间质表皮转化因子通过PI3K/AKT/MMPs信号通路促进肺腺癌细胞的侵袭转移
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摘要
间质表皮转化因子(mesenchymal to epithelial transition factor,MET)在多种癌症中异常表达,影响肿瘤的发生发展,但MET影响肺腺癌的分子机制并不明确。本研究收集3例淋巴结转移的肺腺癌组织(lung adenocarcinoma tissues,LAD)和3例无淋巴结转移的肺腺癌组织,用于微阵列基因芯片分析。结果显示,与无淋巴结转移的肺腺癌组织相比,有淋巴结转移的肺腺癌组织中有1 314条mRNAs表达上调,400条mRNAs表达下调。其中,MET在有淋巴结转移的肺腺癌组织中表达显著升高。随机选取8个差异表达基因,对收集的潍坊医学院附属医院2014年2月至2017年2月间有淋巴结转移的肺腺癌组织和无淋巴结转移的肺腺癌组织各30例通过qRT-PCR实验进行微阵列基因芯片验证。结果显示,所选mRNAs的表达与微阵列结果一致,验证了微阵列基因芯片结果的准确性。通过Western印迹进一步检测MET的表达。结果显示,相较于正常肺上皮细胞,肺腺癌细胞中MET的表达显著升高。利用质粒转染,敲减肺腺癌细胞A549中的MET,Transwell侵袭实验结果显示,敲减MET后肺腺癌细胞的侵袭能力明显降低;对各细胞组进行EGF(epidermal growth factor)处理并检测PI3K/AKT/MMPs信号通路,Western印迹检测结果显示,敲减MET后,肺腺癌细胞中基质金属蛋白酶-2(matrix metalloproteinase 2,MMP-2)和MMP-9的表达显著下降,AKT的磷酸化水平也显著下降。上述结果表明,MET可通过激活PI3K/AKT信号通路进而增加MMPs的表达促进肺腺癌的侵袭转移。
Mesenchymal to epithelial transition factor( MET) is abnormally expressed in many cancers,but the molecular mechanism of MET affecting lung adenocarcinoma is not clear. In this study,3 cases of lung adenocarcinoma tissues( LAD) with lymph node metastasis and 3 cases of LAD without lymph node metastasis were collected for microarray gene chip analysis. The results showed that 1 314 mRNAs were up-regulated and 400 mRNAs were down-regulated in LADs with lymph node metastasis,and the expression of MET was significantly increased in lung adenocarcinoma tissues with lymph node metastasis,compared with those without lymph node metastasis. Eight differentially expressed genes were randomly selected,and qRT-PCR was used to verify the results of microarray gene chip in 30 cases of LADs with lymph node metastasis collected from February 2014 to February 2017 in Affiliated Hospital of Weifang Medical University. The results showed that the expression of the selected mRNAs was consistent with the microarray data,which verified the accuracy of the microarray gene chip results. The expression of MET was further detected by Western blot,and the results showed that the expression of MET in lung adenocarcinoma cells was significantly higher than that in normal lung epithelial cells. Transwell invasion experiment results showed that the invasion ability of A549 lung epithelial cells was significantly reduced after knockdown of MET. After treated with EGF( epidermal growth factor),results showed that the expression levels of matrix metalloproteinase 2( MMP-2) and MMP-9 in lung adenocarcinoma cells were significantly decreased after the knockout of MET,and the phosphorylation level of AKT was also significantly decreased. These results indicated that MET can promote the invasion and metastasis of lung adenocarcinoma cells by activating the PI3 K/AKT signaling pathway and thereby increasing the expression of MMPs.
Mesenchymal to epithelial transition factor( MET) is abnormally expressed in many cancers,but the molecular mechanism of MET affecting lung adenocarcinoma is not clear. In this study,3 cases of lung adenocarcinoma tissues( LAD) with lymph node metastasis and 3 cases of LAD without lymph node metastasis were collected for microarray gene chip analysis. The results showed that 1 314 mRNAs were up-regulated and 400 mRNAs were down-regulated in LADs with lymph node metastasis,and the expression of MET was significantly increased in lung adenocarcinoma tissues with lymph node metastasis,compared with those without lymph node metastasis. Eight differentially expressed genes were randomly selected,and qRT-PCR was used to verify the results of microarray gene chip in 30 cases of LADs with lymph node metastasis collected from February 2014 to February 2017 in Affiliated Hospital of Weifang Medical University. The results showed that the expression of the selected mRNAs was consistent with the microarray data,which verified the accuracy of the microarray gene chip results. The expression of MET was further detected by Western blot,and the results showed that the expression of MET in lung adenocarcinoma cells was significantly higher than that in normal lung epithelial cells. Transwell invasion experiment results showed that the invasion ability of A549 lung epithelial cells was significantly reduced after knockdown of MET. After treated with EGF( epidermal growth factor),results showed that the expression levels of matrix metalloproteinase 2( MMP-2) and MMP-9 in lung adenocarcinoma cells were significantly decreased after the knockout of MET,and the phosphorylation level of AKT was also significantly decreased. These results indicated that MET can promote the invasion and metastasis of lung adenocarcinoma cells by activating the PI3 K/AKT signaling pathway and thereby increasing the expression of MMPs.
