| Objective: Uterine corpus endometrial carcinoma(UCEC)is a common gynecological malignant tumor.In recent years,the incidence of UCEC is on the rise and displays a younger trend worldwide,which seriously affects women’s health.Therefore,further analysis of the molecular mechanism of endometrial carcinoma formation and development,and the search for appropriate diagnostic markers and therapeutic targets are of great significance.With the development of high-throughput sequencing technologies,the functions of noncoding RNAs have been gradually revealed.Although they do not encode proteins,they play important roles in transcription,post-transcriptional and post-translational regulation of coding genes.Natural antisense transcripts(NATs),an important member of the noncoding RNA family,are RNA molecules transcribed by antisense strands and completely or partially overlapped with sense strand genes.Recent studies have shown that NATs can be abnormally expressed in a variety of tumors,usually affecting sense strand genes in cis or regulating the expression of downstream target genes in trans,and regulating the tumorigenesis and development of tumors.Recent studies have shown that NATs are abnormally expressed and play an important role in endometrial carcinoma.However,the current research on NATs in endometrial carcinoma is still in budding stage,and a large number of studies are still needed to confirm the relationship between NATs and the formation and development of endometrial carcinoma and to clarify the mechanism.In this study,we screened the transcriptome data of endometrial carcinoma tissues and paracancer tissues from TCGA database for bioinformatics analysis.Combined with literature review and functional experiments,we finally choosed SOX9-AS1.SOX9-AS1 is abnormally high expressed in endometrial carcinoma.Here,we will focus on exploring the role and mechanism of SOX9-AS1 in the proliferation,apoptosis,migration and invasion of endometrial carcinoma.Methods: 1.The expression of SOX9-AS1 and SOX9 in endometrial carcinoma tissues was analyzed by TCGA database.ENCODE database was used to analyze histone enrichment in SOX promoter region.2.The subcellular localization of SOX9-AS1 was determined by nucleo-cytoplasmic separation experiment.3.Cell culture and transfection:ISHIKAWA cells with low SOA9-AS1 expression were stably transfected with SOX9-AS1 plasmid,and SOX9-AS1 overexpression cell line was constructed.The HEC1 B cells with high SOX9-AS1 expression were transfected with antisense oligonucleotide(ASO)and SOX9-AS1 lowexpression was constructed.HEC1 B cells were stably transfected with SOX9 plasmid to construct SOX9 overexpression cell line.HEC1 B cells were transfected with si-SOX9 to construct SOX9 transient low expression cell lines.4.Cell proliferation capacity was measured in the SOX9-AS1 high and transient low expression cell lines and SOX9 transient low expression cell lines and their control groups by CCK8 method,and the effects of SOX9-AS1 and SOX9 on the proliferation capacity of endometrial carcinoma cells were analyzed.Annexin V-FITC/PI was used to stain SOX9-AS1 high and transient low expression cell lines and SOX9 transient low expression cell lines and their control groups.Flow cytometry was used to analyze the effects of SOX9-AS1 and SOX9 on apoptosis of endometrial carcinoma cells.6.Scratch test: The above SOX9-AS1 high and transient low expression cell lines and SOX9 transient low expression cell lines and their control groups were tested for scratch test,and the effect of SOX9-AS1 and SOX9 on the migration ability of endometrial carcinoma cells was analyzed by measuring the wound healing rate.7.Transwell experiment: The effects of SOX9-AS1 and SOX9 on the invasion ability of endometrial carcinoma cells were determined by Transwell experiment in the above SOX9-AS1 high and transient low expression cell lines and SOX9 transient low expression cell lines and their control groups.8.Quantitative reverse transcription polymerase chain reaction(q RT-PCR): SOX9-AS1 and SOX9 m RNA expression in 10 pairs of endometrial carcinoma tissues and normal endometrial tissues were detected.After silencing SOX9-AS1,expression of SOX9-AS1 and SOX9 was detected.After overexpressing SOX9-AS1,expression of SOX9-AS1 and SOX9 was detected.After