沉默CDC37抑制结肠癌细胞Lovo和Ls174T侵袭迁移
|
摘要
前期研究发现周期分裂蛋白37(cell division cycle 37,CDC37)的异常表达可能与结直肠腺癌的转移有关,但是其作用机制尚不清楚。本研究通过实时定量PCR和免疫印迹实检测CDC37在不同肠癌细胞中的m NRA和蛋白质表达水平,结果发现CDC37在结肠癌Lovo和Ls174T细胞中表达较高。siRNA靶向沉默CDC37表达后,细胞侧向迁移能力、垂直迁移能力、侵袭活性均显著降低。实时定量PCR和免疫印迹实验结果发现,敲除CDC37后,MMP9和MMP2的m NRA和蛋白质表达水平均显著下调。以上研究结果表明,CDC37通过调节MMP9和MMP2等侵袭转移关键分子的表达在结肠癌细胞侵袭转移过程中发挥关键作用。
It has been shown that abnormal expression of CDC37( cell division cycle 37) might be related with the metastasis of colorectal adenocarcinoma in our previous study. But it is unknown how CDC37 regulates the metastasis of colorectal adenocarcinoma. Herein,we found that the mRNA and protein expression of CDC37 in Lovo and Ls174 T cells were higher than that in the other colorectal adenocarcinoma cells through real-time PCR and Western Blot. In addition,our results showed that the lateral migration ability of Lovo and Ls174 T cells were decreased after silencing CDC37 via wound healing assay. Moreover,Transwell assay showed that the vertical migration ability of Lovo and Ls174 T cells was decreased after knocking-down CDC37. Also we found that the invasion activity of the Lovo and Ls174 T cells was reduced after knocking-down CDC37 via Transwell assay. Furthermore,the mRNA and protein expression of MMP9 and MMP2 were significantly decreased after silencing CDC37 in Lovo and Ls174 T cells. Together,these results suggest that CDC37 plays an important role in invasion and migration process through regulating MMP9 and MMP2 in the development of colorectal adenocarcinoma.
It has been shown that abnormal expression of CDC37( cell division cycle 37) might be related with the metastasis of colorectal adenocarcinoma in our previous study. But it is unknown how CDC37 regulates the metastasis of colorectal adenocarcinoma. Herein,we found that the mRNA and protein expression of CDC37 in Lovo and Ls174 T cells were higher than that in the other colorectal adenocarcinoma cells through real-time PCR and Western Blot. In addition,our results showed that the lateral migration ability of Lovo and Ls174 T cells were decreased after silencing CDC37 via wound healing assay. Moreover,Transwell assay showed that the vertical migration ability of Lovo and Ls174 T cells was decreased after knocking-down CDC37. Also we found that the invasion activity of the Lovo and Ls174 T cells was reduced after knocking-down CDC37 via Transwell assay. Furthermore,the mRNA and protein expression of MMP9 and MMP2 were significantly decreased after silencing CDC37 in Lovo and Ls174 T cells. Together,these results suggest that CDC37 plays an important role in invasion and migration process through regulating MMP9 and MMP2 in the development of colorectal adenocarcinoma.