引文
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[11]张国新,徐新伟,孟斌,等.miR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化[J].中国生物化学与分子生物学报(Zhang GX,Xu XW,Meng B,et al.miR-186-5p inhibits epithelial mesenchymal transition in lung adenocarcinoma cells through modulating PTTG1[J]Chin J Biochem Mol Biol),2017,33(4):380-385
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[13]Wu YL,Zhang L,Kim DW,et al.Phase Ib/II study of capmatinib(INC280)plus gefitinib after failure of epidermal growth factor receptor(EGFR)inhibitor therapy in patients with EGFR-mutated,MET factor-dysregulated non-small-cell lung cancer[J].Clin Oncol,2018,36(31):3101-3109
[14]Gherardi E,Birchmeier W,Birchmeier C,et al.Targeting METin cancer:rationale and progress[J].Nat Rev Cancer,2012,12(2):89-103
[15]Granito A,Guidetti E,Gramantieri L.c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma[J].J Hepatocell Carcinoma,2015,2:29-38
[16]Szewczyk B,Skrzypek K,Majka M.Targeting MET receptor in rhabdomyosarcoma:rationale and progress[J].Curr Drug Targets,2017,18(1):98-107
[17]Niemann HH,Jger V,Butler PJ,et al.Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB[J].Cell,2007,130(2):235-246
[18]Miekus K,Pawlowska M,Sekua M,et al.MET receptor is a potential therapeutic target in high grade cervical cancer[J].Oncotarget,2015,6(12):10086-10101
[19]Li C,Wu JJ,Hynes M,et al.c-Met is a marker of pancreatic cancer stem cells and therapeutic target[J].Gastroenterology,2011,141(6):2218-2227
[20]Zeng L,Liao Q,Zou Z,et al.Long non-coding RNA XLOC_006753 promotes the development of multidrug resistance in gastric cancer cells through the PI3K/AKT/m TOR signaling pathway[J].Cell Physiol Biochem,2018,51(3):1221-1236
[21]Zhang G,Li H,Sun R,et al.Long non-coding RNA ZEB2-AS1promotes the proliferation,metastasis and epithelial mesenchymal transition in triple-negative breast cancer by epigenetically activating ZEB2[J].J Cell Mol Med,2019,doi:10.1111/jcmm.14213.[Epub ahead of print]
[22]Mu QJ,Li HL,Yao Y,et al.Chromodomain helicase/ATPase DNA-binding protein 1-like gene(CHD1L)expression and implications for invasion and metastasis of breast cancer[J].PLoS One,2015,10(11):e0143030
[23]Li H,Zhang B,Liu Y,et al.EBP50 inhibits the migration and invasion of human breast cancer cells via LIMK/cofilin and the PI3K/Akt/m TOR/MMP signaling pathway[J].Med Oncol,2014,31(9):162
[24]Fouad H,Salem H,Ellakwa DE,et al.MMP-2 and MMP-9 as prognostic markers for the early detection of urinary bladder cancer[J].J Biochem Mol Toxicol,2019,33(4):e22275
[2]Torre LA,Siegel RL,Jemal A.Lung Cancer Statistics[J].Adv Exp Med Biol,2016,893:1-19
[3]Li H,Yin C,Zhang B,et al.PTTG1 promotes migration and invasion of human non-small cell lung cancer cells and is modulated by miR-186[J].Carcinogenesis,2013,34(9):2145-2155
[4]Meng C,Tang C,Liang J.Progress of biomarkers in diagnosis of bone metastases of lung cancer[J].Chin J Lung Cancer,2018,21(8):615-619
[5]Skead G,Govender D.Gene of the month:MET[J].J Clin Pathol,2015,68(6):405-409
[6]Zeng ZS,Weiser MR,Kuntz E,et al.c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases[J].Cancer Lett,2008,265(2):258-269
