Sphk1基因对间充质干细胞诱导结肠癌RKO细胞增殖和迁移的影响
|
摘要
目的:探讨鞘氨醇激酶1(sphigosine kinase 1,SphK1)基因下调对间充质干细胞(mesenchymal stem cell,MSC)诱导的结肠癌RKO细胞增殖和迁移的影响。方法:采用MSC条件培养液(MSC-CM)和对照培养液(Control-CM)分别干预RKO细胞,CCK-8法检测细胞的增殖能力,Transwell实验检测细胞的迁移能力,Western blotting法检测Ki-67抗原(Ki-67)、MMP-2/9、肿瘤干细胞标志物CD44和CD133蛋白的表达。用慢病毒shRNA载体转染RKO细胞抑制SphK1的表达,观察抑制SphK1的表达对MSC-CM诱导的RKO细胞增殖、迁移以及MMP-2/9、CD44和CD133蛋白表达的影响。结果:MSC-CM可时间依赖性促进RKO细胞的增殖(P<0.05),并明显增强细胞的迁移能力(P<0.01)和细胞中Ki-67、MMP-2/9、CD44和CD133蛋白的表达(均P<0.05或P<0.01)。慢病毒shRNA转染明显抑制RKO细胞SphK1的表达,抑制SphK1的表达可显著抑制MSC-CM对RKO细胞增殖和迁移的促进作用(均P<0.05或P<0.01),且明显抑制MSC-CM诱导的Ki-67、MMP-2/9、CD44和CD133蛋白的表达。结论:MSC-CM可促进RKO细胞的增殖和迁移,下调SphK1可通过抑制MMP-2/9、CD44和CD133的表达逆转MSC-CM诱导的细胞增殖和迁移。
Objective: To investigate the effect of sphingosine kinase 1(SphK1) knockdown on the proliferation and migration of colon cancer RKO cells induced by mesenchymal stem cells(MSCs). Methods: RKO cells were treated with MSCs conditioned medium(MSC-CM) or control medium(Control-CM), respectively. Cell proliferation was detected by CCK-8 assay. Cell migration ability was tested by Transwell chamber assay. The proteins expression of Ki-67, MMP-2/9, CD44 and CD133 was detected by Western blotting.Then, the expression of SphK1 in RKO cells was suppressed by targeted gene lentivirus shRNA vector transfection. The effects of SphK1 knockdown on the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM were observed. Results: The RKO cells proliferation was promoted by MSC-CM in a time-dependent manner; moreover(P<0.05), the migration ability of cells was significantly enhanced after being treated with MSC-CM(P<0.01). In addition, MSC-CM significantly increased the protein expressions of Ki-67, MMP-2/9, CD44 and CD133(all P<0.05 or P<0.01). Lentiviral Sh RNA vector transfection could significantly inhibit the expression of SphK1. Down-regulation of SphK1 significantly inhibited the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM(all P<0.05 or P<0.01). Conclusion: MSC-CM promotes the proliferation and migration of colon cancer RKO cells. Down-regulation of SphK1 reverses the cell proliferation and migration induced by MSC-CM via inhibiting the expression of MMP-2/9, CD44 and CD133.
Objective: To investigate the effect of sphingosine kinase 1(SphK1) knockdown on the proliferation and migration of colon cancer RKO cells induced by mesenchymal stem cells(MSCs). Methods: RKO cells were treated with MSCs conditioned medium(MSC-CM) or control medium(Control-CM), respectively. Cell proliferation was detected by CCK-8 assay. Cell migration ability was tested by Transwell chamber assay. The proteins expression of Ki-67, MMP-2/9, CD44 and CD133 was detected by Western blotting.Then, the expression of SphK1 in RKO cells was suppressed by targeted gene lentivirus shRNA vector transfection. The effects of SphK1 knockdown on the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM were observed. Results: The RKO cells proliferation was promoted by MSC-CM in a time-dependent manner; moreover(P<0.05), the migration ability of cells was significantly enhanced after being treated with MSC-CM(P<0.01). In addition, MSC-CM significantly increased the protein expressions of Ki-67, MMP-2/9, CD44 and CD133(all P<0.05 or P<0.01). Lentiviral Sh RNA vector transfection could significantly inhibit the expression of SphK1. Down-regulation of SphK1 significantly inhibited the proliferation, migration and protein expressions of Ki-67, MMP-2/9, CD44 and CD133 of RKO cells induced by MSC-CM(all P<0.05 or P<0.01). Conclusion: MSC-CM promotes the proliferation and migration of colon cancer RKO cells. Down-regulation of SphK1 reverses the cell proliferation and migration induced by MSC-CM via inhibiting the expression of MMP-2/9, CD44 and CD133.
