Construction And Functional Analysis Of CeRNA Regulatory Network In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer is a common malignant tumor of digestive tract.There were more than 570,000 new cases of esophageal cancer(EC)and more than 500,000 deaths each year in the world,which,ranking the seventh and sixth in all malignant tumors,respectively.The incidence and death of EC in China accounted for more than half of the world.Over 90%of EC in China is esophageal squamous cell carcinoma(ESCC).The treatment of EC mainly includes surgery,radiotherapy,chemotherapy and immunotherapy.Recently,the level of surgical treatment and other comprehensive treatments of EC has rapidly evolved.However,the overall five-year survival rate is only 15-20%because it is already the advanced stage at the time of diagnosis and the high recurrence rate postoperative.Therefore,in-depth study of the possible molecular mechanism of esophageal cancer in the development process,explore specific molecular markers,and seek efficient therapeutic targets are of great significance to improve the level of diagnosis and treatment of esophageal cancer and improve its long-term survival rate.NcRNA include microRNA(miRNA),long noncoding RNA(lncRNAs)and recently discovered circular RNA(circRNA).ncRNAs play crucial roles in carcinogenicity.The regulatory of lncRNAs and circRNA with extensive functions has attracted extensive attention.LncRNAs are a class of RNA molecules with more than 200 nucleotides and no protein coding function.It interacts with DNA,RNA and protein through base complementation and secondary structure of stem loop.It can affect gene expression in epigenetic,transcriptional and post transcriptional levels.It plays a regulatory role through chromatin modification,transcriptional activation and inhibition,intranuclear transport and other mechanisms,affecting genome activity,miRNA stability and RNA protein complex formation.LncRNAs can interact with TFs and act as enhancers or inhibitors of gene expression.circRNA represent a new type of ncRNAs composed of covalently closed continuous rings.Recent studies have shown that miRNA,lncRNAs and circRNA can act as competing endogenous RNAs(ceRNA)to regulate gene expression through common miRNA response elements(MRes).Dysregulated ceRNAs have been shown to exist in a variety of cancers.NcRNAs targeted therapy has entered a phase Ⅰ clinical trial,such as miR-34 in the treatment of liver cancer.We analyzed the differentially expressed miRNA,lncRNAs and circRNA using high throughput sequencing in ESCC.GO and KEGG were used to annotate,enrich and analyze the functions of lncRNAs and circRNA in the regulation of core TFs.The ncRNAs related to TFs were analyzed,and the key TFs and related pathways were screened out.We constructed a ceRNA network to describe the functions of lncRNAs and circRNA in the regulation of core TFs.At the same time,the ceRNA network of key pathways was constructed,and the dysregulated ceRNAs were screened out,which provided a basis for further clinical development It is a potential new therapeutic target.Finally,we further verified the ncRNA by cell function test.Part Ⅰ:Differential expression of non coding RNAin ESCCObjective:The cDNA library of esophageal squamous cell carcinoma was constructed,the full transcriptome sequencing and data collection were performed,and the differentially expressed ncRNAs were screened.Methods:1.Three pairs of esophageal carcinoma and adjacent tissues were collected.The total RNA samples were extracted and purified.2.The cDNA library was constructed.3.Transcriptome sequencing and data collection were performed.4.The analysis of differentially expressed genes(including miRNA,lncRNAs and circRNA)was performed by deseq2,and the standard was:Fold Change≥ 2.0,FDR<0.05.The differentially expressed ncRNAs in esophageal carcinoma and its adjacent tissues were screened.5.Six pairs of esophageal cancer tissues were collected.Five lncRNAs and five circRNA were selected to verify the accuracy of sequencing results by QRT PCR.Results:1.RNA was extracted from three pairs of samples,cDNA library was constructed,and all genes of three pairs of samples were detected.Gene data were uploaded to geo(login code:GSE157373).2.A total of 17127 miRNA,11456 lncRNAs and 7340 circRNA were detected.A total of 3265 miRNA,1084 lncRNAs and 38 circRNA were identified as differentially expressed(Fold Change≥ 2.0,FDR<0.05).Among them,27 RNAs and 1859 miRNA were up-regulated,while 11 miRNA and 1858 miRNA were down regulated.3.Five lncRNAs and five circRNA were selected to verify the results of RNA SEQ,and the PCR results were compared with the sequencing results,which confirmed that the trend of the two results was consistent.4.In ESCC,269 TFs were found to be differentially expressed in HMG,PAX and stat families.6.The differentially expressed lncRNAs were distributed on all chromosomes.The distribution was as follows:antisense(n=196),exon(n=6),intergenic(n=920)and intron(n=95).LncRNAs between genes were the largest category,including 540 up-regulated and 380 down regulated lncRNAs.Conclusion1.There are many differentially expressed miRNA,lncRNAs and circRNA in esophageal carcinoma.2.There are many TFs in esophageal carcinoma,which belong to HMG,PAX and STAT families.3.The differentially expressed lncRNAs were distributed on all chromosomes.Among them,lncRNAs was the most differentially expressed gene.Part Ⅱ:Functional analysis of differential non coding RNA and construction of CeRNA networkObjectiveTo investigate the possible role of 3265 miRNA,1084 lncRNAs and 38 circRNA in the development of esophageal cancer.Methods1.The difference genes of ESCC were analyzed by go enrichment.2.The go analysis of TFs,including biological process,cell components and molecular functions,was carried out.The GO term tree of TFs