【作者】 杨涛；

【导师】 叶章群； 徐华；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2017， 博士

【摘要】 目的探索LncRNA PVT1在肾癌中的作用及其机制研究,探讨PVT1作为肾癌预后中生物标志物的意义。方法分析TCGA肾透明细胞癌RNA-seq数据,挑选出感兴趣的lncRNA PVT1,在肾癌组织及细胞系中进行定量PCR检测PVT1表达,基因富集分析(GSEA)PVT1高表达和低表达样本中KEGG通路的富集情况,对786-0和ACHN肾癌细胞系分别干扰和过表达PVT1后进行细胞增殖实验、Edu、细胞集落形成、流式测细胞周期等功能实验,检测PVT1对肾癌细胞786-O和ACHN细胞增殖功能的影响,Transwell迁移及侵袭实验以及划痕试验检测PVT1对肾癌细胞系的迁移及侵袭能力的影响,western blot检测周期及EMT相关蛋白的表达,裸鼠皮下荷瘤实验检测PVT1对786-0肾癌细胞系异位成瘤能力的影响,结合临床数据分析PVT1对肾癌预后及肿瘤分级、分期的影响。结果筛选出差异表达的有命名的lncRNA 159个,其中上调的82个,下调的77个。上调的基因中包含PVT1、MALAT1、XIST、CRNDE等常见的lncRNA。定量PCR检测表明PVT1在肾癌细胞系及肾癌组织中高表达,GSEA富集分析表明细胞周期通路相关基因集在PVT1高表达样本中明显富集,过表达PVT1明显促进细胞增殖,迁移和侵袭,促进裸鼠异位成瘤的能力,干扰PVT1后则抑制相关功能,western blot检测表明干扰PVT1后CDK6,CCND2,p-Rb,N-cadherin及vimentin的表达下调,E-cadherin,p21及P16表达上调。过表达PVT1则相反。Kaplan-Meier生存分析表明PVT1高表达患者总生存及无进展生存时间更短,单变量和多变量Cox回归分析表明PVT1高表达和差的预后相关。结论PVT1在肾透明细胞癌中高表达,并促进肾癌细胞增殖、迁移及侵袭能力,促进裸鼠异位荷瘤的形成,PVT1表达水平和临床预后及肿瘤分级、分期相关,是一个潜在的肾透明细胞癌治疗及预后的生物标志物。目的证明PVT1及其剪切转录本PVT1AE4通过竞争性吸附miR-200家族,调控BMI1、ZEB1及ZEB2的表达,从而影响肾癌细胞增殖、迁移及侵袭能力。方法分析TCGA肾透明细胞癌RNA-seq及miRNA-seq数据,筛选出与lncRNAPVT1表达负相关并结合生物信息学分析找出与PVT1有相应结合位点的miRNA,构建含有相应靶序列的双荧光素酶报告质粒与相应的miRNA mimics或对照mimic共转染肾癌细胞系,检测双荧光素酶的活性。抗AG02RNA免疫共沉淀检测转染不同miRNA mimics的786-0细胞中AG02结合的内源性PVT1含量,定量PCR检测肾癌组织中PVT1与miR-200c及相关靶基因的相关性,共转染PVT1过表达质粒和miR-200c mimic进行功能回复实验。半定量PCR检测新转录本PVT1AE4在肾癌组织中的表达情况,MTS、集落形成及Transwell侵袭试验检测PVT1AE4在肾癌中的功能。最后共转染PVT1AE4过表达质粒和miR-200c mimic进行功能回复实验。结果miR-20b/106b-5p/203a及miR-200s在肾癌中与PVT1表达负相关,并且与PVT1有相应的结合位点,双荧光素酶实验证明miR-200s尤其是miR-200c能够明显降低双荧光素酶活性,抗AG02 RNA免疫共沉淀检测转染miR-200s mimics后与AG02结合的PVT1含量较对照mimic明显升高,miR-200cmimic效果最明显。MTS、集落形成及Transwell迁移及侵袭实验表明miR-200c能够明显抑制肾癌细胞增殖,迁移及侵袭能力。定量PCR检测表明50对肾癌组织中PVT1和BMI1、ZEB1和ZEB2表达正相关,miR-200c表达和PVT1、BMI1、ZEB1及ZEB2表达负相关,相关功能回复实验表明,PVT1能上调BMI1、ZEB1和ZEB2的mRNA及蛋白水平,共转染miR200c mimic后能够部分抵消这种上调,并且部分抵消PVT1对肾癌细胞增殖,迁移及侵袭能力的促进作用,另外发现PVT1ΔE4作为PVT1 一个新的转录本在肾癌组织中明显高表达,并且具有促癌作用,同样也能与miR-200c结合。结论lncRNAPVT1及其剪切转录本PVT1ΔE4在肾癌中具有明显的促癌作用,其作用机制可能是通过PVT1作为竞争性内源RNA吸附miR-200s来调控BMI1、ZEB1及ZEB2表达从而调控肾癌细胞增殖、迁移及侵袭能力。更多还原