引文
[1]CALON A,ESPINET E,PALOMO-PONCE S,et al.Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation[J].Cancer Cell,2012,22(5):571-584.DOI:10.1016/j.ccr.2012.08.013.
[2]ALBA-CASTELLON L,OLIVERA-SALGUERO R,MESTREFARRERA A,et al.Snail1-dependent activation of cancer-associated fibroblast controls epithelial tumor cell invasion and metastasis[J].Cancer Res,2016,76(21):6205-6217.DOI:10.1158/0008-5472.CAN-16-0176.
[3]TSAI K S,YANG S H,LEI Y P,et al.Mesenchymal stem cells promote formation of colorectal tumors in mice[J].Gastroenterology,2011,141(3):1046-1056.DOI:10.1053/j.gastro.2011.05.045.
[4]SHINAGAWA K,KITADAI Y,TANAKA M,et al.Mesenchymal stem cells enhance growth and metastasis of colon cancer[J].Int J Cancer,2010,127(10):2323-2333.DOI:10.1002/ijc.25440.
[5]VADAS M,XIA P,MCCAUGHAN G,et al.The role of sphingosine kinase 1 in cancer:oncogene or non-oncogene addiction?[J].Biochim Biophys Acta,2008,1781(9):442-447.DOI:10.1016/j.bbalip.2008.06.007.
[6]覃琳,刘诗权,覃蒙斌,等.敲低鞘氨醇激酶1增强人结肠癌RKO细胞对顺铂的敏感性[J].细胞与分子免疫学杂志,2017,33(5):623-629.DOI:10.13423/j.cnki.cjcmi.008134.
[7]XU W R,ZHANG X R,QIAN H,et al.Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro[J].Exp Biol Med,2004,229(7):623-631.
[8]冯吉,陈东凤.骨髓间充质干细胞的旁分泌作用[J].重庆医学,2011,40(13):1332-1333.DOI:10.3969/j.issn.1671-8348.2011.13.038.
[9]SPAETH E L,DEMBINSKI J L,SASSER A K,et al.Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression[J/OL].PLo S One,2013,8(3):e4992[2017-08-26].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609724/.DOI:10.1371/journal.pone.0004992.
[10]HUANG D,DU X,YUAN R,et al.Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2ubiquitination and degradation[J].Biochem Biophys Res Commun,2014,453(1):49-56.DOI:10.1016/j.bbrc.2014.09.061.
[11]TSURU A,SETOGUCHI T,MATSUNOSHITA Y,et al.Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression[J].Br J Cancer,2015,112(7):1232-1240.DOI:10.1038/bjc.2015.84.
[12]WANG Y,CHU Y,YUE B,et al.Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway[J].Oncotarget,2017,8(14):23803-23816.DOI:10.18632/oncotarget.15866.
[13]DING Y,XU D,FENG G,et al.Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and-9[J].Diabetes,2009,58(8):1797-1806.DOI:10.2337/db09-0317.
[14]MALLARD B W,TIRALONGO J.Cancer stem cell marker glycosylation:nature,function and significance[J].Glycoconj J,2017,34(4):441-452.DOI:10.1007/s10719-017-9780-9.