was constructed according to the association of GO term.3.The key signal pathways of TFs enrichment in ESCC were further screened.4.Based on TFs and circRNA/lncRNAs,the co expression network of genes was constructed,and the core lncRNAs and circRNA involved in transcription factor regulation were screened5.To construct a CeRNA network of key pathways for different TFs enrichment,and screen ncRNA which may play a key role in ESCC.Result:1.The first 20 go genes are mainly related to cell cycle,DNA replication cell cycle,DNA strand extension,mitotic nuclear division,Gl/Sconversion of mitotic cell cycle,etc.they are mainly related to cell cycle,DNA replication,fine cell adhesion,cell division and positive regulation of transcription.Up regulated genes are related to cell cycle,cell division and DNA replication.Down regulated genes are related to cell adhesion,calcium homeostasis and cGMP mediated signaling.2.The results of go and KEGG analysis showed that the up regulated TFs was related to MYB,RB-E2F and snRNA activator protein complexes,which was related to cell cycle and division.Down regulated TFs is involved in the LD1/2 and histone deacetylase complexes and the exogenous components of the mitochondrial outer membrane related to the epigenetic regulation and function of mitochondria.3.KEGG analysis shows that there are many pathways involved.We selected hedgehog signal pathway,JAK-STAT signal pathway,TGF-βsignal pathway and MAPK signal pathway according to the classical signal pathway network related to tumor occurrence and development.4.Through network construction,we can see that lncRNAs/circRNA and TFs show distinct co expression patterns in tumor tissues and adjacent normal tissues.Many lncRNAs and cirRNAs participate in the occurrence and development of ESCC.5.We constructed four paths of CeRNA network.The results showed that:11)In TGF-βsignal pathway,SMAD9 is the target of has mir-7703,while loc283856,loc101928509,circSORBs1 and circAMY2b are ceRNA of has mir-7703.2)In MAPK signal path,has mir-4763-3p targets NFZTC4 and JUND,and can compete with loc102724312,loc101927046,SNOTD115,DNMLP46,loc102724312 and circAKAP12.3)In the CeRNA network of JAK STAT signal pathway,we found that the STAT1 and PIAS3 of TFs regulated by has Mir 6197-5p and has mir-4695-5p were significantly up regulated.At the same time,loc105373310,linc00898,wtapp1,lhfpl3-as2,loc339260,circTP63,circdhtkdl and circlmnbl were the up regulated ceRNAs of these two miRNA.4)In th e Hedgehog signal path,GKI/2act is the target of has mir-3667-3p,while 1krt18p58 and circdhtkdl are ceRNAs of has mir-3667-3p.ZIC2 is the target of has-mir-6756-5p,and loc105378763,loc107894526,loc105375690 and circTP63,med15p8 compete with each other.6、CircTP63 is involved in many important pathways,and we speculate that it may play a role in the development of esophageal cancer.Conclusion1.The top 20 go were mainly related to cell cycle,DNA replication,cell adhesion,cell division and positive regulation of transcription.Up regulated genes are related to cell cycle,cell division and DNA replication,Down regulated genes are related to cell adhesion,calcium homeostasis and cGMP mediated signaling.2.The results of go and KEGG analysis showed that the up regulated TFs was related to myb,rb-e2f and snRNAs activator protein complexes.Down regulated TFs was involved in the LSD 1/2 and histone deacetylase complexes and the epigenetic regulation with mitochondria.3.KEGG analysis showed that the inflammatory related pathways such as hedgehog signal pathway,JAK STAT signal pathway,tgf-psignal pathway and MAPK signal pathway were obviously out of balance in ESCC.4.LncRNAs and circRNA showed different co expression patterns in tumor tissues and adj acent normal tissues.Part Ⅲ:Functional verification of circRNA TP63 in ESCCObjectiveObjective to verify the effect of circTP63 on the proliferation,apoptosis and migration of Eca109 cells,and to preliminarily study the mechanism of circTP63 in Eca109.Methods1.Two siRNAs of circTP63 were screened,and two groups of Eca109 cells were constructed by lipofection of si-circTP63-1 and si-circTP63-2.2.The silencing effect of si-circTP63 was analyzed by real-time PCR.3.The proliferation,apoptosis and migration of Eca109 cells transfected with si-circTP63-1 and si-circTP63-2 were studied by CCK-8,flow cytometry and Transwell assay.5.Western blot was used to detect the migration and apoptosis of esophageal squamous cell carcinoma cells.Results:1.Si-circTP63-1 and si-circTP63-2 can down regulate the expression of circTP63,and have no effect on the host gene.2.After silencing the expression of circTP63,cell proliferation decreased.3.Apoptosis test showed that:compared with the blank control group,the apoptosis rate of si-circTP63-1 and si-circTP63-2 in the silence group was significantly increased.4.Trans well assay showed that the number of Eca109 migration cells decreased significantly after transfection of si-circTP63-1 and si-circTP63-2 for 48 h,which indicated that the migration ability of Eca109 cells decreased after inhibition of circTP63,indicating that the invasion ability of Eca 109 cells decreased after inhibition of circTP63.5.Western blot analysis showed that JAK2 had no significant change,p-stat1 and STAT1 protein levels were down regulated,and STAT3 protein level was not affected.This suggests that the mechanism of circTP63 regulating Eca109 cells is related to JAK STAT signaling pathway.Conclusion1.It is the first time that circTP63 plays an important role in esophageal squamous cell carcinoma.2.The apoptosis rate of Eca 109 cells inhibited by circTP63 was significantly increased.3.The invasion ability of Eca109 cells inhibited by circTP63 was decreased.4.CircTP63 can promote the occurrence,development and metastasis of esophageal squamous cell carcinoma by activating jak-STAT1.