The Biological Function And Molecular Mechanaism Of Long Non-coding RNA DLEU2 In Cervical Cancer

Cervical cancer is one of the most common female malignant tumors in the world.The incidence and mortality of CC are increasing year by year,and tend to be younger.Long-chain non-coding RNA,a type of endogenous RNA with a transcript longer than 200 nt,plays an important role in the tumor progression.Based on the sequencing data in GEO and TCGA databases,we used bioinformatics methods to screen lncRNA DLEU2,which is one of the new genes,is highly expressed in cervical cancer tissues and is located on chromosome 13q14.Combined clinical tissue sample verification and clinical correlation analysis,we confirmed that lncRNA DLEU2 was highly expressed in cervical cancer tissues and was closely related to tumor size.These results suggested that lncRNA DLEU2 might promote the occurrence and development of cervical cancer.However,the biological function and molecular mechanism of lncRNA DLEU2 in cervical cancer have not been elucidated.So,we mainly focus on the biological function and molecular mechanism of lncRNA DLEU2 in cervical cancer cells in this paper.Part 1 The expression and clinical significance of lncRNA DLEU2 incervical cancerObjective: To explore the expression and clinical significance of lncRNA DLEU2 in cervical cancer.Methods: We downloaded the cervical cancer datasets(GSE63514,GSE9750 and TCGA-CESC)from GEO and TCGA databases respectively and sued R package to screen out the differentially expressed lncRNA.Then the clinical pathologic feature and biological function of lncRNA DLEU2 were predicted by bioinformatics method.The q RT-PCR was performed to verified the lncRNA DLEU2 expression between cervical cancer tissues and normal cervical tissues.Results: We found 3 differentially expressed lncRNAs from the GSE63514,GSE9750 and TCGA-CESC databases.By analyzing the literature,lncRNA DLEU2 was picked up for further research.The clinical correlation analysis showed that lncRNA DLEU2 was closely related to topography in cervical cancer.Its function was predicted by GO and KEGG enrichment analysis.GO analysis results showed that lncRNA DLEU2 was related to RNA splicing,DNA metabolic process,DNA replication and cell cycle.The KEGG analysis results showed that lncRNA DLEU2 was significant correlated with cell cycle,spliceosome and p53 signaling pathway.Conclusion: The expression level of lncRNA DLEU2 was significantly upregulated in cervical cancer tissues.Lnc RNA DLEU2 was closely related to tumor topography,and might be mainly involved in cell cycle.Part 2 Lnc RNA DLEU2 promoted the proliferation and migration incervical cancer cellObjective: To investigate the expression and subcellular localization of lncRNA DLEU2 in cervical cancer cell,and to explore the effects of lncRNA DLEU2 on growth,colony formation,migration in cervical cancer cells.Methods: q RT-PCR was performed to verify the expression level in He La,Si Ha and Ca Ski cells.FISH was used to predict the subcellular location in cervical cells.We performed the loss-of-function and gain-of-function assays in cervical cancer cells to understand the function of lncRNA DLEU2 in cervical cancer progression.CCK8 assay,colony formation assay and cell migration assay were performed to verify the effects of lncRNA DLEU2 on growth,colony formation,migration in cervical cancer cells.Results: The results of q RT-PCR and FISH were demonstrated that lncRNA DLEU2 was highly expressed and was distributed in both cytoplasm and nucleus in cervical cancer cells.The CCK8 results showed that the cell growth rate was significantly lower after silencing lncRNA DLEU2 in He La,Si Ha and Ca Ski cells;on the contraty,the cell growth rate was higher when lncRNA DLEU2 was overexpressed in He La,Si Ha and Ca Ski cells.The result of colony formation assay showed that the size and number of colony were both lower when lncRNA DLEU2 knockdown in cervical cancer cells,and vice versa.The results of cell migration assay showed that He La,Si Ha and Ca Ski cells with low expression of