[15]LUO J,OK LEE S,LIANG L,et al.Infiltrating bone marrow mesenchymal stem cells increase prostate cancer stem cell population and metastatic ability via secreting cytokines to suppress androgen receptor signaling[J].Oncogene,2014,33(21):2768-2778.DOI:10.1038/onc.2013.233.
[16]WANG S S,GAO X L,LIU X,et al.CD133+cancer stem-like cells promote migration and invasion of salivary adenoid cystic carcinoma by inducing vasculogenic mimicry formation[J].Oncotarget,2016,7(20):29051-29062.DOI:10.18632/oncotarget.8665.
[17]LIU S Q,HUANG J A,QIN M B,et al.Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and u PA via MAPK pathways[J].Int J Colorectal Dis,2012,27(12):1569-1578.DOI:10.1007/s00384-012-1510-y.
[18]LONG J,XIE Y,YIN J,et al.SphK1 promotes tumor cell migration and invasion in colorectal cancer[J].Tumour Biol,2016,37(5):6831-6836.DOI:10.1007/s13277-015-4542-4.
[19]JU T,GAO D,FANG Z Y.Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543[J].Biochem Biophys Res Commun,2016,470(3):728-734.DOI:10.1016/j.bbrc.2016.01.053.
[20]STAYROOK K R,MACK J K,CERABONA D,et al.TGFbeta-mediated induction of SphK1 as a potential determinant in human MDAMB-231 breast cancer cell bone metastasis[J/OL].Bonekey Rep,2015,4:719[2017-08-26].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495778/.DOI:10.1038/bonekey.2015.88.
[21]BEACH J A,ASPURIA P J,CHEON D J,et al.Sphingosine kinase1 is required for TGF-beta mediated fibroblastto-myofibroblast differentiation in ovarian cancer[J].Oncotarget,2016,7(4):4167-4182.DOI:10.18632/oncotarget.6703.
[22]KAWAHARA S,OTSUJI Y,NAKAMURA M,et al.Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells[J].Anticancer Drugs,2013,24(5):473-483.DOI:10.1097/CAD.0b013e32835f705f.
[23]CHATTERJEE I,HUMTSOE J O,KOHLER E E,et al.Lipid phosphate phosphatase-3 regulates tumor growth viaβ-catenin and Cyclin-D1 signaling[J].Mol Cancer,2011,10(1):51.DOI:10.1186/1476-4598-10-51.
[2]ALBA-CASTELLON L,OLIVERA-SALGUERO R,MESTREFARRERA A,et al.Snail1-dependent activation of cancer-associated fibroblast controls epithelial tumor cell invasion and metastasis[J].Cancer Res,2016,76(21):6205-6217.DOI:10.1158/0008-5472.CAN-16-0176.
[3]TSAI K S,YANG S H,LEI Y P,et al.Mesenchymal stem cells promote formation of colorectal tumors in mice[J].Gastroenterology,2011,141(3):1046-1056.DOI:10.1053/j.gastro.2011.05.045.
[4]SHINAGAWA K,KITADAI Y,TANAKA M,et al.Mesenchymal stem cells enhance growth and metastasis of colon cancer[J].Int J Cancer,2010,127(10):2323-2333.DOI:10.1002/ijc.25440.
[5]VADAS M,XIA P,MCCAUGHAN G,et al.The role of sphingosine kinase 1 in cancer:oncogene or non-oncogene addiction?[J].Biochim Biophys Acta,2008,1781(9):442-447.DOI:10.1016/j.bbalip.2008.06.007.
[6]覃琳,刘诗权,覃蒙斌,等.敲低鞘氨醇激酶1增强人结肠癌RKO细胞对顺铂的敏感性[J].细胞与分子免疫学杂志,2017,33(5):623-629.DOI:10.13423/j.cnki.cjcmi.008134.
[7]XU W R,ZHANG X R,QIAN H,et al.Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro[J].Exp Biol Med,2004,229(7):623-631.
