Long Noncoding RNA UCA1 Targets MiR-582-5p And Contributes To The Progression And Drug Resistance Of Bladder Cancer Cells Through ATG7-mediated Autophagy Inhibition

The incidence of bladder cancer is currently increasing every year.Researchers have conducted a lot of research to explore the inhibitory effect of targeted therapy on bladder cancer development.The purpose of this study was to investigate the long noncoding RNA(lncRNA)of urothelial carcinoma embryonic antigen 1 UCA1(Urothelial carcinoma associated 1)in bladder cancer,which targets miR-582-5p and downregulates ATG7 expression to regulate cancer cell autophagy.Inhibition of bladder cancer development and drug resistance.In this paper,the timing and quantitative reverse transcription polymerase chain reaction was used to detect the m RNA(Messenger RNA)expression level.The development and inhibition of cancer cells were determined by detecting the expression of the corresponding proteins,as well as the luciferase reporter gene assay and immunocytochemical assay.UCA1 expression is up-regulated in bladder cancer tissues and cells.Short hairpin RNA(Short hairpin RNA)removal of UCA1 inhibits cell proliferation,invasion,migration and drug resistance.Further research shows that UCA1 can directly interact with miR-582-5p,and there is a negative correlation between miR-582-5p and UCA1.In addition,we found that ATG7 is a target of miR-582-5p and that ATG7 expression can be down-regulated by miR-582-5p overexpression or reduction of UCA1.When studying the introduction of UCA1 shRNA,cancer cell autophagy was inhibited.In addition,in vivo experiments have further demonstrated the role of UCA1 in bladder cancer,including tumor growth,invasion and migration,and reducing UCA1 can inhibit the above activities.These results provide evidence for a novel UCA1 interaction regulatory network in bladder cancer,that is,UCA1 acts on miR-582-5p and then ATG7 expression,thereby affecting autophagy and drug resistance.Our research provides new evidence for the treatment of bladder cancer.Part 1: Study on the interaction between UCA1 and miR-582-5p in bladder cancer tissuesObjective:In order to study the role of UCA1 and miR-582-5p in the progression of bladder cancer,the expression levels of UCA1 and miR-582-5p in bladder cancer tissues and para-cancer normal tissues were analyzed,and whether there was a direct interaction between them was discussed.Method:Detection of UCA1 and miR-582-5p in human normal cells SV-HUC-1 and bladder cancer-derived cells HT-1376,T24,J82,5637 and EJ by quantitative PCR(qPCR),and analysis of UCA1 in cancer cells And miR-582-5p content;miR-582-5p expression levels in UCA1 shRNA-transfected T24 cells were measured by quantitative real-time polymerase chain reaction(q RT-PCR);)Or its mutant sequence(UCA1-mut)was subcloned into the p MIR luciferase reporter gene,then co-transfected with miR-582-5p-type controls into T24 cells,and observed the miRNA recognition sites of miR-582-5p and UCA1 Whether there is a connection between points.Result:Compared with paracancer normal tissues,UCA1 was highly expressed in bladder cancer tissues,while miR-582-5p was poorly expressed.In the same tissue cells,UCA1 and miR-582-5p were negatively correlated in bladder cancer.q RT-PCR results showed that UCA1 interacts with miR-582-5p and down-regulates its expression in T24 cells.The wild-type sequence of UCA1(uca1-wt)or its mutated sequence(uca1-mut)was subcloned into the p MIR luciferase reporter gene,and there was a direct interaction between miR-582-5p and the miRNA recognition site of UCA1.Conclusion:There was a negative correlation between UCA1 and miR-582-5p in bladder cancer cells and adjacent normal tissues,and there was a direct interaction between miR-582-5p and the miRNA recognition site of UCA1.Part 2 Effects of UCA1 and miR-582-5p on the growth and invasion of bladder cancer cellsObjective:To investigate the role of UCA1 and miR-582-5p in cancer cell tissues,and the effect on the growth,invasion and migration of cancer cells.Method:Human bladder cancer cell lines T24 and 5637 cells were divided into four groups: control group,UCA1 shRNA,miR-582-5p inhibitor and shRNA + inhibitor group.Use a pipette tip to scrape a monolayer of confluent cells into the middle of the