lncRNA SNHG16在结直肠癌组织和细胞中表达及其调控结肠癌细胞中GPAM表达的机制
|
摘要
目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)SNHG16在结直肠癌(colorectal cancer,CRC)组织和细胞中的表达及其通过海绵吸附miR-128-3p调控结肠癌细胞线粒体甘油3磷酸酰基转移酶基因(mitochondrial glycerol-3-phosphate acyltransferase,GPAM)表达的分子机制。方法:收集2014年1月至2017年1月甘肃省人民医院肛肠科手术切除的60例CRC患者的癌及癌旁组织标本,以及结直肠癌细胞系SW480、SW620、HCT116、Caco-2、DLD-1、HT29和结肠上皮细胞CCD841,用q PCR法检测CRC组织和细胞系中SNHG16的表达,分析SNHG16表达与CRC患者临床病例特征的关系。分别用miR-128-3p模拟物、miR-128-3p抑制剂、SNHG16敲降载体转染SW480细胞后,用qPCR法检测细胞中miR-128-3p及SNHG16 mRNA的表达,用Western blotting法检测GPAM蛋白的表达,用CCK-8法、克隆形成实验及细胞凋亡实验、Transwell小室法检测细胞的增殖、凋亡及侵袭。用双荧光素酶报告基因法和RNA免疫共沉淀实验验证SNHG16和miR-128-3p mRNA靶向结合。构建小鼠SW480细胞移植瘤模型,观察敲降SNHG16对移植瘤生长的影响。结果:CRC组织及细胞系中SNHG16高表达(均P<0.01),其表达水平与CRC淋巴结转移、Duke’s分期及患者生存期相关(均P<0.01)。敲降SNHG16可显著抑制SW480细胞的增殖及侵袭能力,并诱导细胞凋亡(均P<0.01);敲降SNHG16后小鼠移植瘤瘤体显著小于对照组(P<0.05)。双荧光素酶报告基因检测及RNA免疫沉淀反应结果显示,miR-128-3p与SNHG16相互作用,且在CRC患者中miR-128-3p与SNHG16负相关(P<0.01)。SNHG16通过内源性竞争海绵吸附miR-128-3p影响其下游靶基因GPAM的表达。结论:SNHG16在CRC细胞中可通过海绵吸附miR-128-3p调控GPAM表达,SNHG16及miR-128-3p可作为CRC诊断及治疗的潜在靶点。
Objective: To investigate the expression of long non-coding RNA SNHG16(lncRNA SNHG16) in colorectal cancer(CRC)tissues and cells, and to explore the mechanism of its regulation on the expression of mitochondrial glycerol-3-phosphate acyltransferase(GPAM) via sponging miR-128-3 p. Methods: Sixty pairs of colorectal cancerous tissues and para-cancerous tissues that resected from CRC patients, who underwent surgery in the Department of Anorectal Surgery, Gansu Provincial People's Hospital during Jan.2014 and Jan. 2017, were collected for this study; In addition, CRC cell lines(SW480, SW620, HCT116, Caco-2,DLD-1, HT29) and colonic epithelial cell line CCD841 were also collected for the study. The expression of SNHG16 in collected tissues and cell lines was determined by Real-time quantitative PCR(qPCR), and its correlation to the clinicopathological features of CRC patients was also analyzed. SW480 cells were transfected with miR-128-3 p mimic, miR-128-3 p inhibitor, and si-SNHG16, respectively, and then the mRNA expressions of miR-128-3 p and SNHG16 were detected by qPCR, the protein expression of GPAM was determined by Western blotting,and the cell proliferation, apoptosis and invasion were detected by CCK-8 assay, colony formation assay, cell apoptosis assay and Transwell chamber assay, respectively. The binding between SNHG16 and miR-128-3 p was validated with dual luciferase reporter gene assay and RNA Immunoprecipitation assay. For in vivo experiment, mouse model of SW480 cell exnograft was constructed, and the ef-fect of SNHG16 knockdown on the growth of exnograft was observed. Results: SNHG16 was found to highly expressed in human CRC tissues and cell lines(all P<0.01), and SNHG16 expression level was associated with lymph node metastasis, Duke's stage and patients' survival(all P<0.01). Knockdown of SNHG16 significantly inhibited CRC cell proliferation and invasion, and induced apoptosis(all P<0.01); After SNHG16 knockdown, the volume of exnograft was obviously reduced(P<0.05). Dual luciferase reporter gene assay and RNA Immunoprecipitation assay validated the interaction between miR-128-3 p and SNHG16, and they were negatively correlated with each other in CRC patients(P<0.01). The SNHG16 regulated the expression of its down-stream gene GPAM via endogenously sponging miR-128-3 p. Conclusion: SNHG16 regulates GPAM expression in CRC cells by sponging miR-128-3 p, and SNHG16 and miR-128-3 p may serve as potential targets for the diagnosis and treatment of CRC.
