MiR-148a-3p通过靶向SRPK2抑制结肠癌细胞转移
|
摘要
目的:MicroRNA (miRNA/miR)参与结肠癌各种生物学过程。目前,miR-148a-3p在结肠癌中的作用还不完全清楚。本研究旨在探讨miR-148a-3p对结肠癌细胞转移及侵袭能力的影响及机制。方法:实时定量PCR (qRT-PCR)检测结肠癌细胞系中miR-148a-3p及SRPK2 mRNA的表达,Western blot检测SRPK2蛋白的表达。使用miR-148a-3p mimic,miR-148a-3p inhibitor调节HCT-116及SW480细胞中miR-148a-3p的水平。通过划痕及transwell实验检测miR-148a-3p对结肠癌细胞迁移及侵袭能力的影响。生物信息学分析预测miR-148a-3p的靶点,荧光素酶报告实验验证。Western blot及qRT-PCR检测miR-148a-3p对结肠癌细胞上皮-间质转化及SRPK2表达的影响。结果:与正常结直肠粘膜细胞FHC相比,结肠癌细胞系中miR-148a-3p水平降低(P<0.05)。结肠癌细胞中miR-148a-3p过表达可以明显抑制结肠癌细胞转移及侵袭能力(P<0.05)。相反的,敲低miR-148a-3p结肠癌细胞转移及侵袭能力增强(P<0.05)。荧光素酶报告系统结果提示SRPK2是miR-148a-3p的直接作用靶点。过表达miR-148a-3p可以抑制SRPK2在结肠癌中的表达(P<0.05),但是敲低miR-148a-3p时SRPK2表达明显增高(P<0.05)。结论:MiR-148a-3p可能通过靶向SRPK2抑制结肠癌细胞的转移及侵袭能力。MiR-148a-3p可能成为诊断和治疗结肠癌的靶点之一。
Objective: To investigate the effects of miR-148 a-3 p on colon cancer cell metastasis and invasion. Methods: The expressions of miR-148 a-3 p and SRPK2 mRNA in colon cancer cell lines were detected by quantitative real-time PCR(qRT-PCR). The expression of SRPK2 protein was detected by Western blot. Mi R-148 a-3 p mimic, mi R-148 a-3 p inhibitor was used to regulate the level of mi R-148 a-3 p in HCT-116 and SW480 cells. The effects of miR-148 a-3 p on the migration and invasion of colon cancer cells were examined by woundhealing and transwellassay. The target of miR-148 a-3 p was predicted by bioinformatics analysis and was verified by luciferase reporter assay. The effects of miR-148 a-3 p on epithelial-mesenchymal transition and SRPK2 expression in colon cancer cells were detected by Western blot and q RT-PCR. Results: Mi R-148 a-3 p levels were reduced in CRC cell lines as compared to normal colon cell FHC(P<0.05).Overexpression of miR-148 a-3 p in colon cancer cells significantly inhibited the metastasis and invasion of colon cancer cells(P<0.05). In contrast, knockdown of miR-148 a-3 p showed an increased metastasis and invasion of colon cancer cells(P<0.05). SRPK2 is a direct target of miR-148 a-3 p. Overexpression of miR-148 a-3 p inhibited the expression of SRPK2 in colon cancer(P<0.05), while the expression of SRPK2 was significantly increased in miR-148 a-3 p CRC cells(P<0.05). Conclusion: Mi R-148 a-3 p can inhibit the metastasis and invasion of colon cancer cells by targeting SRPK2. Mi R-148 a-3 pmight be one of the targets for the diagnosis and treatment of colon cancer.
Objective: To investigate the effects of miR-148 a-3 p on colon cancer cell metastasis and invasion. Methods: The expressions of miR-148 a-3 p and SRPK2 mRNA in colon cancer cell lines were detected by quantitative real-time PCR(qRT-PCR). The expression of SRPK2 protein was detected by Western blot. Mi R-148 a-3 p mimic, mi R-148 a-3 p inhibitor was used to regulate the level of mi R-148 a-3 p in HCT-116 and SW480 cells. The effects of miR-148 a-3 p on the migration and invasion of colon cancer cells were examined by woundhealing and transwellassay. The target of miR-148 a-3 p was predicted by bioinformatics analysis and was verified by luciferase reporter assay. The effects of miR-148 a-3 p on epithelial-mesenchymal transition and SRPK2 expression in colon cancer cells were detected by Western blot and q RT-PCR. Results: Mi R-148 a-3 p levels were reduced in CRC cell lines as compared to normal colon cell FHC(P<0.05).Overexpression of miR-148 a-3 p in colon cancer cells significantly inhibited the metastasis and invasion of colon cancer cells(P<0.05). In contrast, knockdown of miR-148 a-3 p showed an increased metastasis and invasion of colon cancer cells(P<0.05). SRPK2 is a direct target of miR-148 a-3 p. Overexpression of miR-148 a-3 p inhibited the expression of SRPK2 in colon cancer(P<0.05), while the expression of SRPK2 was significantly increased in miR-148 a-3 p CRC cells(P<0.05). Conclusion: Mi R-148 a-3 p can inhibit the metastasis and invasion of colon cancer cells by targeting SRPK2. Mi R-148 a-3 pmight be one of the targets for the diagnosis and treatment of colon cancer.
