微小RNA-506-3p调控UHRF1抑制大肠癌细胞转移的机制
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摘要
目的探讨微小RNA-506-3p(miR-506-3p)通过调控含PHD及指环结构域的类泛素蛋白1(UHRF1)的表达,进而影响大肠癌细胞转移能力的机制。方法利用生物信息学的方法预测并筛选UHRF1上游可能发挥调控作用的miRNA-506-3p;在大肠癌细胞及组织中分别检测miR-506-3p及UHRF1的表达水平并分析其相关性;运用荧光素酶报告基因技术检测miR-506-3p与UHRF1 3'非翻译区(3'UTR)特异性结合的情况;荧光定量PCR(qRTPCR)及Western blot检测大肠癌细胞转染miR-506-3p类似物(mimics)后对UHRF1表达水平的影响;构建UHRF1过表达的大肠癌稳转细胞株,分别转染miR-506-3p mimics及其对照序列(mimics control)后,利用Transwell实验检测处理前后大肠癌细胞转移能力的变化。结果相对于正常大肠上皮细胞及癌旁组织而言,miR-506-3p在大肠癌细胞及组织中的表达均下调(均P<0. 05),且与UHRF1的表达水平呈负相关(r=-0. 456,P=0. 044); miR-506-3p通过其"种子序列"与UHRF1 3'UTR特异性结合(P<0. 01),并减少了UHRF1的mRNA及蛋白表达(均P<0. 01);转染miR-506-3p mimics抑制了大肠癌LoVo细胞的转移能力(P<0. 01),而在此基础上增加UHRF1的表达则逆转了miR-506-3p的抑制作用(P<0. 01)。结论 miR-506-3p通过调控UHRF1抑制了大肠癌细胞的转移能力,在大肠癌中发挥抑癌基因的作用。
Objective To explore the effect of microRNA-506-3 p( miR-506-3 p) on metastasis of colorectal cancer( CRC) and its relation to UHRF1( ubiquitin-like,containing PHD and ring finger domain 1). Methods Bioinformatics screening was performed to predict UHRF1 as a potential target of miR-506-3 p. The expression of miR-506-3 p and UHRF1 was detected by qRT-PCR in 20 pairs of CRC and adjacent non-tumoral tissue samples,CRC cell lines LoVo,SW480,HT29,Caco-2 and normal intestinal epithelial cell line NCM460. Luciferase reporter assays,qRT-PCR,Western blotting and Transwell assays were used to evaluate the effects of miR-506-3 p on the regulation of UHRF1 in CRC cells. Results Bioinformatics analysis indicated that UHRF1 was a putative target of miR-506-3 p. The expression of miR-506-3 p decreased in CRC cell lines and in CRC tissues compared to adjacent non-cancerous tissues,and it was negatively correlated with UHRF1 expression. Luciferase reporter assay indicated that miR-506-3 p directly bound to 3'-UTR of UHRF1. Over-expression of miR-506-3 p decreased the expression of UHRF1. In vitro migration assay showed that synthetic miR-506-3 p suppressed UHRF1 expression and reduced metastasis of CRC cells. Conclusion miR-506-3 p suppresses metastasis of CRC through targeting UHRF1 and it may play an important role as a tumor suppressor in CRC.
Objective To explore the effect of microRNA-506-3 p( miR-506-3 p) on metastasis of colorectal cancer( CRC) and its relation to UHRF1( ubiquitin-like,containing PHD and ring finger domain 1). Methods Bioinformatics screening was performed to predict UHRF1 as a potential target of miR-506-3 p. The expression of miR-506-3 p and UHRF1 was detected by qRT-PCR in 20 pairs of CRC and adjacent non-tumoral tissue samples,CRC cell lines LoVo,SW480,HT29,Caco-2 and normal intestinal epithelial cell line NCM460. Luciferase reporter assays,qRT-PCR,Western blotting and Transwell assays were used to evaluate the effects of miR-506-3 p on the regulation of UHRF1 in CRC cells. Results Bioinformatics analysis indicated that UHRF1 was a putative target of miR-506-3 p. The expression of miR-506-3 p decreased in CRC cell lines and in CRC tissues compared to adjacent non-cancerous tissues,and it was negatively correlated with UHRF1 expression. Luciferase reporter assay indicated that miR-506-3 p directly bound to 3'-UTR of UHRF1. Over-expression of miR-506-3 p decreased the expression of UHRF1. In vitro migration assay showed that synthetic miR-506-3 p suppressed UHRF1 expression and reduced metastasis of CRC cells. Conclusion miR-506-3 p suppresses metastasis of CRC through targeting UHRF1 and it may play an important role as a tumor suppressor in CRC.
引文
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[2]韩俊毅,赵中辛,胡海.高龄结直肠癌手术治疗的生存分析[J].同济大学学报(医学版),2016,37(5):107-113.
[3]ZHANG Q,QIAO L,WANG X,et al.UHRF1 epigenetically down-regulates UbcH8 to inhibit apoptosis in cervical cancer cells[J].Cell Cycle,2018,17(3):300-308.
[4]SAIDI S,POPOV Z,JANEVSKA V,et al.Overexpression of UHRF1 gene correlates with the major clinicopathological parameters in urinary bladder cancer[J].Int Braz J Urol,2017,43(2):224-229.
[5]WANG F,YANG Y Z,SHI C Z,et al.UHRF1 promotes cell growth and metastasis through repression of p16(ink4 a)in colorectal cancer[J].Ann Surg Oncol,2012,19(8):2753-2762.
[6]ZHANG Y,LIN C,LIAO G,et al.MicroRNA-506suppresses tumor proliferation and metastasis in colon cancer by directly targeting the oncogene EZH2[J].Oncotarget,2015,6(32):32586-32601.
[7]GUO S,YANG P,JIANG X,et al.Genetic and epigenetic silencing of mircoRNA-506-3p enhances COTL1oncogene expression to foster non-small lung cancer progression[J].Oncotarget,2017,8(1):644-657.
[8]GROTHEY A,SCHMOLL H J.New chemotherapy approaches in colorectal cancer[J].Curr Opin Oncol,2001,13(4):275-286.
[9]沙杰,范理宏.miRNA-146b-3p在早期非小细胞肺癌中的诊断价值[J].同济大学学报(医学版),2018,39(2):73-78.
[10]TOIYAMA Y,HUR K,TANAKA K,et al.Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer[J].Ann Surg,2014,259(4):735-743.
[11]ZHOU J,ZHANG M,HUANG Y,et al.MicroRNA-320b promotes colorectal cancer proliferation and invasion by competing with its homologous microRNA-320a[J].Cancer Lett,2015,356(2 Pt B):669-675.
[12]MA H,PAN J S,JIN L X,et al.MicroRNA-17~92inhibits colorectal cancer progression by targeting angiogenesis[J].Cancer Lett,2016,376(2):293-302.
[13]XIE H,REN X,XIN S,et al.Emerging roles of circRNA_001569 targeting miR-145 in the proliferation and invasion of colorectal cancer[J].Oncotarget,2016,7(18):26680-26691.
[14]WOTSCHOFSKY Z,GUMMLICH L,LIEP J,et al.Integrated microRNA and mRNA signature associated with the transition from the locally confined to the metastasized clear cell renal cell carcinoma exemplified by miR-146-5p[J].PLoS One,2016,11(2):e0148746.