miR-194对人非小细胞肺癌细胞系A549增殖、凋亡的影响及其机制
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摘要
目的观察微小RNA-194(miR-194)对人非小细胞肺癌(NSCLC)细胞系A549增殖、凋亡的影响,并探讨其机制。方法取A549细胞和人正常肺上皮细胞16HBE,采用qRT-PCR法检测两种细胞miR-194。常规培养A549细胞,经脂质体分别转染miR-194抑制物、miR-194模拟物(mimics)、对照48 h(分别计为A、B、C组),采用qRT-PCR验证转染效果,MTS法检测细胞增殖能力,平板克隆实验检测细胞克隆形成能力,流式细胞仪检测细胞凋亡能力,Western blotting法检测细胞JAK2、pSTAT3、活化的Caspase-3蛋白。结果 A549、16HBE细胞中miR-194相对表达量分别为1. 00±0. 10、8. 17±0. 14,两者比较,P <0. 05。A、B、C组miR-194相对表达量分别为0. 31±0. 11、8. 35±0. 31、1. 00±0. 10,A、B组分别与C组比较,P均<0. 05。A组转染24、48、72 h的OD值分别为0. 494±0. 069、0. 843±0. 084、1. 111±0. 109,B组分别为0. 355±0. 026、0. 510±0. 049、0. 706±0. 087,C组分别为0. 427±0. 022、0. 664±0. 068、0. 906±0. 060,A、B组分别与C组比较,P均<0. 05。A、B、C组细胞克隆数目分别为(232. 12±10. 82)、(43. 67±10. 97)、(127. 33±6. 43)个,A、B组分别与C组比较,P均<0. 05。与C组比较,A组细胞凋亡现象及数目明显减少,B组细胞凋亡现象及数目明显增多; A、B、C组细胞凋亡率分别为4. 58%±1. 01%、21. 45%±1. 78%、10. 23%±1. 65%,A、B组分别与C组比较,P均<0. 05。A组JAK2、pSTAT3、活化的Caspase-3蛋白相对表达量分别为5. 97±0. 24、4. 52±0. 18、0. 11±0. 04,B组分别为0. 34±0. 03、0. 21±0. 02、7. 21±0. 79,C组分别为1. 00±0. 10、1. 00±0. 10、1. 00±0. 10,A、B组分别与C组比较,P均<0. 05。结论 miR-194能抑制A549细胞的增殖、克隆形成能力,并诱导细胞凋亡,其机制可能与调控JAK2/STAT3信号通路并促进活化的Caspase-3蛋白表达有关。
Objective To observe the effects of microRNA-194( miR-194) on the proliferation and apoptosis of human non-small-cell lung cancer( NSCLC) cell line A549,and to explore its mechanism. Methods The miR-194 of A549 cells and human normal pulmonary epithelial cells 16 HBE was detected by qRT-PCR. A549 cells were cultured routinely,and were transfecteded with miR-194 inhibitor,miR-194 mimics and controls by liposome for 48 h,( recorded as groups A,B and C,respectively). The transfection effect was verified by qRT-PCR. MTS assay was used to detect cell proliferation,plate cloning assay was used to detect the ability of cell cloning,the apoptotic ability was detected by flow cytometry,and JAK2,pSTAT3,and activated Caspase-3 protein was detected by Western blotting. Results The relative expression of miR-194 in A549 cells and 16 HBE cells was 1. 00 ± 0. 10 and 8. 17 ± 0. 14,respectively,with statistically significant difference( P < 0. 05). The relative expression of miR-194 in the groups A,B and C was 0. 31 ± 0. 11,8. 35 ± 0. 31,and1. 00 ± 0. 10,and statistically significant difference was found between groups A,B and group C( P < 0. 05). OD at 24,48 and 72 h after transfection was 0. 494 ± 0. 069,0. 843 ± 0. 084,and 1. 111 ± 0. 109,respectively,in the group A,0. 355 ± 0. 026,0. 510 ± 0. 049,and 0. 706 ± 0. 087,respectively,in the group B,and 0. 427 ± 0. 022,0. 664 ± 0. 068,and 0. 906 ± 0. 060,respectively,in the group C,with statistically significant difference( all P < 0. 05). The number of cell clones in the groups A,B and C were 232. 12 ± 10. 82,43. 67 ± 10. 97,and 127. 33 ± 6. 43,and statistically significant difference was found between groups A,B and group C( P < 0. 05). Compared with group C,the apoptosis and number of cells in the group A was significantly reduced,while the apoptosis and number of cells in the group B significantly increased. The apoptotic rates of groups A,B and C were 4. 58% ± 1. 01%,21. 45% ± 1. 78%,and 10. 23% ± 1. 65%,and statistically significant difference was found between groups A,B and group C( P < 0. 05). The relative expression levels of JAK2,pSTAT3 and activated C aspase-3 protein were 5. 97 ± 0. 24,4. 52 ± 0. 18,and 0. 11 ± 0. 04,respectively,in the group A,0. 34 ± 0. 03,0. 21 ± 0. 02,and 7. 21 ± 0. 79,respectively,in the group B,and 1. 00 ± 0. 10,1. 00 ±0. 10,and 1. 00 ± 0. 10,respectively,in the group C,and statistically significant difference was found between groups A,B and group C( all P < 0. 05). Conclusions The miR-194 can inhibit the proliferation,clone formation,and induce apoptosis of A549 cells. Its mechanism may be related to regulating JAK2/STAT3 signaling pathway and promoting the expression of activated Caspase-3 protein.