引文
[1]Siegel R L,Miller K D,Jemal A.Cancer statistics,2018[J].CA Cancer JClin,2018,68(1):7
[2]Garden O J,Rees M,Poston G J,et al.Guidelines for resection of colorectal cancer liver metastases[J].Gut,2006,55(Suppl 3):1
[3]Gallagher D J,Kemeny N.Metastatic colorectal cancer:from improved survival to potential cure[J].Oncology,2010,78(3/4):237
[4]Andre T,Bondi C,Navarro M,et al.Improved overall survival with oxaliplatin,fluorouracil,and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial[J].J Clin Oncol,2009,27(19):3109
[5]Bartel D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281
[6]Yan W,Yang W,Liu Z,et al.Characterization of microRNA expression in primary human colon adenocarcinoma cells(SW480)and their lymph node metastatic derivatives(SW620)[J].Onco Targets Ther,2018,11(47):4701
[7]Munkacsy G,Szasz M A,Menyhart O.Gene expression-based prognostic and predictive tools in breast cancer[J].Breast Cancer,2015,22(3):245
[8]Xu C,Zheng Y,Lian D,et al.Analysis of microRNA expression profile identif ies novel biomarkers for non-small cell lung cancer[J].Tumori,2015,101(1):104
[9]G uo J,Miao Y,Xiao B,et al.Differential expression of microRNAspecies in human gastric cancer versus non-tumorous tissues[J].JGastroenterol Hepatol,2009,24(4):652
[10]Baffa R,Fassan M,Volinia S,et al.MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets[J].J Pathol,2009,219(2):214
[11]Katar S,Baran O,Evran S,et al.Expression of miRNA-21,miRNA-107,miRNA-137 and miRNA-29b in meningioma[J].Clin Neurol Neurosurg,2017,156:66
[12]Akao Y,Nakagawa Y,Naoe T.MicroRNA-143 and-145 in colon cancer[J].DNA Cell Biol,2007,26(5):311
[13]Schlormann W,Naumann S,Renner C,et al.Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21and Cyclin D2 in human colon adenoma cells[J].Genes Nutr,2015,10(6):50
[14]Borralho P M,Simoes A E,Gomes S E,et al.miR-143 overexpression impairs growth of human colon carcinoma xenografts in mice with induction of apoptosis and inhibition of proliferation[J].PloSone,2011,6(8):e23787
[15]Liffers S T,Munding J B,Vogt M,et al.MicroRNA-148a is downregulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B[J].Lab Invest,2011,91(10):1472
[16]Zheng B,Liang L,Wang C,et al.MicroRNA-148a suppresses tumor cell invasion and metastasis by downregulating ROCK1 in gastric cancer[J].Clin Cancer Res,2011,17(24):7574
[17]Wang S H,Li X,Zhou L S,et al.microRNA-148a suppresses human gastric cancer cell metastasis by reversing epithelial-to-mesenchymal transition[J].Tumour Biol,2013,34(6):3705
[18]Shi H,Chen X,Jiang H,et al.miR-148a suppresses cell invasion and migration in gastric cancer by targeting DNA methyltransferase1[J].Oncol Lett,2018,15(4):4944
[19]Chen Y,Min L,Zhang X,et al.Decreased miRNA-148a is associated with lymph node metastasis and poor clinical outcomes and functions as a suppressor of tumor metastasis in non-small cell lung cancer[J].Oncol Rep,2013,30(4):1832
[20]Xu Y,Chao L,Wang J,et al.miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells[J].Oncol Lett,2017,14(4):4736
[21]Li H P,Huang H Y,Lai Y R,et al.Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma[J].Oncotarget,2014,5(17):7610
[22]Jang S W,Yang S J,Ehlen A,et al.Serine/arginine protein-specific kinase 2 promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1[J].Cancer Res,2008,68(12):4559
[23]Wang J,Wu H F,Shen W,et al.SRPK2 promotes the growth and migration of the colon cancer cells[J].Gene,2016,586(1):41
[24]Volinia S,Calin G A,Liu C G,et al.A microRNA expression signature of human solid tumors defines cancer gene targets[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(7):2257