引文
[1]Sarris ME,Moulos P,Haroniti A,et al.Smyd3 is a transcriptional potentiator of multiple cancer-promoting genes and required for liver and colon cancer development[J].Cancer cell,2016,29(3):354-366
[2]Tan SH,Barker N.Stemming colorectal cancer growth and metastasis:HOXA5 forces cancer stem cells to differentiate[J].Cancer Cell,2015,28(6):683-685
[3]Verba KA,Wang RY,Arakawa A,et al.Atomic structure of Hsp90-CDC37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase[J].Science,2016,352(6293):1542-1547
[4]Keramisanou D,Aboalroub A,Zhang Z,et al.Molecular mechanism of protein kinase recognition and sorting by the Hsp90kinome-specific coahaperone CDC37[J].Mol Cell,2016,62(2):260-271
[5]Datta R,Bansal T,Rana S,et al.Hsp90/CDC37 assembly modulates TGFβreceptor-II to act as a profibrotic regulator of TFGβsignaling during cardiac hypertrophy[J].Cell Signal,2015,27(12):2410-2424
[6]Wang Z,Wei W,Sun CK,et al.Suppressing the CDC37cochaperone in hepatocellular carcinoma cells inhibits cell cycle progression and cell growth[J].Liver Int,2015,35(4):1403-1415
[7]Stepanova L,Yang G,De Mayo F,et al.Induction of human CDC37 in prostate cancer correlates with the ability of targeted CDC37 expression to promote prostatic hyperplasia[J].Oncogene,2000,19(18):2186-2193
[8]刘家有,熊东,王卓娜,等.CDC37在结直肠腺癌中的表达及临床意义的研究[J],实用肿瘤杂志(LIU Jia-You,Xiong Dong,WANG Zhuo-Na,et al.Expression of Cdc37 in colorectal adenocarninoma and its clinical significane[J].J Practical Oncol),2014,29(5):436-439
[9]Tu HC,Schwitalla S,Qian Z,et al.LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans[J].Genes Dev,2015,29(10):1074-1086
[10]朱剑军,蒋琴,袁粒星,等.靶向FBXL20基因的siRNA片段的筛及其验证[J],中国生物化学与分子生物学报(ZHU JianJun,Jiang Qin,YUAN Li-Xing,et al.Screening and Verification of siRNA Targeted for the FBXL20 Gene[J].Chin J Biochem Mol Biol)2011,27(3):237-243
[11]李春艳,高宁,侯颖春.经典Wnt信号通路与人类肿瘤[J],中国生物化学与分子生物学报(LI Chun-Yan,GAO Ning,HOU Ying-Chun.The canonical Wnt Signaling Pathway in Human Cancer[J].Chin J Biochem Mol Biol)2011,30(5):447-452
[12]Chen Y,Zhang YZ,Zhou ZG,et al.Identification of differently expressed genes in human colorectal adenocarcinoma[J].World J Gastroenterol,2006,12(7):1025-1032
[13]Schwarze S R,Fu V X,Jarrard D F.CDC37 enhances proliferation and is necessary for normal human prostate epithelial cell survival[J].Cancer Res,2003,63:4614-4619
[14]Qin G,Luo M,Chen J,et al.Reciprocal activation between MMP-8 and TGF-β1 stimulates EMT and malignant progression of hepatocellular carcinoma[J].Cancer Lett,2016,374(1):85-95
[15]Botkjaer KA,Kwok HF,Terp MG,et al.Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo[J].Oncotarget,2016,7(13):16773-16792
[16]Zeng Y,Yao X,Chen L.Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-βautocrine loop[J].Oncotarget,2016.doi:10.18632/oncotarget.11450
[17]杨万广,王万鹏,张谢夫.ALDH1A2,MMP2和E-cadherin在结肠癌组织中的表达及相关性[J],世界华人消化杂志(WanGuang Yang,Wan-Peng Wang,Xie-Fu Zhang.Significance of ALDH1A2,MMP2 and E-cadherin expression in colonic cancer[J].World Chin J Digestology),2013,21(4):362-366
[18]Lagares-Tena L,Garcia-Monclus S,Lopez-Alemany R,et al.Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9expression through MAPK/ERK pathway[J].Oncotarget,2016.Doi:10.18632/oncotarget.10872
[19]Huang H,Wu K,Ma J,et al.Dopamine D2 receptor suppresses gastric cancer cell invasion and migration via inhibition of EGFR/AKT/MMP-13 pathway[J].Int Immunopharmacol,2016,39:113-120
