Enhancer RNA–eP2RY2 Induced By Estrogen Promotes Malignant Behaviors Of Bladder Cancer

Purpose: Enhancers are transcriptional regulatory elements that increase target gene expression.It has reported that enhancers could transcribe into functional RNAs with stimulation,that are enhancer RNAs(e RNAs).Increasing evidence showed e RNAs participated in various disease processes including malignant tumors.The purinergic receptor P2Y2(P2RY2)is a typical representative of G-protein coupled purinergic receptors and is widely distributed in normal tissues.There are gender differences in bladder cancer,indicating that estrogen plays an important role in bladder cancer.The P2RY2 enhancer RNA(e P2RY2)is an estrogen-responsive e RNA.This study focused on the relationship between e P2RY2 and estrogen and its role in bladder cancer.Methods: Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the relative expression level of e P2RY2 in bladder cancer tissues and corresponding adjacent tissues.The production of e P2RY2 in bladder cancer cells was induced by estrogen and the change of its expression was detected by RT-q PCR.The small interfering RNA and clustered regular interspaced short palindromic repeats /CRISPR associated 13a(CRISPR/Cas13a)system were used to knock down e P2RY2 of bladder cancer cells and RT-q PCR was used to compare the knockdown efficiency of the two.The proliferation of bladder cancer cells was examined by EDU kit and CCK-8kit.The invasion of bladder cancer cells was detected by transwell assay,and the migration of bladder cancer cells was detected by wound-healing assay.The apoptosis of bladder cancer cells was detected by the caspase 3 enzyme-linked immunosorbent assay assay and flow cytometry.All data are expressed as mean ± standard deviation and all statistical analyses were performed by SPSS 17.Results: This study found that e P2RY2 was up-regulated in bladder cancer tissues compared with adjacent tissues,and the high expression of e P2RY2 was associated with histological grade,TNM stage and depth of invasion of patients with bladder cancer.In bladder cancer cells,the knockdown efficiency of e P2RY2 by CRISPR-Cas13 a system was significantly higher than that of small interfering RNA,and the expression of e P2RY2 in bladder cancer cells increased after estrogen treatment.In addition,estrogen promoted the proliferation,invasion and migration of bladder cancer cells,inhibited the apoptosis of bladder cancer cells.Moreover,after knocking down e P2RY2 with CRISPR-Cas13 a system,the proliferation,invasion and migration of bladder cancer cells were inhibited,and the apoptosis of bladder cancer cells increased.More importantly,this study demonstrated that down-regulation of e P2RY2 impaired the promotion effect of estrogen on the malignant potential of bladder cancer cells.Conclusion: In summary,estrogen and e P2RY2 play a role in facilitating bladder cancer,and estrogen may promote the progression of bladder cancer by inducing the production of e P2RY2.In addition,the CRISPR-Cas13 a system is more applicable for the knockdown study of e P2RY2 in tumor field than the small interfering RNA.This article presents a new perspective on the mechanism of action of estrogen in bladder cancer,and clarifies the molecular pathways that contribute to gender differences in bladder cancer,which provide a new target for early prevention and treatment of bladder cancer.