[25]Schetter A J,Leung S Y,Sohn J J,et al.MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma[J].JAMA,2008,299(4):425
[26]Lodes M J,Caraballo M,Suciu D,et al.Detection of cancer with serum miRNAs on an oligonucleotide microarray[J].PloS One,2009,4(7):e6229
[27]Baltruskeviciene E,Schveigert D,Stankevicius V,et al.Down-regulation of miRNA-148a and miRNA-625-3p in colorectal cancer is associated with tumor budding[J].BMC cancer,2017,17(1):607
[28]Tsai H L,Yang I P,Huang C W,et al.Clinical significance of microRNA-148a in patients with early relapse of stage II stage and IIIcolorectal cancer after curative resection[J].Translational Research,2013,162(4):258
[29]Li N,Mao D,Cao Y,et al.Downregulation of SIRT6 by miR-34c-5p is associated with poor prognosis and promotes colon cancer proliferation through inhibiting apoptosis via the JAK2/STAT3 signaling pathway[J].Int J Oncol,2018,doi:10.3892/ijO.2018.4304
[30]Gong L,Ren M,Lv Z,et al.miR-92b-3p Promotes Colorectal Carcinoma Cell Proliferation,Invasion,and Migration by Inhibiting FBXW7 In Vitro and In Vivo[J].DNA Cell Biol,2018,37(5):501
[31]Wei L J,Li J A,Bai D M,et al.miR-223-RhoB signaling pathway regulates the proliferation and apoptosis of colon adenocarcinoma[J].Chem Biol Interact,2018,289:9
[32]Meng X,Fu R.miR-206 regulates 5-FU resistance by targeting Bcl-2 in colon cancer cells[J].Onco Targets Ther,2018,11:1757
[33]Meng Q,Chen Y,Lian B,et al.miR218 promotes apoptosis of SW1417 human colon cancer cells by targeting cFLIP[J].Oncol Rep,2018,40(2):916
[34]Wang H Y,Lin W,Dyck J A,et al.SRPK2:a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells[J].J Cell Biol,1998,140(4):737
[35]Zhuo Y J,Liu Z Z,Wan S,et al.Enhanced expression of SRPK2contributes to aggressive progression and metastasis in prost ate cancer[J].Biomed Pharmacother,2018,102(5):31
[2]Garden O J,Rees M,Poston G J,et al.Guidelines for resection of colorectal cancer liver metastases[J].Gut,2006,55(Suppl 3):1
[3]Gallagher D J,Kemeny N.Metastatic colorectal cancer:from improved survival to potential cure[J].Oncology,2010,78(3/4):237
[4]Andre T,Bondi C,Navarro M,et al.Improved overall survival with oxaliplatin,fluorouracil,and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial[J].J Clin Oncol,2009,27(19):3109
[5]Bartel D P.MicroRNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281
[6]Yan W,Yang W,Liu Z,et al.Characterization of microRNA expression in primary human colon adenocarcinoma cells(SW480)and their lymph node metastatic derivatives(SW620)[J].Onco Targets Ther,2018,11(47):4701
[7]Munkacsy G,Szasz M A,Menyhart O.Gene expression-based prognostic and predictive tools in breast cancer[J].Breast Cancer,2015,22(3):245
[8]Xu C,Zheng Y,Lian D,et al.Analysis of microRNA expression profile identif ies novel biomarkers for non-small cell lung cancer[J].Tumori,2015,101(1):104
[9]G uo J,Miao Y,Xiao B,et al.Differential expression of microRNAspecies in human gastric cancer versus non-tumorous tissues[J].JGastroenterol Hepatol,2009,24(4):652
[10]Baffa R,Fassan M,Volinia S,et al.MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets[J].J Pathol,2009,219(2):214
[11]Katar S,Baran O,Evran S,et al.Expression of miRNA-21,miRNA-107,miRNA-137 and miRNA-29b in meningioma[J].Clin Neurol Neurosurg,2017,156:66
[12]Akao Y,Nakagawa Y,Naoe T.MicroRNA-143 and-145 in colon cancer[J].DNA Cell Biol,2007,26(5):311
[13]Schlormann W,Naumann S,Renner C,et al.Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21and Cyclin D2 in human colon adenoma cells[J].Genes Nutr,2015,10(6):50
