miR-1180-5p通过激活CDKN1A基因表达抑制前列腺癌细胞增殖、迁移和侵袭
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摘要
目的:研究微小RNA-1180-5p(miR-1180-5p)对前列腺癌细胞株VCAP和LNCaP恶性生物行为的影响及可能的作用机制。方法:合成ds Control(ds Control组)和miR-1180-5p(miR-1180-5p组),分别转染至两个前列腺癌细胞株VCAP和LNCaP。采用qPCR和Western blotting分析转染后各组细胞CDKN1A、Cyclin D1和CDK6 mRNA及蛋白的表达变化,采用流式细胞术、MTT法、平板克隆实验和Transwell实验分别检测细胞周期分布、增殖活力、克隆形成能力、细胞迁移和侵袭能力。结果:qPCR结果显示,相比ds Control,转染miR-1180-5p后VCAP和LNCaP细胞中CDKN1A mRNA表达明显上调(P<0.01);Cyclin D1和CDK6 mRNA表达明显下调(P<0.05或P<0.01)。Western blotting与qPCR结果相符。转染miR-1180-5p后VCAP和LNCaP位于G0/G1期的细胞比例增加(P<0.01),而位于S期和G2/M期的细胞比例减少(P<0.05),细胞周期被阻滞在G0/G1期。转染miR-1180-5p后,两种前列腺癌细胞增殖活力较ds Control组明显降低(P<0.05),miR-1180-5p组两种细胞的克隆数量明显较少(P<0.01),同时miR-1180-5p组两组细胞迁移和侵袭能力均下降(P<0.01)。结论:miR-1180-5p能显著激活前列腺癌细胞中CDKN1A基因的表达,从而抑制前列腺癌细胞的增殖、迁移和侵袭等恶性生物行为。
Objective: To study the effects of micro RNA-1180-5p(miR-1180-5p) on malignant biological behaviors of prostate cancer VCAP and LNCaP cells and the possible mechanisms. Methods: ds Control(ds Control group) and miR-1180-5p(miR-1180-5p group)were constructed and then transfected into two prostate cancer cell lines VCAP and LNCaP. qPCR and Western blotting were used to analyze the changes in mRNA and protein expressions of CDKN1A, Cyclin D1 and CDK6 after transfection. Cell cycle distribution, proliferation activity, clone formation capacity, cell migration and invasion ability were detected by flow cytometry, MTT assay, colony culture assay and Transwell assay, respectively. Results: qPCR results showed that compared with ds Control, CDKN1A mRNA levels in VCAP and LNCaP cells transfected with miR-1180-5p were up-regulated significantly, while the mRNA expressions of Cyclin D1 and CDK6 were significantly down-regulated(all P<0.01). Western blotting result was consistent with that of qPCR. The percentage of cells in G0/G1 phase was increased after transfection of miR-1180-5p(P<0.05), but the proportion of cells in S phase and G2/M phase was decreased and the cell cycle was arrested at G0/G1 phase(P<0.05). The proliferation activity of the two prostate cancer cells was significantly lower than that of the ds Control group after miR-1180-5p transfection(P<0.05), and the number of colonies in the miR-1180-5p group was significantly lower than that in the ds Control group(P<0.01). In the meanwhile, the cell migration and invasion ability in miR-1180-5p group was decreased(P<0.01). Conclusion: miR-1180-5p can significantly activate CDKN1A gene expression in prostate cancer cells and further inhibit the proliferation, migration and invasion of prostate cancer cells.
Objective: To study the effects of micro RNA-1180-5p(miR-1180-5p) on malignant biological behaviors of prostate cancer VCAP and LNCaP cells and the possible mechanisms. Methods: ds Control(ds Control group) and miR-1180-5p(miR-1180-5p group)were constructed and then transfected into two prostate cancer cell lines VCAP and LNCaP. qPCR and Western blotting were used to analyze the changes in mRNA and protein expressions of CDKN1A, Cyclin D1 and CDK6 after transfection. Cell cycle distribution, proliferation activity, clone formation capacity, cell migration and invasion ability were detected by flow cytometry, MTT assay, colony culture assay and Transwell assay, respectively. Results: qPCR results showed that compared with ds Control, CDKN1A mRNA levels in VCAP and LNCaP cells transfected with miR-1180-5p were up-regulated significantly, while the mRNA expressions of Cyclin D1 and CDK6 were significantly down-regulated(all P<0.01). Western blotting result was consistent with that of qPCR. The percentage of cells in G0/G1 phase was increased after transfection of miR-1180-5p(P<0.05), but the proportion of cells in S phase and G2/M phase was decreased and the cell cycle was arrested at G0/G1 phase(P<0.05). The proliferation activity of the two prostate cancer cells was significantly lower than that of the ds Control group after miR-1180-5p transfection(P<0.05), and the number of colonies in the miR-1180-5p group was significantly lower than that in the ds Control group(P<0.01). In the meanwhile, the cell migration and invasion ability in miR-1180-5p group was decreased(P<0.01). Conclusion: miR-1180-5p can significantly activate CDKN1A gene expression in prostate cancer cells and further inhibit the proliferation, migration and invasion of prostate cancer cells.