Objective: To investigate the expression of long non-coding RNA SNHG16(lncRNA SNHG16) in colorectal cancer(CRC)tissues and cells, and to explore the mechanism of its regulation on the expression of mitochondrial glycerol-3-phosphate acyltransferase(GPAM) via sponging miR-128-3 p. Methods: Sixty pairs of colorectal cancerous tissues and para-cancerous tissues that resected from CRC patients, who underwent surgery in the Department of Anorectal Surgery, Gansu Provincial People's Hospital during Jan.2014 and Jan. 2017, were collected for this study; In addition, CRC cell lines(SW480, SW620, HCT116, Caco-2,DLD-1, HT29) and colonic epithelial cell line CCD841 were also collected for the study. The expression of SNHG16 in collected tissues and cell lines was determined by Real-time quantitative PCR(qPCR), and its correlation to the clinicopathological features of CRC patients was also analyzed. SW480 cells were transfected with miR-128-3 p mimic, miR-128-3 p inhibitor, and si-SNHG16, respectively, and then the mRNA expressions of miR-128-3 p and SNHG16 were detected by qPCR, the protein expression of GPAM was determined by Western blotting,and the cell proliferation, apoptosis and invasion were detected by CCK-8 assay, colony formation assay, cell apoptosis assay and Transwell chamber assay, respectively. The binding between SNHG16 and miR-128-3 p was validated with dual luciferase reporter gene assay and RNA Immunoprecipitation assay. For in vivo experiment, mouse model of SW480 cell exnograft was constructed, and the ef-fect of SNHG16 knockdown on the growth of exnograft was observed. Results: SNHG16 was found to highly expressed in human CRC tissues and cell lines(all P<0.01), and SNHG16 expression level was associated with lymph node metastasis, Duke's stage and patients' survival(all P<0.01). Knockdown of SNHG16 significantly inhibited CRC cell proliferation and invasion, and induced apoptosis(all P<0.01); After SNHG16 knockdown, the volume of exnograft was obviously reduced(P<0.05). Dual luciferase reporter gene assay and RNA Immunoprecipitation assay validated the interaction between miR-128-3 p and SNHG16, and they were negatively correlated with each other in CRC patients(P<0.01). The SNHG16 regulated the expression of its down-stream gene GPAM via endogenously sponging miR-128-3 p. Conclusion: SNHG16 regulates GPAM expression in CRC cells by sponging miR-128-3 p, and SNHG16 and miR-128-3 p may serve as potential targets for the diagnosis and treatment of CRC.
引文
[1]AYKUT B,OCHS M,RADHAKRISHNAN P,et al.EMX2 gene expression predicts liver metastasis and survival in colorectal cancer[J/OL].BMC Cancer,2017,17(1):555[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568393/.DOI:10.1186/s12885-017-3556-2.
[2]OKUGAWA Y,TOIYAMA Y,TODEN S,et al.Clinical significance of SNORA42 as an oncogene and a prognostic biomarker in colorectal cancer[J].Gut,2017,66(1):107-117.DOI:10.1136/gutjnl-2015-309359.
[3]SLATTERY M L,HERRICK J S,MULLANY L E,et al.The co-regulatory networks of tumor suppressor genes,oncogenes,and miR-NAs in colorectal cancer[J].Genes Chromosomes Cancer,2017,56(11):769-787.DOI:10.1002/gcc.22481.
[4]KIM S W.The role of microRNAs in colorectal cancer[J].Korean JGastroenterol,2017,69(4):206-211.DOI:10.4166/kjg.2017.69.4.206.
[5]CHEN D L,CHEN L Z,LU Y X,et al.Long noncoding RNA XISTexpedites metastasis and modulates epithelial-mesenchymal transition in colorectal cancer[J/OL].Cell Death Dis,2017,8(8):e3011[2018-08-02].http://www.ncbi.nlm.nih.gov/pmc/journals/1437/.DOI:10.1038/cddis.2017.421.