[14]Borralho P M,Simoes A E,Gomes S E,et al.miR-143 overexpression impairs growth of human colon carcinoma xenografts in mice with induction of apoptosis and inhibition of proliferation[J].PloSone,2011,6(8):e23787
[15]Liffers S T,Munding J B,Vogt M,et al.MicroRNA-148a is downregulated in human pancreatic ductal adenocarcinomas and regulates cell survival by targeting CDC25B[J].Lab Invest,2011,91(10):1472
[16]Zheng B,Liang L,Wang C,et al.MicroRNA-148a suppresses tumor cell invasion and metastasis by downregulating ROCK1 in gastric cancer[J].Clin Cancer Res,2011,17(24):7574
[17]Wang S H,Li X,Zhou L S,et al.microRNA-148a suppresses human gastric cancer cell metastasis by reversing epithelial-to-mesenchymal transition[J].Tumour Biol,2013,34(6):3705
[18]Shi H,Chen X,Jiang H,et al.miR-148a suppresses cell invasion and migration in gastric cancer by targeting DNA methyltransferase1[J].Oncol Lett,2018,15(4):4944
[19]Chen Y,Min L,Zhang X,et al.Decreased miRNA-148a is associated with lymph node metastasis and poor clinical outcomes and functions as a suppressor of tumor metastasis in non-small cell lung cancer[J].Oncol Rep,2013,30(4):1832
[20]Xu Y,Chao L,Wang J,et al.miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells[J].Oncol Lett,2017,14(4):4736
[21]Li H P,Huang H Y,Lai Y R,et al.Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma[J].Oncotarget,2014,5(17):7610
[22]Jang S W,Yang S J,Ehlen A,et al.Serine/arginine protein-specific kinase 2 promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1[J].Cancer Res,2008,68(12):4559
[23]Wang J,Wu H F,Shen W,et al.SRPK2 promotes the growth and migration of the colon cancer cells[J].Gene,2016,586(1):41
[24]Volinia S,Calin G A,Liu C G,et al.A microRNA expression signature of human solid tumors defines cancer gene targets[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(7):2257
[25]Schetter A J,Leung S Y,Sohn J J,et al.MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma[J].JAMA,2008,299(4):425
[26]Lodes M J,Caraballo M,Suciu D,et al.Detection of cancer with serum miRNAs on an oligonucleotide microarray[J].PloS One,2009,4(7):e6229
[27]Baltruskeviciene E,Schveigert D,Stankevicius V,et al.Down-regulation of miRNA-148a and miRNA-625-3p in colorectal cancer is associated with tumor budding[J].BMC cancer,2017,17(1):607
[28]Tsai H L,Yang I P,Huang C W,et al.Clinical significance of microRNA-148a in patients with early relapse of stage II stage and IIIcolorectal cancer after curative resection[J].Translational Research,2013,162(4):258
[29]Li N,Mao D,Cao Y,et al.Downregulation of SIRT6 by miR-34c-5p is associated with poor prognosis and promotes colon cancer proliferation through inhibiting apoptosis via the JAK2/STAT3 signaling pathway[J].Int J Oncol,2018,doi:10.3892/ijO.2018.4304
[30]Gong L,Ren M,Lv Z,et al.miR-92b-3p Promotes Colorectal Carcinoma Cell Proliferation,Invasion,and Migration by Inhibiting FBXW7 In Vitro and In Vivo[J].DNA Cell Biol,2018,37(5):501
[31]Wei L J,Li J A,Bai D M,et al.miR-223-RhoB signaling pathway regulates the proliferation and apoptosis of colon adenocarcinoma[J].Chem Biol Interact,2018,289:9
[32]Meng X,Fu R.miR-206 regulates 5-FU resistance by targeting Bcl-2 in colon cancer cells[J].Onco Targets Ther,2018,11:1757
[33]Meng Q,Chen Y,Lian B,et al.miR218 promotes apoptosis of SW1417 human colon cancer cells by targeting cFLIP[J].Oncol Rep,2018,40(2):916
[34]Wang H Y,Lin W,Dyck J A,et al.SRPK2:a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells[J].J Cell Biol,1998,140(4):737
[35]Zhuo Y J,Liu Z Z,Wan S,et al.Enhanced expression of SRPK2contributes to aggressive progression and metastasis in prost ate cancer[J].Biomed Pharmacother,2018,102(5):31