引文
[1]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2015[J].CA Cancer J Clin,2015,65(1):5-29.DOI:10.3322/caac.21254.
[2]KARIMIAN A,AHMADI Y,YOUSEFI B.Multiple functions of p21 in cell cycle,apoptosis and transcriptional regulation after DNA damage[J].DNA Repair(Amst),2016,42:63-71.DOI:10.1016/j.dnarep.2016.04.008.
[3]WANG C,CHEN Z,GE Q,et al.Up-regulation of p21(WAF1/CIP1)by mi RNAs and its implications in bladder cancer cells[J].FEBS Lett,2014,588(24):4654-4664.DOI:10.1016/j.febslet.2014.10.037.
[4]GE Q,WANG C,CHEN Z,et al.The suppressive effects of mi R-1180-5p on the proliferation and tumorigenicity of bladder cancer cells[J].Histol Histopathol,2017,32(1):77-86.DOI:10.14670/HH-11-772.
[5]WANG C,GE Q,CHEN Z,et al.A new double stranded RNA suppresses bladder cancer development by upregulating p21(Waf1/CIP1)expression[J].Biomed Res Int,2015,2015:304753.DOI:10.1155/2015/304753.
[6]PLACE R F,LI L C,POOKOT D,et al.Micro RNA-373 induces expression of genes with complementary promoter sequences[J].Proc Natl Acad Sci U S A,2008,105(5):1608-1613.DOI:10.1073/pnas.0707594105.
[7]LI L C,OKINO S T,ZHAO H,et al.Small ds RNAs induce transcriptional activation in human cells[J].Proc Natl Acad Sci U S A,2006,103(46):17337-17342.DOI:10.1073/pnas.0607015103.
[8]BARATA P,SOOD A K,HONG D S.RNA-targeted therapeutics in cancer clinical trials:Current status and future directions[J].Cancer Treat Rev,2016,50:35-47.DOI:10.1016/j.ctrv.2016.08.004.
[9]ZHU L,JIANG H,SHEONG F K,et al.Understanding the core of RNA interference:The dynamic aspects of Argonaute-mediated processes[J].Prog Biophys Mol Biol,2016.DOI:10.1016/j.pbiomolbio.2016.09.008.
[10]GUO D,BARRY L,LIN S S,et al.RNAa in action:from the exception to the norm[J].RNA Biol,2014,11(10):1221-1225.DOI:10.4161/15476286.2014.972853.
[11]ZHENG L,WANG L,GAN J,et al.RNA activation:promise as a new weapon against cancer[J].Cancer Lett,2014,355(1):18-24.DOI:10.1016/j.canlet.2014.09.004.
[12]WANG X,WANG J,HUANG V,et al.Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation[J].Biochem J,2012,443(3):821-828.DOI:10.1042/BJ20111491.
[13]KREIS N N,LOUWEN F,YUAN J.Less understood issues:p21(Cip1)in mitosis and its therapeutic potential[J].Oncogene,2015,34(14):1758-1767.DOI:10.1038/onc.2014.133.
[14]YEO D,HE H,BALDWIN G S,et al.The role of p21-activated kinases in pancreatic cancer[J].Pancreas,2015,44(3):363-369.DOI:10.1097/MPA.0000000000000276.
[15]WILCZYNSKA A,GIT A,ARGASINSKA J,et al.CPEB and mi R-15/16 Co-Regulate Translation of Cyclin E1 m RNA during Xenopus Oocyte Maturation[J].PLo S One,2016,11(2):e146792.DOI:10.1371/journal.pone.0146792.
