Study On The Mechanism Of The Expression ΔNp63α And The Molecular Mechanism Of Lnc-LIF-AS Promoting The Expression Of LIF In Cervical Cancer Cells

BackgroundCervical carcinoma(CaCx)is the second most prevalent cancer among women.Currently,according to the estimation of World Health Organization,there are 529.800 new cases diagnosed annually in worldwide,with a high incidence of cervical cancer in developping countries especially in China.Therefore,study on mechanisms of cervical carcinogenesis is an crutial factor for the theraphy of cervical cancer.The persistent infection of Human Papillomavirus(HPV)is considered as the major etiologic contributor to the development of cervical cancer.The E6 and E7 gene products are the key HPV proteins that initiate tumors by deregulating the host cell growth cycle by inactivating p53 and hypophosphorylating retinoblastoma protein(pRB).As a member of p53 family,p63 plays an crutial role in the epithelial differentiation.However,the mechanism on tumorigenesis is unclear.The main subtypes of p63 is ΔNp63α in normal cervical epithelial basal cells and cervical cancer tissues.Study has reported that ΔNp63α is a bidirectional regulator of the ΔNp63αtranscription in human liver cancer cell line Hep3 B.However,the specific transcriptional regulation mechanism and function of ΔNp63α in cervical cancer cells are not clear.Therefore we are to investigate the regulation of ΔNp63α in the development of cervical cancer,and hope to provide a new therapeutic targets for cervical cancer.Although human papillomavirus(HPV)infection has been found in most of the cervical cancer cases,additional genetic and epigenetic changes are required fordisease progression.lncRNAs were broadly described as RNAs over 200 nucleotides(nt)in length,which possessed a lot of structural features of the mRNAs,transcripted by RNA polymerase II(RNA polII),with no open reading frame.Abnormal expression of Lnc RNAs was associated with a variety of human diseases,such as cancer,spinocerebellar ataxia and cardiovascular disease.Studies have shown that increased HOTIA gene in cervical cancer is related with the poor prognosis.In order to study the function of ΔNp63α in the development of cervical cancer,our group previously constructed a SiHa cell line stably overexpressing ΔNp63α: SiHa/Con SiHa/ΔNp63α.The microarray hybridization of cell lines SiHa/Con SiHa/ΔNp63α were analyzed for ΔNp63α mRNA and lncRNA.Among them,Lnc-LIF-AS and LIF were downregulated by ΔNp63α simultaneously.The results of previous studies showed thatΔNp63α can directly inhibit the expression of LIF,and indirectly inhibit the expression of LIF by inhibiting the expression of Lnc-LIF-AS.However,the mechanism of the regulation and the functions of ΔNp63α in cervical cancer are yet not clear.Therefore this article will study on the role of ΔNp63α in cervical cancer cells from the following three parts.Part One: Study on the transcriptional regulation mechanism of ΔNp63α in cervical cancer cells Objective To study on the transcriptional regulation mechanism of ΔNp63α in cervical cancer cells Methods(1)The most possible sequence of ΔNp63αpromoter was analysised with Bioinformatics and constructed successfully whitch was inserted into luciferase reporter vector pGL3-Basic.(2)The role of ΔNp63α on the ΔNp63α promoter activity was detected by Luciferase Assay.(3)The regulation of ΔNp63α promotor activity by STAT3 was detected by Luciferase Assay.(4)The effect of P-STAT3 on the expression ofΔNp63α in cervical cancer cell lins was detected by Western Blot.(5)The expression level of ΔNp63α and STAT3 was detected by Western Blot.(6)The effect of ΔNp63α on the expression of P-STAT3 in cervical cancer cell lins and HEK293 T cells was detected by Western Blot.Result(1)A plasmid containing the ΔNp63α Promoter was constructed successfully identified by sequencing technique.(2)The luciferase reporter gene revealed that ΔNp63α can positively regulated the activity of ΔNp63α.(3)The luciferase reporter gene revealed that STAT3 can positively regulated the activity of ΔNp63α.(4)STAT3 stimulant and inhibitor can activate and inhibit the expression of ΔNp63αdetected by Western Blot.(5)Overexpression of ΔNp63α negatively control the phosphorylation level of STAT3 and knockdown of ΔNp63α can activate the expression of P-STAT3,but it is not in the HEK293 T cells.Conclusion:(1)ΔNp63α and STAT3 exerts a Positive Effect on the ΔNp63α promoter activity.(2)STAT3 transactivates the level of ΔNp63α protein.(3)ΔNp63α negtively regulate the ecpression of P-STAT3Part Two: ΔNp63α positive regulate cell differentiation Objectve(1)To study the relationship between ΔNp63α and LIF in cervical cancer tussues.(2)To study the relationship between ΔNp63α and cell differentiation protein in cervical cancer tussues.Methed(1)The expression between ΔNp63α and LIF was detected by immunohistochemical.(2)The relationship between the expression of LIF and the prognosis of cervical cancer patients was analyzed by TCGA data base.(3)The differentiation markers in ME-180 cells was detected by western blot.Result(1)The expression level of LIF was inversely associated with LIF expression in cervical tissues.(2)High levels of LIF expression conferred significantly shorter survival among cervical patients in the TCGA cervical cancer RNAseq data.(3)ΔNp63α has positive related with epithelium differentiation in cervical cancer cells.Part Three: The molecular mechanism of Lnc-LIF-AS promoting the expression of LIF in cervical cancer Objective(1)To study whether the Lnc-LIF-AS regulates the expression LIF by their overlapping sequences.(2)Predict miRNAs that Lnc-LIF-AS may adsorb,and further study the effect of miRNA on LIF expression(3)To study on mechanism of the regulation between ΔNp63α LIF and Lnc-LIF-AS.Method(1)The expression of LIF was detected by Q-PCR and Western Blot in SiHa cells,which were transfected with Lnc-LIF-AS or Lnc-LIF-AS-DN vectors.(2)The most possible candidate miRNAs that may combine to Lnc-LIF-AS were analyzed by miRbase.(3)SiHa cells were stimulated with miRNAs mimic,and the expression levels of Lnc-LIF-AS and LIF were detected by Q-PCR.(4)The effect of ΔNp63α on the mRNA levels of miR-644b-3p and miR-579-3p in SiHa cells was detected by Q-PCR.Result(1)Lnc-LIF-AS can accumulate the mRNA levels of LIF through the overlapping 3’UTR of LIF mRNA sequences.(2)miR-644b-3p and miR-579-3p accumulate the expression of Lnc-LIF-AS and LIF mRNA levels.(3)ΔNp63αcan negtively regulate the expression of miR-644b-3p and miR-579-3p mRNA levels.Conclusions(1)The Lnc-LIF-AS regulates the expression LIF by their overlapping sequences.(2)The expression of miR-644b-3p and miR-579-3p can accumulate the expression of Lnc-LIF-AS and LIF mRNA levels.(3)ΔNp63α,miRNAs,LncRNs and LIF forming a complex regulation mechanism in cervical cancers which should be developed further and deeper.