[20]Zhang Y,Li X,Li J,et al.Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors[J].Peptides,2016,83:8-15
[2]Tan SH,Barker N.Stemming colorectal cancer growth and metastasis:HOXA5 forces cancer stem cells to differentiate[J].Cancer Cell,2015,28(6):683-685
[3]Verba KA,Wang RY,Arakawa A,et al.Atomic structure of Hsp90-CDC37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase[J].Science,2016,352(6293):1542-1547
[4]Keramisanou D,Aboalroub A,Zhang Z,et al.Molecular mechanism of protein kinase recognition and sorting by the Hsp90kinome-specific coahaperone CDC37[J].Mol Cell,2016,62(2):260-271
[5]Datta R,Bansal T,Rana S,et al.Hsp90/CDC37 assembly modulates TGFβreceptor-II to act as a profibrotic regulator of TFGβsignaling during cardiac hypertrophy[J].Cell Signal,2015,27(12):2410-2424
[6]Wang Z,Wei W,Sun CK,et al.Suppressing the CDC37cochaperone in hepatocellular carcinoma cells inhibits cell cycle progression and cell growth[J].Liver Int,2015,35(4):1403-1415
[7]Stepanova L,Yang G,De Mayo F,et al.Induction of human CDC37 in prostate cancer correlates with the ability of targeted CDC37 expression to promote prostatic hyperplasia[J].Oncogene,2000,19(18):2186-2193
[8]刘家有,熊东,王卓娜,等.CDC37在结直肠腺癌中的表达及临床意义的研究[J],实用肿瘤杂志(LIU Jia-You,Xiong Dong,WANG Zhuo-Na,et al.Expression of Cdc37 in colorectal adenocarninoma and its clinical significane[J].J Practical Oncol),2014,29(5):436-439
[9]Tu HC,Schwitalla S,Qian Z,et al.LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans[J].Genes Dev,2015,29(10):1074-1086
[10]朱剑军,蒋琴,袁粒星,等.靶向FBXL20基因的siRNA片段的筛及其验证[J],中国生物化学与分子生物学报(ZHU JianJun,Jiang Qin,YUAN Li-Xing,et al.Screening and Verification of siRNA Targeted for the FBXL20 Gene[J].Chin J Biochem Mol Biol)2011,27(3):237-243
[11]李春艳,高宁,侯颖春.经典Wnt信号通路与人类肿瘤[J],中国生物化学与分子生物学报(LI Chun-Yan,GAO Ning,HOU Ying-Chun.The canonical Wnt Signaling Pathway in Human Cancer[J].Chin J Biochem Mol Biol)2011,30(5):447-452
[12]Chen Y,Zhang YZ,Zhou ZG,et al.Identification of differently expressed genes in human colorectal adenocarcinoma[J].World J Gastroenterol,2006,12(7):1025-1032
[13]Schwarze S R,Fu V X,Jarrard D F.CDC37 enhances proliferation and is necessary for normal human prostate epithelial cell survival[J].Cancer Res,2003,63:4614-4619
[14]Qin G,Luo M,Chen J,et al.Reciprocal activation between MMP-8 and TGF-β1 stimulates EMT and malignant progression of hepatocellular carcinoma[J].Cancer Lett,2016,374(1):85-95
[15]Botkjaer KA,Kwok HF,Terp MG,et al.Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo[J].Oncotarget,2016,7(13):16773-16792
[16]Zeng Y,Yao X,Chen L.Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-βautocrine loop[J].Oncotarget,2016.doi:10.18632/oncotarget.11450
[17]杨万广,王万鹏,张谢夫.ALDH1A2,MMP2和E-cadherin在结肠癌组织中的表达及相关性[J],世界华人消化杂志(WanGuang Yang,Wan-Peng Wang,Xie-Fu Zhang.Significance of ALDH1A2,MMP2 and E-cadherin expression in colonic cancer[J].World Chin J Digestology),2013,21(4):362-366
[18]Lagares-Tena L,Garcia-Monclus S,Lopez-Alemany R,et al.Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9expression through MAPK/ERK pathway[J].Oncotarget,2016.Doi:10.18632/oncotarget.10872
[19]Huang H,Wu K,Ma J,et al.Dopamine D2 receptor suppresses gastric cancer cell invasion and migration via inhibition of EGFR/AKT/MMP-13 pathway[J].Int Immunopharmacol,2016,39:113-120
[20]Zhang Y,Li X,Li J,et al.Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors[J].Peptides,2016,83:8-15