Study On The Function And Mechanism Of Long Non-coding RNA STYK1-2 In The Development Of Bladder Cancer

Background Bladder cancer(BC)is one of the most prevalent malignances in the urinary system,with an increasing incidence year by year.Current studies believe that the occurrence and development of BC are related to environmental and epigenetic factors,and the pathophysiological mechanism is not fully clear yet.In-depth exploration of the mechanism of occurrence and development of BC is conducive to promoting the progress of BC diagnosis,treatment and long-term monitoring,thereby improving the prognosis of BC patients.Long non-coding RNA(lnc RNA)is a molecule that is more than 200 nucleotides in length and generally does not encode protein.With the continuous progress of sequencing technology and the deepening of research in recent years,researchers have found that the abnormal expression level of lnc RNA may be closely related to the canceration of normal cells and the proliferation,progression,drug resistance of cancer cells.In the early stage,our research group used high-throughput sequencing to screen out lnc RNA STYK1-2 that was significantly down-regulated in BC tissues.MTS experiments and Transwell experiments found that inhibiting the expression of lnc-STYK1-2 can promote the proliferation,migration and invasion of bladder cancer cells.This study further explored the role of lnc-STYK1-2 in the development of BC and related molecular regulation mechanisms,and provided a theoretical basis for finding possible early diagnosis and prognosis markers and potential therapeutic targets for BC.Methods1.Sh-lnc-STYK1-2 lentivirus was successfully constructed and transfected to BC cell lines 5637 and T24,lnc-STYK1-2 expression levels were detected by q RT-PCR;2.Stably transfected cell lines were used to perform subcutaneous tumor formation in nude mice;3.High-throughput sequencing and bioinformatics analysis were adopted to detect the changes of RNA expression when knock down lnc-STYK1-2;4.To explore the relationships between lnc-STYK1-2 and miR-146b-5p,ITGA2 and miR-146b-5p,dual-luciferase reporter genes assays were performed;5.The rescue experiment was used to confirm that lnc-STYK1-2 regulates cell proliferation,migration and invasion by targeting miR-146b-5p in BC cell lines;(1)miR-146b-5p inhibitors were transfected to lnc-STYK1-2 knockdown cells;(2)CCK-8 assay was applied to detect cell growth curve;(3)Transwell assay was used to analyze cell migration and invasion abilities;6.ITGA2 level and proteins related to Akt/ NF-κB/STAT3 pathway were examined by Western blot assay;7.Using statistical methods to analyze the results above.Results1.lnc-STYK1-2 was significantly downregulated compared with sh-NC,stably transfected 5637-sh-NC,5637-sh-lnc-STYK1-2,T24-sh-NC,T24-sh-lnc-STYK1-2cell lines were successfully constructed;2.Knock down of lnc-STYK1-2 can enhance the growth of subcutaneous tumor formation in nude mice;3.High-throughput sequencing revealed that there were 126 differentially expressed m RNAs,with 17 up-regulated m RNAs and 109 down-regulated m RNAs;KEGG pathway analysis showed that dysregulated genes were enriched on the pathways in cancer and Akt/ NF-κB/STAT3 pathway when knock down lnc-STYK1-2;q RT-PCR experiment suggested that lnc-STYK1-2-miR-146b-5p-ITGA2 may have an interaction;4.Dual-luciferase reporter genes assays showed that miR-146b-5p directly bound to lnc-STYK1-2,and ITGA2 directly bound to miR-146b-5p;5.lnc-STYK1-2 can promote the proliferation,migration and invasion abilities in BC cell lines by sponging miR-146b-5p during the rescue experiment;6.ITGA2,Akt,STST3,p65 expression were decreased and p-Akt,p-STAT3,p-p65 expression were increased when knock down lnc-STYK1-2;while ITGA2,Akt,STST3,p65 expression were increased and p-Akt,p-STAT3,p-p65 expression were decreased when knock down lnc-STYK1-2 with miR-146b-5p depletion.Conclusions1.Decreased lnc-STYK1-2 can promote the growth of BC cells;2.lnc-STYK1-2 and miR-146b-5p,ITGA2 and miR-146b-5p had a competitive binding relationship with each other;3.lnc-STYK1-2 can promote the proliferation,migration and invasion abilities in BC by regulating miR-146b-5p/ITGA2 directly,and simultaneously regulating Akt/ NF-κB/STAT3 pathway indirectly.