Mechanism Exploration Of Hac-circ-0005777 Sponge Adsorbing MiR-1305 In Bladder Cancer Promoting Disease Progression

Objective:In this study,the clinical samples of patients with bladder cancer(BC)were collected to screen for differentially expressed molecules.Through the experiments in vitro and in vivo,we studied the Mechanism exploration of Hac-circ-0005777 sponge adsorbing miR-1305 in bladder cancer promoting disease.We aimed to eProvide experimental basis for discovering molecular markers for diagnosis,treatment and prognosis of bladder cancer.Methods:1.RT-PCR was performed to screen for the differentially expressed CircRNA in 47 cases of BC,and the expression levels in clinical staging and grading of BC were analyzed;2.Agarose gel experiment and RNaseR experiment confirmed that the differentially expressed Hac-circ-0005777 was CircRNA;3.Verified the sequence identity of Hac-circ-0005777 by sequencing and website prediction;4.RT-PCR was used to verify the efficiency of Hac-circ-0005777 interference and overexpression.CCK8 assay was used to detect cell viability,cloning assay was used to detect the ability of cloning,wound healing teat and metastasis test were used to verify bladder cancer cell metastasis.In vivo experiments verified the turnorigenicity of Hac-circ-0005777;5.The localization of Hac-circ-0005777 in the cells was determined by nuclear plasma separation and FISH fluorescence experimen;6..Circ Bank,Circ MIR,Circ Interaome prediction and RNA Pudown experiment screened the miRNA with the highest binding possibility of Hac-circ-0005777;7.MiRDB,Targescan and other websites predicted downstream binding molecules,and heat maps suggested binding downstream molecules;8.Protein expression was determined by Western Blot.Results:1.RT-PCR suggested that:Hac-circ-0005777 is low expressed in the adjacent tissues,highly expression in cancer tissues,highly expressed in Ta/Tis/T1-2 stage,and low stage,with statistically significant differences(P<0.05);2.Agarose gel electrophoresis and RNaseR experiment confirmed that:Hac-circ-0005777 was CircRNA,sequencing and website prediction Hac-circ-0005777 sequence was the same;3.In vitro experiments confirmed that Hac-circ-0005777 Si and miR1305 inhibited the development of BC,Hac-circ-0005777 promoted the development of BC.Animal experiments showed that Hac-circ-0005777 promoted the formation of BC and the difference was statistically significant(P<0.05);4.Nuclear plasma separation and FISH fluorescence experiment suggested that Hac-circ-0005777 was located in the cytoplasm.5.Circ Bank,Circ MIR,Circ Interaome prediction and RNA Pudown test screening were the most likely to bind Hac-circ-0005777 to miRNA1305.6.Prediction and heat map of miRDB,Targescan and other websites suggested that the downstream molecule of hac-circ-0005777 is TGF-?2;7.Western Blot showed that Hac-circ-0005777 up-regulated the expression of TGF-?2 while miRNA1305 down-regulated that.Conclusions:1.Hac-circ-0005777 up-regulateed TGF-?2 through sponge adsorption of miRNA1305 and associated with cancer progression.2.Hac-circ-0005777 promoted the occurrence and development of BC,miRNA 1305 inhibited the progression of bladder cancer;3.Hac-circ-0005777 up-regulateed the expression of TGF-? 2,miRNA1305 down-regulated that;4.Hac-circ-0005777 was lowly expressed adjacent to cancer,highly expressed in cancer,and highly expressed in lower grade and stage;5.Hac-circ-0005777 was a CircRNA,located in the cytoplasm.