well.After incubation,cell migration was captured with a DM2500 bright-field microscope(Lecka,Wetzlar,Germany),and the migration distance was measured by Image J software.The invasion ability of T24 and 5637 cells was performed using the Transwell invasion test.The effects of UCA1 and miR-582-5p on the growth and invasion of cancer cells were determined experimentally.Result:UCA1 shRNA and miR-582-5p inhibitors were performed in cell growth,invasion and migration analysis.The results showed that down-regulating UCA1 expression can significantly inhibit the growth of T24 and 5637 cells,while miR-582-5p inhibitors significantly promote T24 and The growth of 5637 cells and down-regulation of UCA1 expression significantly inhibited the invasion of T24 and5637 cells,while miR-582-5p inhibitor significantly promoted the invasion of T24 and 5637 cells.Wound healing results showed that down-regulation of UCA1 expression significantly inhibited Migration of T24 and 5637 cells,while miR-582-5p inhibitor significantly promoted migration of T24 and 5637 cells;a combination of UCA1 shRNA and miR-582-5p inhibitor compared to miR-582-5p inhibitor alone Shows reduced activity.Conclusion:Down-regulation of UCA1 expression and miR-582-5p can inhibit the growth,invasion and migration of cancer cells in cancer tissues.Compared with miR-582-5p inhibitors alone,UCA1 shRNA and miR-582-5p inhibit The combination of agents showed diminished activity,indicating that miR-582-5p and UCA1 expression have a negative correlation with each other,and can jointly regulate the growth,invasion and migration of cancer cells.Part 3 Effects of UCA1 and miR-582-5p on drug resistance in bladder cancer cellsObjective:To investigate the effects of UCA1 and miR-582-5p expression on drug resistance of cancer cells.The effect of down-regulating UCA1 expression on autophagy inhibition was measured by measuring the ratio of micro-related proteins and autophagy substrates,and the targeted autophagy gene ATG7 was determined by the relevant effects of UCA1 targeting miR-582-5p.Method:The protein immunotrace method was used to lyse cells with RIPA lysis buffer(Beyotime Biotechnology Institute).An equal amount of protein sample was separated using 10% SDS-PAGE and transferred to a PVDF membrane(Millipore,Billerica,MA,USA).After incubation with 5% skimmed milk in TBST,the membrane was incubated with rabbits against ATG7(ab133528),LC3 A / B(ab62721),P62(ab155686),and multidrug resistance protein 1(Abcam,Cambridge,MA,USA).Antibodies(Abcam,Cambridge,MA,USA)were incubated with MRP1,ab3368),lung resistance-related proteins(LRP,ab92544),GST(ab19256),and topoisomerase II(TOPOII,ab52934)overnight at 4 ° C.They were then incubated with HRP-conjugated secondary antibodies(ab6721)for 1.5 hours at room temperature.Finally,the blot was observed by electroluminescence(ECL)and detected using a Chemi Doc XRS imaging system.Human bladder cancer cell lines T24 and 5637 cells were divided into four groups: control group,UCA1 shRNA,miR-582-5p inhibitor and shRNA + inhibitor group.A single layer of confluent cells was scraped in the middle of the well with a pipette tip.UCA1 and miR-582-were detected in human normal cells SV-HUC-1 and bladder cancer-derived cells T24,5637 by quantitative PCR(qPCR).Quantitative expression of 5p,and the effects of UCA1 and miR-582-5p on autophagy were analyzed by measuring the ratio of microtubule-associated proteins(autophagy markers)and autophagy substrate levels.Mi R-582-5p was transfected with ATG7-wt Effects of Cells on Luciferase Activity.Result:Compared with the control group,the protein expressions of MRP1,LRP and GST were significantly reduced in the UCA1 shRNA group.But for TOPO-II,UCA1 shRNA has been shown to enhance its expression,while miR-582-5p inhibitors have shown the opposite effect.UCA1 shRNA can down-regulate the expression of MRP1,LRP,GST,and up-regulate the expression of TOPO-II.UCA1 shRNA transfection reduced the level of LC3-II.The reduced LC3-II caused a significant reduction in the LC3-II / LC3-I ratio.Since p62 is a substrate for autophagy degradation,the increase in p62 expression by UCA1 shRNA confirmed the down-regulation of UCA1 Expression inhibits cell autophagy;ATG7 is down-regulated by UCA1 shRNA and up-regulated by miR-582-5p