学进展, 2016,16(20):3898-3901. doi:10.13241/j. cnki. pmb.2016.20.026.
[18]Jiang G, Huang C, Li J, et al. Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145[J]. Mol Cancer Ther,2017, 16(5):924-935. doi:10.1158/1535-7163. MCT-16-0631.
[19]Nan J, Du Y, Chen X, et al. TPCA-1 is a direct dual inhibitor of STAT3 and NF-κB and regresses mutant EGFR-associated human non-small cell lung cancers[J]. Mol Cancer Ther, 2014,13(3):617-629. doi:10.1158/1535-7163. MCT-13-0464.
[20]Wang Y, Chen T, Huang H, et al. miR-363-3p inhibits tumor growth by targeting PCNA in lung adenocarcinoma[J]. Oncotarget, 2017, 8(12):20133-20144. doi:10.18632/oncotarget.15448.
[21]Song S, Jacobson KN, McDermott KM, et al. ATP promotes cell survival via regulation of cytosolic[Ca2+] and Bcl-2/Bax ratio in lung cancer cells[J]. Am J Physiol Cell Physiol,2016, 310(2):C99-C114. doi:10.1152/ajpcell.00092.2015.