[16]SUN L,HUANG Y,WEI Q,et al.Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8(PHF8)and regulates its function in the cell cycle[J].J Biol Chem,2015,290(7):4075-4085.DOI:10.1074/jbc.M114.602532.
[17]SANCHEZ-GONZALEZ C,CIUDAD C J,IZQUIERDO-PULIDO M,et al.Urolithin A causes p21 up-regulation in prostate cancer cells[J].Eur J Nutr,2016,55(3):1099-1112.DOI:10.1007/s00394-015-0924-z.
[2]KARIMIAN A,AHMADI Y,YOUSEFI B.Multiple functions of p21 in cell cycle,apoptosis and transcriptional regulation after DNA damage[J].DNA Repair(Amst),2016,42:63-71.DOI:10.1016/j.dnarep.2016.04.008.
[3]WANG C,CHEN Z,GE Q,et al.Up-regulation of p21(WAF1/CIP1)by mi RNAs and its implications in bladder cancer cells[J].FEBS Lett,2014,588(24):4654-4664.DOI:10.1016/j.febslet.2014.10.037.
[4]GE Q,WANG C,CHEN Z,et al.The suppressive effects of mi R-1180-5p on the proliferation and tumorigenicity of bladder cancer cells[J].Histol Histopathol,2017,32(1):77-86.DOI:10.14670/HH-11-772.
[5]WANG C,GE Q,CHEN Z,et al.A new double stranded RNA suppresses bladder cancer development by upregulating p21(Waf1/CIP1)expression[J].Biomed Res Int,2015,2015:304753.DOI:10.1155/2015/304753.
[6]PLACE R F,LI L C,POOKOT D,et al.Micro RNA-373 induces expression of genes with complementary promoter sequences[J].Proc Natl Acad Sci U S A,2008,105(5):1608-1613.DOI:10.1073/pnas.0707594105.
[7]LI L C,OKINO S T,ZHAO H,et al.Small ds RNAs induce transcriptional activation in human cells[J].Proc Natl Acad Sci U S A,2006,103(46):17337-17342.DOI:10.1073/pnas.0607015103.
[8]BARATA P,SOOD A K,HONG D S.RNA-targeted therapeutics in cancer clinical trials:Current status and future directions[J].Cancer Treat Rev,2016,50:35-47.DOI:10.1016/j.ctrv.2016.08.004.
[9]ZHU L,JIANG H,SHEONG F K,et al.Understanding the core of RNA interference:The dynamic aspects of Argonaute-mediated processes[J].Prog Biophys Mol Biol,2016.DOI:10.1016/j.pbiomolbio.2016.09.008.
[10]GUO D,BARRY L,LIN S S,et al.RNAa in action:from the exception to the norm[J].RNA Biol,2014,11(10):1221-1225.DOI:10.4161/15476286.2014.972853.
[11]ZHENG L,WANG L,GAN J,et al.RNA activation:promise as a new weapon against cancer[J].Cancer Lett,2014,355(1):18-24.DOI:10.1016/j.canlet.2014.09.004.
[12]WANG X,WANG J,HUANG V,et al.Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation[J].Biochem J,2012,443(3):821-828.DOI:10.1042/BJ20111491.
[13]KREIS N N,LOUWEN F,YUAN J.Less understood issues:p21(Cip1)in mitosis and its therapeutic potential[J].Oncogene,2015,34(14):1758-1767.DOI:10.1038/onc.2014.133.
[14]YEO D,HE H,BALDWIN G S,et al.The role of p21-activated kinases in pancreatic cancer[J].Pancreas,2015,44(3):363-369.DOI:10.1097/MPA.0000000000000276.
[15]WILCZYNSKA A,GIT A,ARGASINSKA J,et al.CPEB and mi R-15/16 Co-Regulate Translation of Cyclin E1 m RNA during Xenopus Oocyte Maturation[J].PLo S One,2016,11(2):e146792.DOI:10.1371/journal.pone.0146792.
[16]SUN L,HUANG Y,WEI Q,et al.Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8(PHF8)and regulates its function in the cell cycle[J].J Biol Chem,2015,290(7):4075-4085.DOI:10.1074/jbc.M114.602532.
[17]SANCHEZ-GONZALEZ C,CIUDAD C J,IZQUIERDO-PULIDO M,et al.Urolithin A causes p21 up-regulation in prostate cancer cells[J].Eur J Nutr,2016,55(3):1099-1112.DOI:10.1007/s00394-015-0924-z.