[8]冯吉,陈东凤.骨髓间充质干细胞的旁分泌作用[J].重庆医学,2011,40(13):1332-1333.DOI:10.3969/j.issn.1671-8348.2011.13.038.
[9]SPAETH E L,DEMBINSKI J L,SASSER A K,et al.Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression[J/OL].PLo S One,2013,8(3):e4992[2017-08-26].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609724/.DOI:10.1371/journal.pone.0004992.
[10]HUANG D,DU X,YUAN R,et al.Rock2 promotes the invasion and metastasis of hepatocellular carcinoma by modifying MMP2ubiquitination and degradation[J].Biochem Biophys Res Commun,2014,453(1):49-56.DOI:10.1016/j.bbrc.2014.09.061.
[11]TSURU A,SETOGUCHI T,MATSUNOSHITA Y,et al.Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression[J].Br J Cancer,2015,112(7):1232-1240.DOI:10.1038/bjc.2015.84.
[12]WANG Y,CHU Y,YUE B,et al.Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway[J].Oncotarget,2017,8(14):23803-23816.DOI:10.18632/oncotarget.15866.
[13]DING Y,XU D,FENG G,et al.Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and-9[J].Diabetes,2009,58(8):1797-1806.DOI:10.2337/db09-0317.
[14]MALLARD B W,TIRALONGO J.Cancer stem cell marker glycosylation:nature,function and significance[J].Glycoconj J,2017,34(4):441-452.DOI:10.1007/s10719-017-9780-9.
[15]LUO J,OK LEE S,LIANG L,et al.Infiltrating bone marrow mesenchymal stem cells increase prostate cancer stem cell population and metastatic ability via secreting cytokines to suppress androgen receptor signaling[J].Oncogene,2014,33(21):2768-2778.DOI:10.1038/onc.2013.233.
[16]WANG S S,GAO X L,LIU X,et al.CD133+cancer stem-like cells promote migration and invasion of salivary adenoid cystic carcinoma by inducing vasculogenic mimicry formation[J].Oncotarget,2016,7(20):29051-29062.DOI:10.18632/oncotarget.8665.
[17]LIU S Q,HUANG J A,QIN M B,et al.Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and u PA via MAPK pathways[J].Int J Colorectal Dis,2012,27(12):1569-1578.DOI:10.1007/s00384-012-1510-y.
[18]LONG J,XIE Y,YIN J,et al.SphK1 promotes tumor cell migration and invasion in colorectal cancer[J].Tumour Biol,2016,37(5):6831-6836.DOI:10.1007/s13277-015-4542-4.
[19]JU T,GAO D,FANG Z Y.Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543[J].Biochem Biophys Res Commun,2016,470(3):728-734.DOI:10.1016/j.bbrc.2016.01.053.
[20]STAYROOK K R,MACK J K,CERABONA D,et al.TGFbeta-mediated induction of SphK1 as a potential determinant in human MDAMB-231 breast cancer cell bone metastasis[J/OL].Bonekey Rep,2015,4:719[2017-08-26].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495778/.DOI:10.1038/bonekey.2015.88.
[21]BEACH J A,ASPURIA P J,CHEON D J,et al.Sphingosine kinase1 is required for TGF-beta mediated fibroblastto-myofibroblast differentiation in ovarian cancer[J].Oncotarget,2016,7(4):4167-4182.DOI:10.18632/oncotarget.6703.
[22]KAWAHARA S,OTSUJI Y,NAKAMURA M,et al.Sphingosine kinase 1 plays a role in the upregulation of CD44 expression through extracellular signal-regulated kinase signaling in human colon cancer cells[J].Anticancer Drugs,2013,24(5):473-483.DOI:10.1097/CAD.0b013e32835f705f.
[23]CHATTERJEE I,HUMTSOE J O,KOHLER E E,et al.Lipid phosphate phosphatase-3 regulates tumor growth viaβ-catenin and Cyclin-D1 signaling[J].Mol Cancer,2011,10(1):51.DOI:10.1186/1476-4598-10-51.