ITCH对肺癌A549细胞增殖侵袭和凋亡的影响
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摘要
目的探讨ITCH在肺癌细胞系中的表达以及ITCH沉默对肺癌A549细胞增殖、侵袭、凋亡的影响及其可能机制。方法采用Western blotting检测人正常肺上皮细胞系BEAS-2B和人肺癌细胞系A549、Calu3中ITCH蛋白的表达情况。采用PEI转染试剂分别向A549细胞转染si ITCH(si ITCH组)和si NC(si NC组),采用CCK-8法、Transwell实验和流式细胞术检测两组细胞增殖能力、侵袭能力和凋亡的变化,Western blotting检测两组ITCH、Bcl-2、Bax、Cyclin D1、MMP-2、MMP-9、β-catenin、E-cadherin蛋白表达。结果人肺正常上皮细胞系BEAS-2B中ITCH蛋白表达水平为0. 98±0. 043,显著低于Calu3细胞的1. 92±0. 073和A549细胞的2. 74±0. 151,差异均有统计学意义(P <0. 05)。si ITCH组96 h时细胞增殖倍数为1. 97±0. 021,显著低于si NC组的3. 72±0. 232(P <0. 05); si ITCH组细胞凋亡率为(43. 2±1. 52)%,显著高于si NC组的(2. 3±0. 32)%,差异有统计学意义(P <0. 05)。si ITCH组穿膜细胞数为(297. 2±22. 21)个,显著低于si NC组的(601. 2±3. 21)个,差异有统计学意义(P <0. 05)。与si NC组比较,si ITCH组Bcl-2、Cyclin D1、MMP-2、MMP-9、β-catenin表达水平降低,Bax、E-cadherin表达水平升高。结论 ITCH沉默通过调控MMP、Cyclin D1、Bcl-2/Bax等信号通路抑制肺癌细胞增殖、侵袭并诱导其凋亡。
Objective To investigate the expression of ITCH in lung cancer cells and the effect of ITCH silencing on proliferation,invasion and apoptosis of lung cancer cells A549 and its possible mechanisms. Methods Western blotting was used to detect the expression of ITCH protein in human normal lung epithelial cell lines BEAS-2 B and human lung cancer cell lines A549 and Calu3.Si ITCH( si ITCH group) and si NC( si NC group) were transfected into A549 cells by PEI transfection reagent,respectively. CCK-8 method,Transwell test and flow cytometry were used to detect the changes of cell proliferation,invasion and apoptosis in the two groups. The protein expression of ITCH,Bcl-2,Bax,Cyclin D1,MMP-2,MMP-9,β-catenin and E-cadherin in the two groups were detected by Western blotting assay. Results The expression level of ITCH protein in human lung normal epithelial cell line BEAS-2 B was 0. 98 ± 0. 043,significantly lower than that in Calu3 cell line( 1. 92 ± 0. 073) and A549 cell line( 2. 74 ± 0. 151),and the difference was statistically significant( P < 0. 05). The cell proliferation rate in si ITCH group was 1. 97 ± 0. 021 at 96 h,which was significantly lower than that in si NC group( 3. 72 ± 0. 232),and the difference was statistically significant( P < 0. 05). The apoptotic rate in si ITCH group was( 43. 2 ± 1. 52) %,which was significantly higher than that in si NC group( 2. 3 ± 0. 32) %. The difference was statistically significant( P < 0. 05). The number of perforating cells in si ITCH group was 297. 2 ± 21. 21,which was significantly lower than 601. 2 ± 3. 21 in si NC group( P < 0. 05). Compared with the si NC group,the expression levels of Bcl-2,Cyclin D1,MMP-2,MMP-9 and β-catenin in si ITCH group decreased,while the expression levels of Bax and E-cadherin increased. Conclusion Silencing ITCH inhibits lung cancer proliferation,invasion and induces apoptosis by regulating signaling pathways such as MMP,Cyclin D1 and Bcl-2/Bax.
Objective To investigate the expression of ITCH in lung cancer cells and the effect of ITCH silencing on proliferation,invasion and apoptosis of lung cancer cells A549 and its possible mechanisms. Methods Western blotting was used to detect the expression of ITCH protein in human normal lung epithelial cell lines BEAS-2 B and human lung cancer cell lines A549 and Calu3.Si ITCH( si ITCH group) and si NC( si NC group) were transfected into A549 cells by PEI transfection reagent,respectively. CCK-8 method,Transwell test and flow cytometry were used to detect the changes of cell proliferation,invasion and apoptosis in the two groups. The protein expression of ITCH,Bcl-2,Bax,Cyclin D1,MMP-2,MMP-9,β-catenin and E-cadherin in the two groups were detected by Western blotting assay. Results The expression level of ITCH protein in human lung normal epithelial cell line BEAS-2 B was 0. 98 ± 0. 043,significantly lower than that in Calu3 cell line( 1. 92 ± 0. 073) and A549 cell line( 2. 74 ± 0. 151),and the difference was statistically significant( P < 0. 05). The cell proliferation rate in si ITCH group was 1. 97 ± 0. 021 at 96 h,which was significantly lower than that in si NC group( 3. 72 ± 0. 232),and the difference was statistically significant( P < 0. 05). The apoptotic rate in si ITCH group was( 43. 2 ± 1. 52) %,which was significantly higher than that in si NC group( 2. 3 ± 0. 32) %. The difference was statistically significant( P < 0. 05). The number of perforating cells in si ITCH group was 297. 2 ± 21. 21,which was significantly lower than 601. 2 ± 3. 21 in si NC group( P < 0. 05). Compared with the si NC group,the expression levels of Bcl-2,Cyclin D1,MMP-2,MMP-9 and β-catenin in si ITCH group decreased,while the expression levels of Bax and E-cadherin increased. Conclusion Silencing ITCH inhibits lung cancer proliferation,invasion and induces apoptosis by regulating signaling pathways such as MMP,Cyclin D1 and Bcl-2/Bax.