【Abstract】 PurposeTo investigate the role of LncRNA PVT1 in renal cell carcinoma and its mechanism and explore the significance of PVT1 as a biomarker in the prognosis of renal cell carcinoma.MethodsTCGA RNA-seq expression data of renal clear cell carcinoma were analyzed and the IncRNA PVT1 was picked out.PVT1 expression was detected by quantitative PCR in RCC tissues and cell lines.GSEA analysis on KEGG pathway was performed according to PVT1 expression.Cell proliferation assay,Edu,cell colony formation,flow cytometry,Scratch assay,Transwell migration assay and invasion assay were performed on PVT1 knockdown and overexpression renal cell lines to investigate the effect of PVT1 on proliferation,migration and invasion of renal carcinoma cells.Western blot was used to detect the expression of the cell cycle and EMT related proteins.Nude mice bearing subcutaneous tumor assay was performed to detect the tendency of ectopic tumorigenesis of PVT1 overexpressed or interfered renal carcinoma cell lines.The clinical data were used to analyze the effect of PVT1 on the prognosis and tumor grade and stage.ResultsWe found that PVT1 was upregulated in clear cell renal carcinoma(ccRCC)tissues and cells.The GSEA revealed that genes associated with cell cycle was remarkably enriched in higher PVT1 tumors versus lower tumors.Further experiments revealed that knockdown of PVT 1 remarkably inhibited ccRCC cell proliferation and cell invasion.Western blots indicate that P21,P16 and E-cadherin proteins were upregulated,while Phospho-Rb,CDK6,CCND2,N-cadherin and vimentin were downregulated after PVT1 knocked down while PVT1 overexpression reversed these changes.Last,in vivo study revealed that PVT1 promotes xenograft tumor growth in nude mice.Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival and disease-free survival in renal cell carcinoma patients,PVT1 expression was correlated with clinicopathological factors.ConclusionPVT1 is highly expressed in renal clear cell carcinoma and promotes the proliferation,migration and invasion of renal cell carcinoma and expedites the formation of heterotopic tumor in nude mice.High expression of PVT1 was associated with poor overall survival and disease-free survival in renal cell carcinoma patients.The inhibition of PVT1 expression may be a promising strategy for ccRCC targeted therapy.PurposeTo investigate the possibility of PVT1 and its splicing transcript PVT1ΔE4 acting as ceRNA in renal cell carcinoma and regulating cell proliferation and invasion through interacting with miR200s.MethodsWe analysed the RNA-seq and miRNA-seq of ccRCC in TCGA database to find miRNAs negatively correlated with PVT1 expression and tried to pick out those with binding sites to PVT1 at the same time.Insights of the mechanism of competitive endogenous RNAs(ceRNAs)were gained through luciferase assays and RNA binding protein immunoprecipitation(RIP)and rescue experiments.Correlation between PVT1 and BMI1,ZEB1,ZEB2 and E-cadherin as well as correlation between miR-200c and PVT1,BMI1,ZEB1 and ZEB2 in 50 paired ccRCC tumor tissues were determined with RT-PCR.Last,the expression of PVT1ΔE4 in renal cancer tissues was checked by PCR,and the rescue experiments were also performed to evaluate its ability to binding miR-200s.ResultsmiR-20b/106b-5p/203a and miR-200s are qualified potential miRNAs that can bind PVT1.Further experiments revealed that PVT1 act as a ceRNA to sponge miR-200s especially miR-200c to regulate BMI1,ZEB1 and ZEB2 expression.MTS assays,colony formation and Transwell migration and invasion assay indicated that miR-200c significantly inhibited the proliferation and invasion of renal cancer cells.Besides,we found a positive correlation between PVT1 and BMI1,ZEB1 and ZEB2 while a negtive correlation between miR-200c and PVT1,BMI1,ZEB1 and ZEB2 in our 50 paired renal cancer tissues and their normal counterparts through RT-PCR.Last,a noval transcript of PVT1 with its exon 4 skipped was found,which had the same function as the full length transcript but exhibited a higher expression level than that of the full length transcript.As the 4th exon does not contain the binding site of miR-200s,it can also binding with miR-200s to act as a ceRNA to regulate BMI1,ZEB1 and ZEB2 expression.ConclusionTaken together,our research demonstrates that PVT1 and its splicing variant PVT1ΔE4 could promote renal cancer cell proliferation and invasion partially by competitively binding the miR-200 family and regulating the expression of BMI1,ZEB1 and ZEB2.更多还原