silencing WDR5,m RNA expressions of WDR5 and SOX9 were detected.9.Western blot:After silencing SOX9-AS1,SOX9 protein expression was detected.After silencing WDR5,the protein expression of WDR5 and SOX9 was detected.10.RNA immunodeposition(RIP): RIP experiment was used to detect the direct physical binding between SOX9-AS1 and WDR5.11.Chromatin immunodeposition(Ch IP): The enrichment of H3K4me3 in SOX9 promoter region was detected by Ch IP assay.After silencing SOX9-AS1,the enrichment of H3K4me3 in SOX9 promoter region was detected.12.Tumorigenesis experiment in nude mice: HEC1 B cells were used to complete tumorigenesis in nude mice.After obvious tumor formation,the nude mice were randomly divided into two groups;Intratumoral injection was performed using in Vivo ASO-SOX9-AS1 and ASO-NC.To investigate the effect of SOX9-AS1 on the volume and quality of endometrial carcinoma cells in vivo.Results: 1.Transcriptome analysis of endometrial carcinoma tissues in TCGA database showed that SOX9-AS1 and SOX9 were significantly overexpressed(P<0.001),and the expression of SOX9 was highly positively correlated with SOX9-AS1.Ten pairs of endometrial carcinoma tissues and normal endometrial tissues were detected by q RT-PCR,and the results showed that the expressions of SOX9-AS1 and SOX9 in endometrial carcinoma tissues were abnormally increased compared with normal endometrial tissues,and their expressions were highly positively correlated.2.Cytoplasmic separation experiments showed that SOX9-AS1 was mainly located in the nuclei of endometrial carcinoma cells HEC1 B and ISHIKAWA.3.Functional experiments: Cell proliferation was significantly reduced in SOX9-AS1 and SOX9 transient low expression HEC1 B cell lines(P < 0.001);Apoptosis(including early and late apoptosis)was significantly increased(P<0.001);Cell migration decreased significantly(P<0.01);Cell invasion ability was significantly decreased(P<0.001).There was no difference in proliferation,apoptosis,migration and invasion between so X9-AS1 overexpressed cell lines and control groups.4.SOX9-AS1 exerts its biological roles by regulating SOX9: CCK8 showed that silencing SOX9-AS1 expression could significantly reverse the enhanced effect of increased SOX9 expression on cell proliferation(P<0.01);Apoptosis assay showed that silencing SOX9-AS1 expression could significantly reverse the inhibitory effect of increased SOX9 expression on apoptosis(P<0.05);Scratch assay showed that silencing SOX9-AS1 expression could significantly reverse the promoting effect of increased SOX9 expression on cell migration(P<0.01);Transwell experiment showed that silencing SOX9-AS1 expression could significantly reverse the promoting effect of increased SOX9 expression on cell invasion(P<0.01).5.q RT-PCR and Western Blot results showed that the expression of SOX9 m RNA and protein was significantly decreased after silencing the expression of SOX9-AS1(P<0.001);After overexpression of SOX9-AS1,the m RNA expression of SOX9 did not change significantly.After silencing WDR5 expression,SOX9 m RNA and protein expression were significantly decreased(P<0.001).6.Chromatin immunodeposition(Ch IP)assay showed that H3K4me3 was highly enriched in SOX9 promoter region in HEC1 B cells.After silencing SOX9-AS1,H3K4me3 enrichment level in SOX9 promoter decreased.7.RNA immunodeposition(RIP)showed that SOX9-AS1 was highly enriched on WDR5(P<0.001).8.Results of tumor formation experiment in nude mice: Compared with the control group,after ASO-SOX9-AS1 silencing SOX9-AS1 expression,tumor volume and mass in the experimental group were significantly reduced(P<0.01).Conclusion: 1.The expression level of SOX9-AS1 in endometrial carcinoma is significantly higher than that in normal endometrial tissue.2.SOX9-AS1 can promote the proliferation,migration and invasion of endometrial carcinoma cells and inhibit cell apoptosis.SOX9-AS1 can promote the tumorigenesis of endometrial carcinoma cells in vivo.3.SOX9-AS1 exerts its biological roles by regulating SOX9.4.The abberantexpressed SOX9-AS1 in endometrial carcinoma can enhance H3K4me3 modification in the SOX9 promoter region by binding to WDR5,activating the expression of SOX9,and promoting the occurrence and development of endometrial carcinoma. |
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