Objective To observe the effects of microRNA-194( miR-194) on the proliferation and apoptosis of human non-small-cell lung cancer( NSCLC) cell line A549,and to explore its mechanism. Methods The miR-194 of A549 cells and human normal pulmonary epithelial cells 16 HBE was detected by qRT-PCR. A549 cells were cultured routinely,and were transfecteded with miR-194 inhibitor,miR-194 mimics and controls by liposome for 48 h,( recorded as groups A,B and C,respectively). The transfection effect was verified by qRT-PCR. MTS assay was used to detect cell proliferation,plate cloning assay was used to detect the ability of cell cloning,the apoptotic ability was detected by flow cytometry,and JAK2,pSTAT3,and activated Caspase-3 protein was detected by Western blotting. Results The relative expression of miR-194 in A549 cells and 16 HBE cells was 1. 00 ± 0. 10 and 8. 17 ± 0. 14,respectively,with statistically significant difference( P < 0. 05). The relative expression of miR-194 in the groups A,B and C was 0. 31 ± 0. 11,8. 35 ± 0. 31,and1. 00 ± 0. 10,and statistically significant difference was found between groups A,B and group C( P < 0. 05). OD at 24,48 and 72 h after transfection was 0. 494 ± 0. 069,0. 843 ± 0. 084,and 1. 111 ± 0. 109,respectively,in the group A,0. 355 ± 0. 026,0. 510 ± 0. 049,and 0. 706 ± 0. 087,respectively,in the group B,and 0. 427 ± 0. 022,0. 664 ± 0. 068,and 0. 906 ± 0. 060,respectively,in the group C,with statistically significant difference( all P < 0. 05). The number of cell clones in the groups A,B and C were 232. 12 ± 10. 82,43. 67 ± 10. 97,and 127. 33 ± 6. 43,and statistically significant difference was found between groups A,B and group C( P < 0. 05). Compared with group C,the apoptosis and number of cells in the group A was significantly reduced,while the apoptosis and number of cells in the group B significantly increased. The apoptotic rates of groups A,B and C were 4. 58% ± 1. 01%,21. 45% ± 1. 78%,and 10. 23% ± 1. 65%,and statistically significant difference was found between groups A,B and group C( P < 0. 05). The relative expression levels of JAK2,pSTAT3 and activated C aspase-3 protein were 5. 97 ± 0. 24,4. 52 ± 0. 18,and 0. 11 ± 0. 04,respectively,in the group A,0. 34 ± 0. 03,0. 21 ± 0. 02,and 7. 21 ± 0. 79,respectively,in the group B,and 1. 00 ± 0. 10,1. 00 ±0. 10,and 1. 00 ± 0. 10,respectively,in the group C,and statistically significant difference was found between groups A,B and group C( all P < 0. 05). Conclusions The miR-194 can inhibit the proliferation,clone formation,and induce apoptosis of A549 cells. Its mechanism may be related to regulating JAK2/STAT3 signaling pathway and promoting the expression of activated Caspase-3 protein.
引文
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[18]Miao J,Wang W,Wu S,et al.miR-194 suppresses proliferation and migration and promotes apoptosis of osteosarcoma cells by targeting CDH2[J].Cell Physiol Biochem,2018,45(5):1966-1974.
[19]李灵毅,骆曼.MiR-194对恶性黑色素瘤细胞增殖和凋亡的影响及机制[J].中国现代医学杂志,2017,27(21):31-36.
[20]Bao J,Zou JH,Li CY,et al.miR-194 inhibits gastric cancer cell proliferation and tumorigenesis by targeting KDM5B[J].Eur Rev Med Pharmacol Sci,2016,20(21):4487-4493.
[21]Tayeh Z.Extract in combination with cisplatin/etoposide/doxorubicin suppresses lymphoma cell growth through induction of Caspase-3 dependent apoptosis[J].Int J Mol Sci,2018,19(8):99-110.
[22]Sun Y,Zhou P,Chen S,et al.The JAK/STAT3 signaling pathway mediates inhibition of host cell apoptosis by chlamydia psittaci infection[J].Pathogens Dis,2017,2(1):88-96.
[23]Cheng R,Liu YJ,Cui JW,et al.Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells[J].Oncotarget,2017,8(18):30252-30264.
[24]Ouyang J,Pan X,Lin H,et al.GKN2 increases apoptosis,reduces the proliferation and invasion ability of gastric cancer cells through down-regulating the JAK/STAT signaling pathway[J].Am J Transl Res,2017,9(2):803-811.
[25]李雷雷,郭彬,郭佳培,等.肿瘤相关巨噬细胞通过JAK2/STAT3途径调控肝癌细胞凋亡的机制研究[J].现代预防医学,2017,44(13):2406-2410.
