The Roles Of Long Non-coding RNA EIF1AX-AS1 In Human Endometrial Carcinoma Cells

BackgroundEndometrial cancer kills approximately 76,000 women worldwide each year,and increasing disease mortality and newly diagnosed cases make endometrial cancer a significant factor affecting women’s health.Therefore,it is urgent and important to study the specific mechanisms of endometrial cancer development and to identify markers for early diagnosis of endometrial cancer.Studies have shown that long-stranded non-coding RNA(lnc RNA)is not transcriptional garbage,but plays an important role in various biological processes such as cell growth,differentiation,apoptosis and migration.lnc RNA can affect the encoding and expression of tumor-related genes through various pathways such as transcription and translation,and also plays an important role in the development of tumor invasion and metastasis.However,there are many lnc RNAs in endometrial cancer whose roles and mechanisms are still not elucidated.Therefore,in this study,we propose to screen,identify and analyze the differentially expressed lnc RNAs in endometrial cancer cells by transcriptome sequencing to explore their biological functions and mechanisms in endometrial cancer cells and provide new ideas for the treatment and prevention of endometrial cancer.Purpose1.To screen and identify differentially expressed lnc RNAs in endometrial cancer cells,and clarify the full length of EIF1AX-AS1 and its location and expression in endometrial cancer cell lines.2.To clarify the biological function of EIF1AX-AS1 in endometrial cancer cells.3.To identify the downstream target genes of EIF1AX-AS1 and reveal the mechanism of the biological functions of the cells in endometrial cancer cells.Content1.To screen and verify the differentially expressed lnc RNA in endometrial cancer cells.(1)Through transcriptome sequencing technology to select the lnc RNA that are differentially expressed in normal endometrial and endometrial cancer cells(2)Using RT-q PCR technology to verify the transcriptome sequencing results.2.The full length of EIF1AX-AS1 and its localization and expression in endometrial cancer cell lines.(1)To explore the localization of EIF1AX-AS1 in endometrial cancer cells by FISH technique.(2)Using RACE technology to define its full length.(3)Combined with the technology of plasmid construction,to study whether it expresses protein or not.3.To investigate the effects of silencing and overexpression of EIF1AX-AS1 on the function of endometrial cancer cells.(1)To study the effect of knocking down the expression of EIF1AX-AS1 on cell function.(2)To study the effect of overexpression of EIF1AX-AS1 on cell function.4.To reveal the specific mechanism of EIF1AX-AS1 regulating target genes.(1)To study the effect of low or overexpression of EIF1AX-AS1 on the expression of EIF1 AX in endometrial cancer cells.(2)Verify whether EIF1AX-AS1 affects cell function through EIF1 AX.(3)Use bioinformatics to screen RNA binding proteins that interact with EIF1AX-AS1.And use RNA pull down technology to verify.(4)Analyze the results of RIP and RNA pull down,and study the relationship between EIF1AX-AS1,EIF1 AX and the RNA binding protein.5.To clarify the specific mechanism of the influence of EIF1AX-AS1 target genes on cell function.(1)Use protein immunoprecipitation(Co-IP)combined with mass spectrometry to analyze proteins that can interact with EIF1AX(2)Verify whether EIF1 AX binds to the protein and affects cell function.Method1.The differentially expressed lnc RNA and m RNAs,in high-grade and poorly differentiated endometrial carcinoma and paracancerous tissues were screened by full transcriptome sequencing,and the sequencing results were analyzed by differential analysis,GO enrichment analysis and KEGG pathway analysis.According to the multiple of difference and P value,the most meaningful lnc RNA was screened out.2.The location of EIF1AX-AS1 in endometrial cancer cell lines was determined by FISH,and the full length of EIF1AX-AS1 was determined by rapid amplification of c DNA ends.3.EIF1AX-AS1 si RNA or pc DNA3.1-EIF1AX-AS1,transfection was used to detect the effect of lnc RNA on proliferation and apoptosis of endometrial cancer cells by CCK-8,flow cytometry,JC-1,RT-q PCR and Western Blot techniques.4.To explore how EIF1AX-ASl regulates the expression of EIF1 AX by RNA pull down and RNA co-immunoprecipitation(RIP),and to find the RNA binding proteins and target genes that directly interact with EIF1AX-ASl.5.Through Co-IP combined with mass spectrometry analysis technology,the protein interacting with EIF1 AX was revealed.Result1.Through the analysis of transcriptome sequencing data and clinical samples,it was found that the expression of EIF1AX-ASl was down-regulated in endometrial carcinoma.2.In the sequencing results of endometrial carcinoma tissues,it was found that there was a significant negative correlation between the expression of EIF1 AX and the expression of EIF1AX-ASl.It is suggested that there may be an association between EIF1AX-ASl and EIF1 AX.3.The rapid amplification of DNA ends by(RACE)showed that EIF1AX-AS1 was a polyadenylate tail transcript composed of 1466 bases,which was not consistent with the sequence predicted by LNCipedia and other databases.The results of FISH that EIF1AX-AS1 was located in the cytoplasm.4.Cell function test showed that overexpression of EIF1AX-AS1 or knockdown of EIF1 AX expression could inhibit proliferation and promote apoptosis of endometrial carcinoma.In addition,the expression of EIF1AX-AS1 was silenced and the expression of EIF1 AX m RNA was increased,but the overexpression was on the contrary.The results of actinomycin D assay showed the same results.It is suggested that EIF1AX-AS1 can reduce the stability of EIF1 AX m RNA and promote its degradation.5.RNA pull down results showed that EIF1AX-AS1 combined with PCBP1,and RIP experiments showed that PCBP1 combined with EIF1AX-AS1 and EIF1 AX m RNA.Silencing the expression of PCBP1 can inhibit the degradation of EIF1 AX m RNA by EIF1AX-AS1,suggesting that PCBP1 is involved in the regulation of EIF1 AX m RNA stability by EIF1AX-AS1.6.The results of Co-IP combined with mass spectrometry showed that EIF1 XA interacted with YBX-1.EIF1 AX may regulate C-MYC by combining with YBX-1affecting apoptosis.ConclusionEIF1AX-AS1 is lowly expressed in endometrial cancer tissues;it can reduce EIF1 AX expression by interacting with PCBP1 and participating in EIF1 AX m RNA degradation.In turn,the reduced expression of EIF1 AX,which depends on its translation with YBX-1,decreases the expression of C-MYC,ultimately inhibiting the proliferation of endometrial cancer cell lines and promoting their apoptosis.