Long Noncoding RNA GCAT1 Regulates The Proliferation And Invasion Of Glandular Cystitis Through MiR-155-5p/PTEN

BackgroundGlandular Cystitis(GC)is an inflammatory disease of the bladder which is easy to relapse,and its pathological manifestations are hyperplasia and metaplasia of transitional epithelium.Cystitis glandularis was once thought to have a low incidence,but with the popularity of cystoscopy and the increase in the rate of pathological biopsies,the incidence of cystitis glandularis continues to increase.There is no clear causal relationship between cystitis glandularis and bladder cancer,but some cystitis glandularis has a tendency to become cancerous.Its regulatory mechanism is not clear.Long noncoding RNAs(Lnc RNAs)are RNAs less than 200 nt that do not encode proteins and have diverse biological functions.In our study,it was found that Lnc RNA GCAT1 may be involved in the occurrence and development of cystitis glandularis.Part I: Low GCAT1 expression may be a risk factor for recurrence of cystitis glandularisPurposes:Exploring the role of GCAT1 in recurrence of GCMethods:A retrospective study of 190 patients from our center was conducted,and univariate and multivariate logistic regression analysis was used to explore the risk factors for GC recurrence.Differentially expressed genes were screened out by sequencing in 6 subjects(3 GC patients and 3 control subjects).Differentially expressed genes were analyzed using KEGG and GO,while GCAT1 was confirmed as a target gene.Validation sequencing was performed on 10 patients,and Fisher’s test was used to determine the effect of low expression of GCAT1 on recurrence of GC.Results: Retrospective analysis found urinary tract infection(OR,5.19;95% CI,1.32-20.44),long-term indwelling catheter(OR,5.98;95%CI,1.52-23.59),urinary tract stones(OR,5.38;95 %)CI,1.66-17.51),epithelial metaplasia(OR,5.11;95% CI,1.23-21.24),and atypical hyperplasia(OR,5.10;95% CI,1.72-15.10)were independent risk factors for GC recurrence.The recurrence rate of GC patients with low GCAT1 expression was 60%(3 cases/5 cases),which was higher than that of GC patients with high GCAT1 expression(0 case/5 cases),but there was no statistical difference by Fisher test(P =0.167).Conclusion: Low GCAT1 expression may predict the recurrence of GC.Part II: The role of GCAT1 in cystitis glandularis.Purposes: Explore the function of GCAT1 in GC.Methods:The expression of GCAT1 in GC tissues and normal tissues was detected by q PCR.GC primary cells were isolated and cultured by using the collected GC surgical specimens.Cell models of GCAT1 overexpression and knockdown was constructed using plasmids.The CCK-8 kit was used to detect the cell proliferation ability of the cell model,and the Transwell chamber was used to detect the cell invasion ability.The secretion and expression of TNF-α and IL-1β were detected by ELISA.Result:Compared with normal bladder tissue,the expression of GCAT1 was significantly decreased in GC lesion tissue(P<0.01).The results of CCK-8 assay showed that compared with the negative control group,the cell viability of the GCAT1-high expression line was decreased,while the cell viability of the GCAT1-silenced line was increased(P <0.01).The results of cell invasion assay showed that the cell migration ability of GCAT1-overexpressing strain was weakened,while that of GCAT1-silenced strain was enhanced.The expression of TNF-α and IL-1β increased in GCAT1-overexpressing cells,and decreased in GCAT1-silenced cells.Conclusion: GCAT1 is down-regulated in GC.GCAT1 can inhibit the proliferation of GC and inhibit the expression of inflammation factors.Part III: The mechanism of GCAT1 in cystitis glandularis.Purposes: Explore the mechanism of GCAT1 in GC.Methods:Prediction of GCAT1 targeted miRNAs and genes using bioinformatics methods.The expressions of PTEN and miR-155-5p in GC tissues and normal tissues were detected by q PCR.q RT-PCR and Western blot were used to detect the expression of miR-155-5p and PTEN in the pathological tissues of normal people and GC patients.q RT-PCR and Western blot were used to detect the expression of miR-155-5p and PTEN in the primary GC cell models.CCK-8 kits and Transwell chambers were used to detect cell viability and proliferation.The secretion and expression of TNF-α and IL-1β were detected by ELISA.The binding sites of miR-155-5p to GCAT1 and PTEN were verified using luciferase experiments.Result:Binding site prediction analysis showed that GCAT1 and PTEN have the same sequence,there may be a binding site between miR-155-5p and GCAT1,and miR-155-5p and PTEN have the same binding site.In the luciferase assay,wild-type GCAT1 and PTEN bind more strongly to miR-155-5P than the mutants after removing the corresponding sequences.q RT-PCR and Western blot showed that PTEN expression was positively correlated with GCAT1 and negatively correlated with miR-155-5p.Inhibition of miR-155-5p or PTEN,GCAT1 cannot play the function of inhibiting cell proliferation and cell viability,nor can it inhibit the secretion of inflammatory factors.After inhibiting PTEN,the inhibition of miR-155-5p could not enhance effect of cell proliferation and cell viability,nor inhibit the secretion of inflammatory factors.Conclusion: Our results demonstrate that GCAT1 inhibits the proliferation and invasion of glandular cystitis.In addition,GCAT1 is shown to achieve this regulation by targeting the miR-155-5p/PTEN axis.