[2]裴振,霍小蕾,田向阳,等.长链非编码RNA LINC00467促进非小细胞肺癌细胞的增殖[J].西安交通大学学报(医学版),2018,39(3):402-408.
[3]Okwuosa TM,Anzevino S.Cardiovascular disease in cancer survivors[J].Postgrad Med J,2017,93(1096):82-90.
[4]Rotow J.Understanding and targeting resistance mechanisms in NSCLC[J].Nat Rev Cancer,2017,17(11):637-658.
[5]Simonson B.MicroRNA therapeutics:the next magic bullet[J].Mini Rev Med Chem,2015,15(6):467-474.
[6]Wang XZ,Hang YK,Liu JB,et al.Over-expression of microR-NA-375 inhibits papillary thyroid carcinoma cell proliferation and induces cell apoptosis by targeting ERBB2[J].J Pharmacol Sci,2016,130(2):78-84.
[7]Guo J,Meng R,Yin Z,et al.A serum microRNA signature as a prognostic factor for patients with advanced NSCLC and its association with tissue microRNA expression profiles[J].Mol Med Rep,2016,13(6):4643-4653.
[8]Mi J,Zou Y,Lin X,et al.Dysregulation of the miR-194-CUL4Bnegative feedback loop drives tumorigenesis in non-small-cell lung carcinoma[J].Mol Oncol,2017,11(3):305-319.
[9]Zhou L,Di Q,Sun B,et al.MicroRNA-194 restrains the cell progression of non-small cell lung cancer by targeting human nuclear distribution protein C[J].Oncol Rep,2016,35(6):3435-3444.
[10]Lachgar A,Tazi MA,Afif M,et al.Lung cancer:incidence and survival in Rabat,Morocco[J].Rev Epidemiol Sante Publique,2016,64(6):391-395.
[11]Zellweger M,Abdelnour-Berchtold E,Krueger T,et al.Surgical treatment of pulmonary metastasis in colorectal cancer patients:current practice and results[J].Crit Rev Oncol Hematol,2018,127(1):105-116.
[12]O'Kane GM.Systemic therapy of lung cancer CNS metastases using molecularly targeted agents and immune checkpoint inhibitors[J].CNS Drugs,2018,32(6):527-542.
[13]李婷,边莉.microRNAs在肺癌中的相关研究及进展[J].昆明医科大学学报,2017,38(7):130-135.
[14]Tao H,Chen ZW,Yang JJ.MicroRNA-29a suppresses cardiac fibroblasts proliferation via targeting VEGF-A/MAPK signal pathway[J].Int J Biol Macromol,2016,88(2):414-423.
[15]Li H,Ouyang R,Wang Z,et al.MiR-150 promotes cellular metastasis in non-small cell lung cancer by targeting FOXO4[J].Sci Rep,2016,6(1):39001-39012.
[16]Gao Y,Guo Y,Wang Z,et al.Analysis of circulating miRNAs 21and 375 as potential biomarkers for early diagnosis of prostate cancer[J].Neoplasma,2016,63(4):623-628.
[17]Zhang W,Qian P,Zhang X,et al.Autocrine/Paracrine human growth hormone-stimulated microRNA 96-182-183 cluster promotes epithelial-mesenchymal transition and invasion in breast cancer[J].J Biol Chem,2015,290(22):13812-13829.
[18]Miao J,Wang W,Wu S,et al.miR-194 suppresses proliferation and migration and promotes apoptosis of osteosarcoma cells by targeting CDH2[J].Cell Physiol Biochem,2018,45(5):1966-1974.
[19]李灵毅,骆曼.MiR-194对恶性黑色素瘤细胞增殖和凋亡的影响及机制[J].中国现代医学杂志,2017,27(21):31-36.
[20]Bao J,Zou JH,Li CY,et al.miR-194 inhibits gastric cancer cell proliferation and tumorigenesis by targeting KDM5B[J].Eur Rev Med Pharmacol Sci,2016,20(21):4487-4493.
[21]Tayeh Z.Extract in combination with cisplatin/etoposide/doxorubicin suppresses lymphoma cell growth through induction of Caspase-3 dependent apoptosis[J].Int J Mol Sci,2018,19(8):99-110.
[22]Sun Y,Zhou P,Chen S,et al.The JAK/STAT3 signaling pathway mediates inhibition of host cell apoptosis by chlamydia psittaci infection[J].Pathogens Dis,2017,2(1):88-96.
[23]Cheng R,Liu YJ,Cui JW,et al.Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells[J].Oncotarget,2017,8(18):30252-30264.
[24]Ouyang J,Pan X,Lin H,et al.GKN2 increases apoptosis,reduces the proliferation and invasion ability of gastric cancer cells through down-regulating the JAK/STAT signaling pathway[J].Am J Transl Res,2017,9(2):803-811.
[25]李雷雷,郭彬,郭佳培,等.肿瘤相关巨噬细胞通过JAK2/STAT3途径调控肝癌细胞凋亡的机制研究[J].现代预防医学,2017,44(13):2406-2410.