[7]Zhou W,Jiang C,Zhan N,et al.Human epidermal growth factor receptor 2,epidermal growth factor receptor,and c-METoverexpression and survival in biliary tract cancer:A metaanalysis[J].J Cancer Res Ther,2018,14(Suppl):S28-S35
[8]Holland JD,Gyorffy B,Vogel R,et al.Combined Wnt/betacatenin,Met,and CXCL12/CXCR4 signals characterize basal breast cancer and predict disease outcome[J].Cell Rep,2013,5(5):1214-1227
[9]Yang Z,Li H,Wang Z,et al.Microarray expression profile of long non-coding RNAs in human lung adenocarcinoma[J].Thorac Cancer,2018,9(10):1312-1322
[10]Zhang B,Li H,Yin C,et al.Dock1 promotes the mesenchymal transition of glioma and is modulated by MiR-31[J].Neuropathol Appl Neurobiol,2017,43(5):419-432
[11]张国新,徐新伟,孟斌,等.miR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化[J].中国生物化学与分子生物学报(Zhang GX,Xu XW,Meng B,et al.miR-186-5p inhibits epithelial mesenchymal transition in lung adenocarcinoma cells through modulating PTTG1[J]Chin J Biochem Mol Biol),2017,33(4):380-385
[12]Zhang B,Yin C,Li H,et al.Nir1 promotes invasion of breast cancer cells by binding to chemokine(C-C motif)ligand 18through the PI3K/Akt/GSK3beta/Snail signalling pathway[J].Eur J Cancer,2013,49(18):3900-3913
[13]Wu YL,Zhang L,Kim DW,et al.Phase Ib/II study of capmatinib(INC280)plus gefitinib after failure of epidermal growth factor receptor(EGFR)inhibitor therapy in patients with EGFR-mutated,MET factor-dysregulated non-small-cell lung cancer[J].Clin Oncol,2018,36(31):3101-3109
[14]Gherardi E,Birchmeier W,Birchmeier C,et al.Targeting METin cancer:rationale and progress[J].Nat Rev Cancer,2012,12(2):89-103
[15]Granito A,Guidetti E,Gramantieri L.c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma[J].J Hepatocell Carcinoma,2015,2:29-38
[16]Szewczyk B,Skrzypek K,Majka M.Targeting MET receptor in rhabdomyosarcoma:rationale and progress[J].Curr Drug Targets,2017,18(1):98-107
[17]Niemann HH,Jger V,Butler PJ,et al.Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB[J].Cell,2007,130(2):235-246
[18]Miekus K,Pawlowska M,Sekua M,et al.MET receptor is a potential therapeutic target in high grade cervical cancer[J].Oncotarget,2015,6(12):10086-10101
[19]Li C,Wu JJ,Hynes M,et al.c-Met is a marker of pancreatic cancer stem cells and therapeutic target[J].Gastroenterology,2011,141(6):2218-2227
[20]Zeng L,Liao Q,Zou Z,et al.Long non-coding RNA XLOC_006753 promotes the development of multidrug resistance in gastric cancer cells through the PI3K/AKT/m TOR signaling pathway[J].Cell Physiol Biochem,2018,51(3):1221-1236
[21]Zhang G,Li H,Sun R,et al.Long non-coding RNA ZEB2-AS1promotes the proliferation,metastasis and epithelial mesenchymal transition in triple-negative breast cancer by epigenetically activating ZEB2[J].J Cell Mol Med,2019,doi:10.1111/jcmm.14213.[Epub ahead of print]
[22]Mu QJ,Li HL,Yao Y,et al.Chromodomain helicase/ATPase DNA-binding protein 1-like gene(CHD1L)expression and implications for invasion and metastasis of breast cancer[J].PLoS One,2015,10(11):e0143030
[23]Li H,Zhang B,Liu Y,et al.EBP50 inhibits the migration and invasion of human breast cancer cells via LIMK/cofilin and the PI3K/Akt/m TOR/MMP signaling pathway[J].Med Oncol,2014,31(9):162
[24]Fouad H,Salem H,Ellakwa DE,et al.MMP-2 and MMP-9 as prognostic markers for the early detection of urinary bladder cancer[J].J Biochem Mol Toxicol,2019,33(4):e22275