[6]YAN L,YAO J,QIU J.miRNA-495 suppresses proliferation and migration of colorectal cancer cells by targeting FAM83D[J/OL].Biomed Pharmacother,2017,96:974-981[2018-08-02].http://sciencedirect.com/science/journal/07533322.DOI:10.1016/j.biopha.2017.11.138.
[7]DENG B,WANG B,FANG J,et al.miRNA-203 suppresses cell proliferation,migration and invasion in colorectal cancer via targeting of EIF5A2[J/OL].Sci Rep,2016,6:28301[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931903/.DOI:10.1038/srep28301.
[8]SAM S,SAM M R,ESMAEILLOU M,et al.Effective targeting survivin,caspase-3 and microRNA-16-1 expression by methyl-3-pentyl-6-methoxyprodigiosene triggers apoptosis in colorectal cancer stem-like cells[J].Pathol Oncol Res,2016,22(4):715-723.DOI:10.1007/s12253-016-0055-8.
[9]FANG L,LI H,WANG L,et al.MicroRNA-17-5p promotes chemotherapeutic drug resistance and tumour metastasis of colorectal cancer by repressing PTEN expression[J/OL].Oncotarget,2014,5(10):2974-2987[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102784/.DOI:10.18632/oncotarget.1614.
[10]WANG H,NIE L,WU L,et al.NR2F2 inhibits Smad7 expression and promotes TGF-β-dependent epithelial-mesenchymal transition of CRC via transactivation of miR-21[J].Biochem Biophys Res Commun,2017,485(1):181-188.DOI:10.1016/j.bbrc.2017.02.049.
[11]ZHOU X,XU X,WANG J,et al.Identifying miRNA/mRNA negative regulation pairs in colorectal cancer[J/OL].Sci Rep,2015,5:12995[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534763/.DOI:10.1038/srep12995.
[12]WU L,SHI B,HUANG K,et al.MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1[J].Oncol Rep,2015,34(5):2797-2805.DOI:10.3892/or.2015.4251.
[13]WEI S,FAN Q,YANG L,et al.Promotion of glycolysis by HO-TAIR through GLUT1 upregulation via m TOR signaling[J].Oncol Rep,2017,38(3):1902-1908.DOI:10.3892/or.2017.5840.
[14]MISAWA A,TAKAYAMA K,URANO T,et al.Androgen-induced long noncoding RNA(lncRNA)SOCS2-AS1 promotes cell growth and inhibits apoptosis in prostate cancer cells[J].J Biol Chem,2016,291(34):17861-17880.DOI:10.1074/jbc.M116.718536.
[15]CHEN T,XIE W,XIE L,et al.Expression of long noncoding RNAlncRNA-n336928 is correlated with tumor stage and grade and overall survival in bladder cancer[J].Biochem Biophys Res Commun,2015,468(4):666-670.DOI:10.1016/j.bbrc.2015.11.013.
[16]ZHOU X,CHEN H,ZHU L,et al.Helicobacter pylori infection related long noncoding RNA(lncRNA)AF147447 inhibits gastric cancer proliferation and invasion by targeting MUC2 and up-regulating miR-34c[J].Oncotarget,2016,7(50):82770-82782[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347731/.DOI:10.18632/oncotarget.13165.
[17]ZHANG X,XIONG Y,TANG F,et al.Long noncoding RNAHNF1A-AS1 indicates a poor prognosis of colorectal cancer and promotes carcinogenesis via activation of the Wnt/β-catenin signaling pathway[J/OL].Biomed Pharmacother,2017,96:877-883[2018-08-02].https://www.sciencedirect.com/science/article/abs/pii/S0753332217343123.DOI:10.1016/j.biopha.2017.10.033.
[18]BROCKMOLLER S F,BUCHER E,MULLER B M,et al.Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase(GPAM)in breast cancer-link to patient survival,hormone receptor status,and metabolic profiling[J].J Proteome Res,2012,11(2):850-860.DOI:10.1021/pr200685r.
[19]WU Q,MENG W Y,JIE Y,et al.lncRNA MALAT1 induces colon cancer development by regulating miR-129-5p/HMGB1 axis[J].JCell Physiol,2018,233(9):6750-6757.DOI:10.1002/jcp.26383.
