Study On The Role And Molecular Mechanism Of Long Noncoding RNA AC114812.8 In Bladder Urothelial Carcinom

Object:Bladder cancer is the ninth most common cancer in the world,with an annual incidence of about 430,000 cases.It has a high mortality rate and complicated pathogenesis without ideal treatment currently.Patients with high-grade muscular invasive bladder cancer can only be forced to undergo radical total cystectomy with adjuvant radiotherapy and chemotherapy.But these treatments significantly reduce the patient’s quality of life and survival time after surgery,and bring heavily physical and psychological burden to the patients.Therefore,we must continue to study the mechanism of bladder cancer in order to develop more effectively treatments of bladder cancer.Long non-codingRNAs(lncRNA s)areRNA molecules longer than 200 bp,which play an important role in the regulation of transcription.Our team has previously identified a series of lnc NRAs that are abnormally expressed in bladder cancer tissues by high-throughput sequencing.Among them,AC114812.8,an lncRNA that is significantly overexpressed in bladder cancer has attracted our attention.The purpose of this study is to reveal the function and mechanism of AC114812.8 in the bladder cancer,and help to develop promising target for the diagnosis and therapy of bladder cancer.Methods:First,we explored the expression level of AC114812.8 in bladder cancer tissues and cells through PCR experiments,and confirmed its subcellular localization through nuclear separation experiments and FISH experiments.Second,we investigated its effect on bladder cancer cell function by knocking down and increasing the expression of AC114812.8.We used the CCK-8 assay and clone formation assay to detect cell proliferation ability.We used the Transwell chamber assay and wound healing assay to detect cell migration ability.Meanwhile,we used the Matrigel chamber experiment to detect cell invasion ability.At the same time,we investigated the effects of AC114812.8on bladder cancer in vivo through animal experiments.Third,we predicted the miRNA downstream of AC114812.8 through bioinformatics analysis and confirmed it by pull down assay and luciferase reporter assay.Then,we predicted the target genes regulated downstream by bioinformatics analysis,and verified them by Western Blot experiments and luciferase reporter assay.Results:Using q RT-PCR,we found that the expression level of AC114812.8 in bladder cancer was significantly increased.Nuclear-cytoplasm separation experiments and FISH experiments showed that AC114812.8 was mainly localized in the cytoplasm.The results of the clone formation assay and the CCK-8 assay showed that the proliferation of bladder cancer cells was significantly weakened after AC114812.8 silencing.On the contrary,overexpression of AC114812.8 significantly promoted the proliferation of bladder cancer cells.Using Transwell chamber assay,wound healing assay,and Matrigel chamber assay,we found that the silencing of AC114812.8 can inhibit the migration and invasion ability of bladder cancer cells.Meanwhile,the migration and invasion ability of bladder cancer cells were significantly enhanced after overexpression of AC114812.8.After revealing the function of AC114812.8 in bladder cancer cells through in vitro experiments,we further explored whether it can effectively regulate bladder cancer in vivo.We found that the tumor volume and weight of the AC114812.8 overexpression group were significantly higher than control group.In addition,we used bioinformatics analysis to predict the miRNAs which AC114812.8can bind to,and found that mi R-371b-5p may be the candidate miRNA.Furthermore,the results of luciferase reporter assay andRNA pull down assay also demonstrated the direct binding between AC114812.8 and mi R-371b-5p.According to the predictions of mi Randa and Target Scan,we predicted the potential downstream target genes of mi R-371b-5p,and we found that mi R-371b-5p may bind to the 3′UTR region of FUT4.Therefore,we performed a luciferase reporter assay,and the results showed that mi R-371b-5p can bind to the FUT4 3′UTR region to function.To verify our results,we conducted a rescue experiment.The results show that mi R-371b-5p can rescue the enhancement effect of AC114812.8 on FUT4 expression level and bladder cancer cell proliferation and migration.Finally,using Western Blot,we analyzed the effect of AC114812.8 on the expression of related genes in EMT process in bladder cancer.The results showed that the expression levels of β-catenin,Slug,N-cadherin and Vimentin in the AC114812.8 overexpression group were significantly increased,indicating that AC114812.8 promoted the EMT process in bladder cancer.Conclusion:Our research shows that AC114812.8 can act as a mi R-371b-5p sponge to inhibit the silencing effect of mi R-371b-5p on the target gene FUT4,thereby regulating FUT4 to promote the progress of bladder cancer.AC114812.8 may be a potential target for the diagnosis and therapy of bladder cancer.