Research On The Role Of TSPAN31 In Tumors And Its Influence On Akt Pathway In Cervical Cancer Based On Bioinformatics

Background:(1)Tetraspanin(TSPANs)family proteins are known to be linked with proliferation,invasion and metastasis in most tumors.For example,TSPAN7 is reported as a potential marker for liver cancer.As a member of the TSPANs family,TSPAN31 has recently been discovered and thus its functions remain unclear.With the help of bioinformatics methods,we aim to study the role of TSPAN31 in several cancers,especially cervical cancer.(2)Our lab previously found that TSPAN31 and CDK4 gene are reverse complementary sequence at the 3'-UTR region.Studies in liver cancer cells found that TSPAN31 promotes tumor invasion and has negative regulatory effect on CDK4;Recent literature showed that in lipomas and liposarcoma,the expression of TSPAN31 and CDK4 increased consistently compared to normal tissue;To find out whether this phenomenon is prevalent in other tumors,we screened out tumors with similar trends through bioinformatics techniques.Then,we chose cervical cancer as a model to study the underlying mechanism.Purpose:As a member of the Tetraspanin protein superfamily,TSPAN31 was only recently discovered and thus its functions are largely unknown.TSPAN31 and CDK4 genes are reverse complementary sequences at their 3'-UTR regions.They are known as a pair of natural antisense transcripts(NATs).NATs are endogenous complementary RNAs produced in the natural cells of organisms.Double-stranded sense-antisense transcripts can be formed in various ways.RNA plays an important role in regulating various physiological and pathological processes such as cell differentiation,tumorigenesis and energy metabolism.Use bioinformatics technology,we analyzed the expression level of TSPAN31 in various tissues and several types of tumors,and the effect of TSPAN31 expression on the prognosis and survival of patients.If all the tumors we studied,we chose cervical cancer to study the effect of TSPAN31 on the growth and metastasis of cervical tumor cells.Method:TCGA,GTEx and related databases were used to analyze the expression level of TSPAN31 in normal tissues,organs and tumor tissues,and the impact of TSPAN31 on the tumor patient survival.In order to define the relationship between TSPAN31 and CDK4 in cervical cancer cells,we overexpressed or silenced TSPAN31 in cervical cancer cells,respectively.Western Blot showed that the expression levels of CDK4 and TSPAN31 were negatively correlated.In order to clarify whether the negative expression correlation is due to partial overlap of their 3' end sequence,we designed the following experiment: By searching TSPAN31 gene on NCBI website,we determined the gene sequences of TSPAN31 and CDK4.We synthesized three TSPAN31plasmids: TSPAN31 full-length plasmid(Tetraspanin31-full length),plasmid expressing only the TSPAN31 3'-UTR(Tetraspanin31-3'UTR),and plasmid that excludes 3'-UTR region complementary to the CDK4 gene(Tetraspanin31--CDS).After transfection of the overexpression plasmids into cervical cancer cells,we detected CDK4 level by Western Blot.Changes in CDK4 expression are often related to cell proliferation.In order to explore the effect of TSPAN31 on cell proliferation in cervical cancer cells,we performed TSPAN31 overexpression and knockdown experiments on cervical cancer cells.Cell clone formation experiments was used to detect the proliferative ability of the treated cells.We calculated the clone formation rate as follows: clone formation rate =(number of clones/number of inoculated cells)× 100%.Many studies on four-span membrane proteins have shown that four-span membrane proteins affect tumor metastasis by activating the AKT pathway,such as TSPAN1,CD81,and CD9.In order to explore whether TSPAN31 activates the AKT pathway in cervical cancer,we designed the following experiment to overexpress the TSPAN31 gene in cervical cancer cells.48 hours post transfection,Western Blot was used to detect TSPAN31,AKT,p-AKT,GSK3? and ?-catenin.Result:(1)We compared TSPAN31 levels of tumor tissues with respect to corresponding normal tissues in TCGA,GTEx and their related databases(p<0.05).We found significant increases of TSPAN31 in diffuse large b-cell lymphoma(DLBC),multiform glioma(GBM),low-grade glioma of the brain Cell tumor(LGG),pancreatic cancer(PAAD)and thymoma(THYM)and decreases in CESC(cervical cancer),UCEC(endometrial cancer)and UCS(uterine sarcoma)(p<0.05).Subsequently,we used the R language to analyze the prognostic survival of the tumor sample data in the TCGA database,which showed decreases in BRCA(breast cancer),HNSC(head and neck squamous cell carcinoma)and LGG(low-grade brain glioma).High TSPAN31 expression increases patient survival in KIRC,while low TSPAN31 expression can increase the survival rate of patients in BRCA?HNSC?LGG,(p<0.05).(2)TSPAN31 and CDK4 are a pair of trans NATs.In cervical cancer,over-expression of TSPAN31 inhibits expression of CDK4,which significantly reduces the expression of CDK4.In order to explore whether the complementary region of the two affects CDK4 expression,we designed an experiment by synthesizing three kinds of TSPAN31 plasmids: TSPAN31 full-length plasmid(Tetraspanin31-full length),plasmid that expresses the TSPAN31 3'-UTR(Tetraspanin31-3'UTR)and plasmid that does not contain the 3'region complementary to the CDK4 gene(Tetraspanin31-CDS).48h post transfection,WB results showed that only Tetraspanin31-3'UTR and Tetraspanin31-full length groups decreased CDK4,whereas Tetraspanin31-CDS group and the control group showed no significant changes in CDK4.(3)In order to verify the effect of TSPAN31 on the AKT pathway,48 hours post transfection of TSPAN31-full plasmid,WB was performed to detect AKT,GSK3?,p-AKT,p-GSK3? and ?-catenin in Hela cells.Results showed that AKT and GSK3?showed little changes,whereas p-AKT,p-GSK3? and ?-catenin increased significantly compared to control(p<0.05),indicating that TSPAN31 promotes AKT phosphorylation and leads to activation of GSK3? and accumulation of ?-catenin.Conclusion:Bioinformatics analysis showed that the expression of TSPAN31 was increased in DLBC,GBM,LGG,PAAD and THYM and decreased in CESC,UCEC and UCS tumors compared to normal tissues(p<0.05).Kaplan-Meier plot showed that high expression of TSPAN31 improves the survival rate of KIRC patients,while low expression of TSPAN31 improves the survival rate of BRCA,HNSC,and LGG patients(p<0.05).In cervical cancer,TSPAN31 and CDK4 are NATs to each other.TSPAN31 down-regulates CDK4 through the action of the 3'complementary region.Overexpression of TSPAN31 can promote the phosphorylation of AKT and GSK3?,leading to an increase in ?-catenin to activate the AKT pathway.