[20]LU W,WANG J,YANG G,et al.Posttranscriptional regulation of Galectin-3 by miR-128 contributes to colorectal cancer progression[J/OL].Oncotarget,2017,8(9):15242-15251[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844768/.DOI:10.18632/oncotarget.14839.
[21]KRELL J,STEBBING J,CARISSIMI C,et al.TP53 regulates miR-NA association with AGO2 to remodel the miRNA-mRNA interaction network[J].Genome Res,2016,26(3):331-341.DOI:10.1101/gr.191759.115.
[22]ZHAO L,LI R,XU S,et al.Tumor suppressor miR-128-3p inhibits metastasis and epithelial-mesenchymal transition by targeting ZEB1in esophageal squamous-cell cancer[J].Acta Biochim Biophys Sin(Shanghai),2018,50(2):171-180.DOI:10.1093/abbs/gmx132.
[23]HUANG C Y,HUANG X P,ZHU J Y,et al.miR-128-3p suppresses hepatocellular carcinoma proliferation by regulating PIK3R1 and is correlated with the prognosis of HCC patients[J].Oncol Rep,2015,33(6):2889-2898.DOI:10.3892/or.2015.3936.
[24]BLOCK I,BURTON M,SORENSEN K P,et al.Association of miR-548c-5p,miR-7-5p,miR-210-3p,miR-128-3p with recurrence in systemically untreated breast cancer[J/OL].Oncotarget,2018,9(10):9030-9042[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823652/.DOI:10.18632/oncotarget.24088.
[25]FRIXA T,SACCONI A,CIOCE M,et al.MicroRNA-128-3p-mediated depletion of Drosha promotes lung cancer cell migration[J].Carcinogenesis,2018,39(2):293-304.DOI:10.1093/carcin/bgx134.
[2]OKUGAWA Y,TOIYAMA Y,TODEN S,et al.Clinical significance of SNORA42 as an oncogene and a prognostic biomarker in colorectal cancer[J].Gut,2017,66(1):107-117.DOI:10.1136/gutjnl-2015-309359.
[3]SLATTERY M L,HERRICK J S,MULLANY L E,et al.The co-regulatory networks of tumor suppressor genes,oncogenes,and miR-NAs in colorectal cancer[J].Genes Chromosomes Cancer,2017,56(11):769-787.DOI:10.1002/gcc.22481.
[4]KIM S W.The role of microRNAs in colorectal cancer[J].Korean JGastroenterol,2017,69(4):206-211.DOI:10.4166/kjg.2017.69.4.206.
[5]CHEN D L,CHEN L Z,LU Y X,et al.Long noncoding RNA XISTexpedites metastasis and modulates epithelial-mesenchymal transition in colorectal cancer[J/OL].Cell Death Dis,2017,8(8):e3011[2018-08-02].http://www.ncbi.nlm.nih.gov/pmc/journals/1437/.DOI:10.1038/cddis.2017.421.
[6]YAN L,YAO J,QIU J.miRNA-495 suppresses proliferation and migration of colorectal cancer cells by targeting FAM83D[J/OL].Biomed Pharmacother,2017,96:974-981[2018-08-02].http://sciencedirect.com/science/journal/07533322.DOI:10.1016/j.biopha.2017.11.138.
[7]DENG B,WANG B,FANG J,et al.miRNA-203 suppresses cell proliferation,migration and invasion in colorectal cancer via targeting of EIF5A2[J/OL].Sci Rep,2016,6:28301[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931903/.DOI:10.1038/srep28301.
[8]SAM S,SAM M R,ESMAEILLOU M,et al.Effective targeting survivin,caspase-3 and microRNA-16-1 expression by methyl-3-pentyl-6-methoxyprodigiosene triggers apoptosis in colorectal cancer stem-like cells[J].Pathol Oncol Res,2016,22(4):715-723.DOI:10.1007/s12253-016-0055-8.
[9]FANG L,LI H,WANG L,et al.MicroRNA-17-5p promotes chemotherapeutic drug resistance and tumour metastasis of colorectal cancer by repressing PTEN expression[J/OL].Oncotarget,2014,5(10):2974-2987[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102784/.DOI:10.18632/oncotarget.1614.
[10]WANG H,NIE L,WU L,et al.NR2F2 inhibits Smad7 expression and promotes TGF-β-dependent epithelial-mesenchymal transition of CRC via transactivation of miR-21[J].Biochem Biophys Res Commun,2017,485(1):181-188.DOI:10.1016/j.bbrc.2017.02.049.
