Circ0054123/miR147a/MCM3 Pathway Regulates Biological Characteristics Of Bladder Cancer Cells

Introduction:Bladder cancer is a malignant tumor that occurs on the bladder mucosa.It is one of the common malignant tumors of urinary system and one of the ten common tumors of the whole body.At present,many breakthroughs have been made in the study of the mechanism and characteristics of bladder cancer.However,the research on the treatment of bladder cancer is still limited to surgery and chemotherapy.There are few studies on using biological characteristics of bladder cancer,such as proliferation and metastasis,to inhibit and treat bladder cancer.In recent studies,there is more and more evidence that MCM family plays an important role in the occurrence and development of tumors.It has been reported that Mcm3 gene is over expressed in most human cancers?such as colon cancer,lung cancer,gastric cancer,renal cancer and breast cancer?.Plus,Mcm3 is considered to be a key factor in cell division.Circular RNA can regulate genes by acting as a micro RNA sponge,thus reducing its ability to target m RNA.However,in bladder cancer,this integrated regulatory pathway is rarely studied.Therefore,this paper aims to explore the influence of circ RNA / mi RNA / Mcm3 pathway on the biological characteristics of bladder cancer cells and its detailed mechanism,to find key molecular markers,and to provide new ideas for the clinical treatment of bladder cancer.Methods : 1.Screening and detection of MCM3 upstream micro RNA Through the analysis of targetscan,mi RBase,mirwalk and other bioinformatics websites,mi RNAs with complementary bases and highly conserved 3 '-UTR region of Mcm3 m RNA were selected,and mi RNAs with low expression abundance and not studied in bladder cancer were selected.30 pairs of bladder cancer cases with clear pathological diagnosis were randomly selected from the specimen bank to extract the total RNA and protein from cancer and adjacent tissues.Cultivate 4 bladder cancer cell lines and epithelial immortalized cell lines,and extract their total RNA and protein,The expression of Mcm3 m RNA and mi RNA was detected by QRT PCR,and the expression of Mcm3 protein was detected by Western blot.To analyze the correlation between Mcm3 and mi RNA expression in cells.Two bladder cancer cell lines were selected for the follow-up study.2.Clarify the regulatory mechanism of mi RNA on Mcm3 and its biological function3.in bladder cancer cell line.The expression of Mcm3 m RNA and protein in bladder cancer cell lines were detected after transfection of mi RNA mimics / inhibitor.According to the prediction sites of bioinformatics,the wild-type and mutant double luciferase reporter gene plasmids were constructed to determine the specific binding of mi RNA to Mcm3 m RNA 3 '-UTR region.The mimetics / inhibitors of mi RNA transfected into bladder cancer cell lines and their corresponding negative controls,RTCA and Ed U experiments were used to detect the proliferation of cells after transfection;Transwell experiment was used to detect the invasion and metastasis of cells;recovery experiment was used to detect the regulation of mi RNA on Mcm3 and the effect of mi RNA on the function of bladder cancer cells through Mcm3.4.Screening and detection of MCM3 upstream micro RNA Based on the prediction of bioinformatics websites such as RNAhybrid,we selected the circ RNA which has complementary bases with the target mi RNA and has not been studied.By using the double luciferase reporter gene experiment,RNA pulldown test and RNA immunoprecipitation test,it is verified that the predicted circ RNA has the same target and can be combined with the mi RNA studied.5.The biological function of circrna in bladder cancer and the regulation of m RNA by mi RNA Circ RNA was silenced in bladder cancer cell lines,Cell proliferation was detected by RTCA and edu,and invasion and metastasis were detected by Transwell.QRT PCR and Western blot were used to detect the effect of silencing circrna on the expression of Mcm3.The regulation of mi RNA by circrna,the expression of Mcm3 by circrna and the effect on the function of bladder cancer cells were detected by the recovery experiment.6.Tumor formation in nude mice 4-6 weeks old,BALB / C nude mice were injected subcutaneously with umuc3 cells to observe the growth of tumor and compare the growth of different groups.The transplanted tumor was immunohistochemistry,The changes of Ki-67 and Mcm3 were detected to determine the effect of circrna and Mcm3 on tumor proliferation.Results: 1.Compared with the corresponding epithelial immortalized cells and paracancerous tissues,Mcm3 was highly expressed in four bladder cancer cell lines and bladder cancer tissues.Through bioinformatics analysis,prediction and double luciferase reporter gene experiment,it was found that mi R147 a specifically combined with 3 '-UTR region of Mcm3 m RNA and negatively regulated the expression level of Mcm3 m RNA and protein.The expression of mi R147 a in bladder cancer tissue and the two selected bladder cancer cell lines T24 and umuc3 was lower than that in the corresponding adjacent tissues and epithelial immortalized cells.2.In vitro,mi R147 a was found to have a negative effect on the proliferation,invasion and metastasis of bladder cancer cells.The effect of mi R147 a on the proliferation,invasion and metastasis of bladder cancer cells was achieved by targeting Mcm3.3.The analysis and prediction of bioinformatics method showed that has_circ₀054123 might be used as an endogenous competitive RNA to adsorb mi R147 a.Through the double luciferase reporter gene test,RNA pulldown test and RNA immunoprecipitation test,it is confirmed that has_circ₀054123 contains the same target as mi R147 a and can be combined with it.Endogenous inhibition of has_circ₀054123 can negatively regulate the increase of mi R147 a.4.Compared with the corresponding paracancerous tissues,has_circ₀054123 was highly expressed in bladder cancer tissues.It was found that has_circ₀054123 could inhibit the proliferation,invasion and metastasis of bladder cancer cells in vitro.This result was also confirmed in vivo experiments in nude mice.After the injection of long-acting chemical modified CIRC-Si,the tumorigenic quality was significantly reduced compared with the control group.5.Endogenous silencing of has_circ₀054123 can positively regulate and down regulate the expression of Mcm3,and the effect of has_circ₀054123 on the regulation of Mcm3 is realized by competitive combination of mi R147 a.