lncRNA DLEU2 migrated significantly slower,while He La,Si Ha and Ca Ski cells with overexpression of lncRNA DLEU2 migrated faster.Conclusion: Lnc RNA DLEU2 was highly expressed in cervical cancer cells and promoted the cell proliferation and migration.Part 3 Lnc RNA DLEU2 promoted cell proliferation via promotingcell-cycle progression and inhibiting the activity of Notch signalingpathwayObjective: To explore the downstream molecular mechanism of lncRNA DLEU2 promoting the proliferation in cervical cancer cell.Methods: Flow cytometry was used to analyze the cell cycle.RNA-Seq was performed to analyze the differentially expressed genes after silencing lnc NRA DLEU2.GO and KEGG analyze were used to predicted the pathway that differentially expressed genes were involved in.Bioinformatics,q RT-PCR and western blot were used to analyze the expression of these key genes.Results: Flow cytometry was used to detect the cell cycle in He La,Si Ha and Ca Ski cells when lncRNA DLEU2 knockdown and overexpression,and the results showed that the number of cervical cancer cells was increased in G1 phase and decreased S phase when lncRNA DLEU2 knockdown;when lncRNA DLEU2 was overexpressed in cervical cancer cells,the number of cervical cancer cells was decreased in G1 phase and increased in S phase.Western blot analysis showed that the expression levels of c-Myc,CDK4,and CDK2 were reduced in cervical cancer cells with lncRNA DLEU2 knockdown,and the expression levels of c-Myc,CDK4,and CDK2 were elevated in cervical cancer cells when lncRNA DLEU2 overexpressed.RNA-Seq results showed that 471 differentially expressed genes were obtained after silencing lncRNA DLEU2,of which 303 were up-regulated and 168 were down regulated.GO analysis showed that these differentially expressed genes were mainly involved in DNA-based transcriptional regulation,regulation of transcription by RNA polymerase II,cell cycle,cell differentiation,cell adhesion,and so on.KEGG pathway analysis results show that these differentially expressed genes are significantly related to proteoglycans in cancer,human cytomegalovirus infection,Notch signaling pathway.q RT-PCR results verified the expression of differentially expressed genes involved in cell cycle and NOTCH signaling pathways(CDKN1A,E2F4,WEE1,NOTCH1,RBPJ),which were consistent with the sequencing results.Western blot analysis showed the protein expression levels of P21,P53 and related signaling pathway genes,and the results confirmed that lncRNA DLEU2 promoted the cell cycle progression and inhibited the activity of Notch signaling pathway by inhibiting P53.Conclusion: Lnc RNA DLEU2 promoted the cell cycle progression and inhibited the activity of Notch signaling pathway by inhibiting P53,thus promoted the proliferation in cervical cancer cells.Part 4 SP1 promoted the expression of lncRNA DLEU2 in cervical cancercellsObjective: To explore the regulatory effect of transcription factor SP1 on lncRNA DLEU2.Methods: The lncRNA DLEU2 promoter region was downloaded from UCSC and NCBI.PROMP and JASPAR were used to predicted the transcription factors that can bind to the lncRNA DLEU2 promoter region.The plasmid pc DNA3.1+SP1 and p GL3.0+DLEU2 were constructed,and the dual luciferase experiment was performed to detect SP1 binding to lncRNA DLEU2 promoter binding region.q RT-PCR was used to verify SP1 promoting the expression of lncRNA DLEU2.Results: The 1000 bp of upstream in lncRNA DLEU2 promoter region was downloaded from UCSC and NCBI.Through analyzing the transcription factor that could bind to lncRNA DLEU2 promoter region by PROMO and JASPAR,we found that there were multiple binding sites between SP1 and lncRNA DLEU2.The results of dual luciferase experiments showed that transcription factor SP1 can bind to the lncRNA DLEU2 promoter region.The result of q RT-PCR indicated that SP1 could promote the expression of lncRNA DLEU2 by transcriptionally activate lncRNA DLEU2.Conclusion: The transcription factor SP1 can activate the expression of lncRNA DLEU2 through binding to linc RNA DLEU2 promoter region.