[11]ZHOU X,XU X,WANG J,et al.Identifying miRNA/mRNA negative regulation pairs in colorectal cancer[J/OL].Sci Rep,2015,5:12995[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534763/.DOI:10.1038/srep12995.
[12]WU L,SHI B,HUANG K,et al.MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1[J].Oncol Rep,2015,34(5):2797-2805.DOI:10.3892/or.2015.4251.
[13]WEI S,FAN Q,YANG L,et al.Promotion of glycolysis by HO-TAIR through GLUT1 upregulation via m TOR signaling[J].Oncol Rep,2017,38(3):1902-1908.DOI:10.3892/or.2017.5840.
[14]MISAWA A,TAKAYAMA K,URANO T,et al.Androgen-induced long noncoding RNA(lncRNA)SOCS2-AS1 promotes cell growth and inhibits apoptosis in prostate cancer cells[J].J Biol Chem,2016,291(34):17861-17880.DOI:10.1074/jbc.M116.718536.
[15]CHEN T,XIE W,XIE L,et al.Expression of long noncoding RNAlncRNA-n336928 is correlated with tumor stage and grade and overall survival in bladder cancer[J].Biochem Biophys Res Commun,2015,468(4):666-670.DOI:10.1016/j.bbrc.2015.11.013.
[16]ZHOU X,CHEN H,ZHU L,et al.Helicobacter pylori infection related long noncoding RNA(lncRNA)AF147447 inhibits gastric cancer proliferation and invasion by targeting MUC2 and up-regulating miR-34c[J].Oncotarget,2016,7(50):82770-82782[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347731/.DOI:10.18632/oncotarget.13165.
[17]ZHANG X,XIONG Y,TANG F,et al.Long noncoding RNAHNF1A-AS1 indicates a poor prognosis of colorectal cancer and promotes carcinogenesis via activation of the Wnt/β-catenin signaling pathway[J/OL].Biomed Pharmacother,2017,96:877-883[2018-08-02].https://www.sciencedirect.com/science/article/abs/pii/S0753332217343123.DOI:10.1016/j.biopha.2017.10.033.
[18]BROCKMOLLER S F,BUCHER E,MULLER B M,et al.Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase(GPAM)in breast cancer-link to patient survival,hormone receptor status,and metabolic profiling[J].J Proteome Res,2012,11(2):850-860.DOI:10.1021/pr200685r.
[19]WU Q,MENG W Y,JIE Y,et al.lncRNA MALAT1 induces colon cancer development by regulating miR-129-5p/HMGB1 axis[J].JCell Physiol,2018,233(9):6750-6757.DOI:10.1002/jcp.26383.
[20]LU W,WANG J,YANG G,et al.Posttranscriptional regulation of Galectin-3 by miR-128 contributes to colorectal cancer progression[J/OL].Oncotarget,2017,8(9):15242-15251[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844768/.DOI:10.18632/oncotarget.14839.
[21]KRELL J,STEBBING J,CARISSIMI C,et al.TP53 regulates miR-NA association with AGO2 to remodel the miRNA-mRNA interaction network[J].Genome Res,2016,26(3):331-341.DOI:10.1101/gr.191759.115.
[22]ZHAO L,LI R,XU S,et al.Tumor suppressor miR-128-3p inhibits metastasis and epithelial-mesenchymal transition by targeting ZEB1in esophageal squamous-cell cancer[J].Acta Biochim Biophys Sin(Shanghai),2018,50(2):171-180.DOI:10.1093/abbs/gmx132.
[23]HUANG C Y,HUANG X P,ZHU J Y,et al.miR-128-3p suppresses hepatocellular carcinoma proliferation by regulating PIK3R1 and is correlated with the prognosis of HCC patients[J].Oncol Rep,2015,33(6):2889-2898.DOI:10.3892/or.2015.3936.
[24]BLOCK I,BURTON M,SORENSEN K P,et al.Association of miR-548c-5p,miR-7-5p,miR-210-3p,miR-128-3p with recurrence in systemically untreated breast cancer[J/OL].Oncotarget,2018,9(10):9030-9042[2018-08-02].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823652/.DOI:10.18632/oncotarget.24088.
[25]FRIXA T,SACCONI A,CIOCE M,et al.MicroRNA-128-3p-mediated depletion of Drosha promotes lung cancer cell migration[J].Carcinogenesis,2018,39(2):293-304.DOI:10.1093/carcin/bgx134.