引文
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[18] Lim SK,Lu SY,Kang SA,et al. Wnt signaling promotes breastcancer by blocking ITCH-mediated degradation of the YAP/TAZtranscriptional coactivator WBP2[J]. Cancer Res,2016,76(21):6278-6289.
[19] Xia K,Zhang Y,Cao S,et al. miR-411 regulated ITCH expres-sion and promoted cell proliferation in human hepatocellular car-cinoma cells[J]. Biomed Pharmacother,2015,70:158-163.
[2] Yan P,Zhu H,Yin L,et al. Integrinαvβ6 promotes lung canc-er proliferation and metastasis through upregulation of IL-8-medi-ated MAPK/ERK signaling[J]. Transl Oncol,2018,11(3):619-627.
[3] Park SM,Choi EY,Bae DH,et al. The LncRNA EPEL pro-motes lung cancer cell proliferation through E2F target activation[J]. Cell Physiol Biochem,2018,45(3):1270-1283.
[4] Jennek S,Mittag S,Reiche J,et al. Tricellulin is a target of theubiquitin ligase Itch[J]. Ann N Y Acad Sci,2017,1397(1):157-168.
[5] Tsui YM,Sze KM,Tung EK,et al. Dishevelled-3 phos-phorylation is governed by HIPK2/PP1 Cα/ITCH axis andthe non-phosphorylated form promotes cancer stemness viaLGR5 in hepatocellular carcinoma[J]. Oncotarget,2017,8(24):39430-39442.
[6] Xu M,Wang DC,Wang X,et al. Correlation between mucin bi-ology and tumor heterogeneity in lung cancer[J]. Semin Cell DevBiol,2017,64:73-78.
[7] Gen Y,Yasui K,Kitaichi T,et al. ASPP2 suppresses invasionand TGF-β1-induced epithelial-mesenchymal transition by inhibi-ting Smad7 degradation mediated by E3 ubiquitin ligase ITCH ingastric cancer[J]. Cancer Lett,2017,398:52-61.
[8] Duan B,Cheng L,Ma Q. Spinal circuits transmitting mechanicalpain and itch[J]. Neurosci Bull,2018,34(1):186-193.
[9] Wong LS,Wu T,Lee CH. Inflammatory and noninflammatoryitch:implications in pathophysiology-directed treatments[J]. IntJ Mol Sci,2017,18(7):1485.
[10]孙洋,郭彦君,周丽娜.特发性肺间质纤维化合并肺癌的研究进展[J].临床肿瘤学杂志,2017,22(7):658-663.
[11]钱倩,郝可可. miRNA在肺腺癌恶性胸腔积液中的表达及临床意义[J].临床肿瘤学杂志,2017,22(9):781-786.
[12] Li Y,Zhang H,Li Y,et al. MiR-182 inhibits the epithelial tomesenchymal transition and metastasis of lung cancer cells by tar-geting the Met gene[J]. Mol Carcinog,2018,57(1):125-136.
[13] Chi Y,Jin Q,Liu X,et al. miR-203 inhibits cell proliferation,invasion,and migration of non-small-cell lung cancer by down-regulating RGS17[J]. Cancer Sci,2017,108(12):2366-2372.
[14] Rajasinghe LD,Pindiprolu RH,Gupta SV. Delta-tocotrienol in-hibits non-small-cell lung cancer cell invasion via the inhibition ofNF-κB,uPA activator,and MMP-9[J]. Onco Targets Ther,2018,11:4301-4314.
[15] Jung CH,Han AR,Chung HJ,et al. Linarin inhibits radiation-induced cancer invasion by downregulating MMP-9 expression viathe suppression of NF-κB activation in human non-small-cell lungcancer A549[J]. Nat Prod Res,2018:1-5.
[16] Losuwannarak N,Sritularak B,Chanvorachote P. Cycloartobilox-anthone induces human lung cancer cell apoptosis via mitochon-dria-dependent apoptotic pathway[J]. In Vivo,2018,32(1):71-78.
[17] Lim JS,Ibaseta A,Fischer MM,et al. Intratumoural heterogene-ity generated by Notch signalling promotes small-cell lung cancer[J]. Nature,2017,545(7654):360-364.
[18] Lim SK,Lu SY,Kang SA,et al. Wnt signaling promotes breastcancer by blocking ITCH-mediated degradation of the YAP/TAZtranscriptional coactivator WBP2[J]. Cancer Res,2016,76(21):6278-6289.
[19] Xia K,Zhang Y,Cao S,et al. miR-411 regulated ITCH expres-sion and promoted cell proliferation in human hepatocellular car-cinoma cells[J]. Biomed Pharmacother,2015,70:158-163.