Screening And Identification Of The Differentially Expressed Genes In Wilms’ Tumor

Background: Wilms’ tumor(WT)is the most common childhood renal malignancy that affects approximately 1 in 10,000 children in populations.Age of diagnosis is between 1 and 3 years and both kidneys are affected in ~5% of children.Most cases of Wilms’ tumors are sporadic,however rare cases of familial WT have also been described.Approximately 10% of WT cases are associated with germline mutations and/or congenital abnormalities,such as Denys–Drash syndrome(DDS),Beckwith–Wiedemann syndrome(BWS),Wilms-Aniridia-Genitourinary anomalies-mental Retardation(WAGR)syndrome and Perlman syndrome.WT is a complex embryonal tumor with conventional triphasic histology(blastemic,epithelial and stromal components).While these three components are typically found,WT may also display heterologous elements such as cartilage,osteoid and neural elements.This heterogeneity implies a complexity to the underlying causes of WT that has fascinated investigators for decades.The Wilms’ tumor 1(WT1),located at chromosome 11p13,was first cloned in 1990 as one of the first tumor suppressor genes in WT.Subsequently,beta-catenin proto-oncogene(CTNNB1)and Wilms’ tumor gene on the X chromosome(WTX)have been identified in tumors.The combined frequency of WT1,CTNNB1,and WTX genetic alterations has been estimated to occur in roughly one-third of WT.Furthermore,WT maintenance and disease progression are associated with the altered expression of multiple other genes,such as p53,MYCN,CITED1,SIX2,TOP2 A,and CRABP2.Specifically,mutations in p53 appear to be a common finding in unfavorable histology(UH)WT and a notorious marker of treatment resistance.A recent whole exome study has indentified mutations in microRNA processing genes including DROSHA and DGCR8.However,the frequency of alterations in DROSHA and DGCR8 are similarly uncommon,leaving a significant fraction of cases without an identified ??driver‘‘ genetic defect.It is well-known that numerous recurrent copy number aberrations and loss of heterozygosity(LOH)events have been described,some of which affect known genes(e.g.11 p LOH and 17 p loss),while the critical genes with other regions(e.g.1q gain,1p loss and 16 q loss)remain elusive.Only a few of these aberrations have known associations with histology or outcome.The documented association between relapse and LOH for 1p and 16 q is being used to stratify patients within the current Children‘s Oncology Group therapeutic protocols to warrant a more intensive drug regimen up front for favorable histology(FH)WT.Meanwhile,loss of genetic material at 4q,11 q,and 14 q has also emerged as features of UHWT and poor prognosis.Therefore,identification and characterization of these genes is of primary importance in understanding the onset and progression of tumor and ultimately leading to recognition of potential markers and specific targets for prevention and individualized treatment of tumor.Over the last few decades,numerous markers,especially the novel ones,have been confirmed and shown variations in the prevalence.However,the results of these studies were inconsistent partly because the sample sizes were usually small,the ethnic backgrounds and the experimental techniques were varied.In order to overcome the limitation of individual studies,we performed this meta-analysis to provide a more precise and comprehensive outcome for genetic tests and a basis for the prevention,early diagnosis and treatment of WT.Subsequently,this project adopted Next generation sequencing technologies to perform transcriptome sequencing and bioinformatics analysis on tumor and non-tumor tissue samples,screened and identified the differentially expressed gene of WT.In this research,the main contents and results are listed as follows:Part 1 Genetic variations frequencies in Wilms’ tumor: a meta-analysis and systematic reviewObjective: This was a meta-analysis and systematic review to determine the global prevalence of genetic variations in Wilms tumors.Methods:A comprehensive search of Pubmed,Embase,Web of science and Cochrane Library was performed including cross-referencing independently by 2 assessors.Meta-analysis was done with Stata 12.0.Results:Mutations in WT1,WTX,and CTNNB1 account for only about one-third of tumors.There is a clear relationship between p53 mutations and anaplastic WT,the histologic subtype with poorer prognosis.Frequency of MYCN mutations are uncommon,leaving a significant fraction of cases without an identified ?driver‘ genetic defect.DROSHA,DGCR8 may have a crucial role in WT tumorigenesis,the frequency of mutations in a broad unbiased WT series still remains to be determined.LOH signatures lie in the low sensitivity.Part 2 RNA expression profiles in Wilms’ tumor by Next generation sequencing technologiesObjective: To elucidate altered RNA expression profiles between Wilms’ tumor and adjacent nontumors by the next generation sequencing technology.Methods:We used Next generation sequencing technologies to discovery differentially expressed genes between Wilms’ tumor and adjacent nontumors.To identify the differentially expressed genes by q-PCR.Results:The whole maps of Wilms’ tumor is set up by next generation sequencing.ASIC1,SCG5,HnRNPL and CACNB4 may be the differentially expressed genes for Wilms’ tumor.ASIC1,SCG5,HnRNPL and CACNB4 are identified by q-PCR.Part 3 Study of hnRNPL in regulation the cell proliferation and apoptosis of Wilms’ tumor and its potential mechanismObjective: To explore hnRNPL in regulation the cell proliferation and apoptosis of Wilms’ tumor and its potential mechanism.Methods:RNA-binding protein immuno-precipitation(RIP)was used to map p53 mRNA binding to hnRNPL,to confirm the direct interaction of hnRNPL with p53 mRNA.To silence of hnRNPL by siRNA in G401 cell line and to study its effct on proliferation and apoptosis of Wilms’ tumor cell.q-PCR and WB detection of hnRNPL、p53 and bcl-2 in G401 cell line expression with silencing hn RNPL.Results:In RIP products,hnRNPL directly binding with p53 mRNA,which prompt us to address the potential biological roles of hnRNPL acting as p53 mRNA-binding protein.siRNA hnRNPL inhibits G401 cell growth by MTT assay and in mouse xenografts and increases cell apoptosis by flow-cytometric analysis.Expression of hnRNPL 、 p53 and bcl-2 mRNA and protein are down-regulated in G401 cell line after siRNA hnRNPL.Conclusion:WT maintenance and disease progression are associated with the altered expression of multiple genes(WT1,WTX,CTNNB1,p53,MYCN,DROSHA and DGCR8),numerous recurrent copy number aberrations and loss of heterozygosity(LOH)events.However,the frequency of alterations are uncommon,leaving a significant fraction of cases without an identified ?driver‘ genetic defect.The relative expression of hnRNPL mRNA was higher than that in normal tissues.The RIP of hn RNPL and p53 mRNA suggests that they bind to each other.siRNA hnRNPL inhibits G401 cell growth and increases cell apoptosis.Expression of hnRNPL、p53 and bcl-2 mRNA and protein are down-regulated significantly in G401 cell line after siRNA hnRNPL.hnRNPL is a hub in p53/Bcl2 signaling pathway,which plays an important role in the proliferation and apoptosis of Wilms’ tumor.