inhibitors;miR-582-5p reduces luciferase activity in ATG7-wt transfected cells,but in ATG-No effect in mut transfected cells.Conclusion:Down-regulation of UCA1 expression can reduce drug resistance in bladder cancer cells,while miR-582-5p inhibitor can improve drug resistance in bladder cancer cells.Down-regulation of UCA1 expression can inhibit autophagy of T24 and5637 cells by targeting down-regulation of miR-582-5p.Mi R-582-5p directly targets and inhibits ATG7,and UCA1 regulates bladder cancer resistance by targeting the autophagy gene ATG7 in miR-582-5p.Part 4 Effect of autophagy on bladder cancer cell development and drug resistanceObjective:UCA1 targeting miR-582-5p inhibits ATG7’s ability to regulate autophagy,combines with the development of cancer cells and drug resistance to explore the correlation.Previous in vitro studies have progressed,and our in vivo studies validate the above conclusions.Method:Human bladder cancer cell lines T24 and 5637 cells were divided into four groups: control group,UCA1 shRNA,miR-582-5p inhibitor and shRNA + inhibitor group.Introduced 10 n M rapamycin(CST,# 9904)for experiments.A single layer of confluent cells was scraped in the middle of the well with a pipette tip.Human normal cells SV-HUC-1 were quantitatively PCR(qPCR).Quantitative expression of UCA1 and miR-582-5p was detected in bladder cancer-derived cells T24,5637.Cells were lysed by RIPA lysis buffer(Beyotime Institute of Biotechnology)using protein immunotracer method.An equal amount of protein sample was separated using 10% SDS-PAGE and transferred to a PVDF membrane(Millipore,Billerica,MA,USA).After incubation with 5% skimmed milk in TBST,the membrane was incubated with rabbits against ATG7(ab133528),LC3 A / B(ab62721),P62(ab155686),and multidrug resistance protein 1(Abcam,Cambridge,MA,USA).Antibodies(Abcam,Cambridge,MA,USA)were incubated with MRP1,ab3368),lung resistance-related proteins(LRP,ab92544),GST(ab19256),and topoisomerase II(TOPOII,ab52934)overnight at 4 ℃.They were then incubated with HRP-conjugated secondary antibodies(ab6721)for 1.5 hours at room temperature.Finally,the blot was observed by electroluminescence(ECL)and detected using a Chemi Doc XRS imaging system.Four-week-old experimental white rats were raised under standard conditions according to the animal experiment protocol,and conventional sterile food and water were randomly fed.A total of 20 animals were equally divided into two groups.One group was injected subcutaneously with 5 × 106 T24 cells transfected with UCA1 shRNA,and the other group was injected with scrambled T24 cells.Tumor volume was measured every 5 days for 30 days.After all mice were sacrificed,tumor volume was calculated by the formula length×width 2×0.5,and UCA1 and miR-582-were detected in human normal cells SV-HUC-1 and bladder cancer-derived cells T24,5637 by quantitative PCR(qPCR).5p quantitative expression.Result:UCA1 shRNA negatively regulates autophagy,as evidenced by reduced LC3-II and increased P62 and LC3-II / LC3-I ratios,while rapamycin plays the opposite role;UCA1 shRNA reduces resistance,which is caused by Proved by reduced expression of related proteins(including MRP1,LRP,and GST);also,rapamycin exhibits opposite activity;UCA1 shRNA significantly reduces cell growth,cell invasion,and migration,and rapamycin is at these activities Shows the opposite effect.Consistent with in vitro results,compared with controls,down-regulating UCA1 expression significantly inhibited tumor growth.Down-regulating UCA1 expression down-regulated the m RNA level of ATG7,but up-regulating the expression level of miR-582-5p.Immunohistochemical results showed that down-regulating UCA1 The expression reduced the expression of Ki67,PCNA,MMP-9 and VEGF in vivo,supplementing the conclusion of miR-582-5p’s anticancer activity.Conclusion:UCA1 targets miR-582-5p,and the inhibition of UCA1 expression leads to an increase in miR-582-5p expression,which inhibits the expression of the autophagy gene ATG7,thereby inhibiting autophagy of bladder cancer cells and inhibiting drug resistance,Inhibit the development of cancer cells.Both in vitro and in vivo studies are in line with experimental conclusions,and in vivo and in vitro studies are in agreement,supplementing the anti-cancer activity of miR-582-5p.