| Objective: Bladder cancer is one of the most common tumors in the world,being the eighth highest incidence of all malignant tumors and the top ten mortality rates.When patients are diagnosed initially,no less than 70% patients are diagnosed as non muscle invasive bladder cancer(NMIBC),and 25% of bladder cancer patients have muscular infiltration at the beginning of diagnosis,which is invasive bladder cancer.These tumors are poorly differentiated or undifferentiated in terms of cytopathological grade,and micro metastases often occur early.Even if comprehensive treatments such as radiotherapy and chemotherapy are adopted,the prognosis is poor.Invasive bladder cancer is primarily treated by surgical resection and neoadjuvant chemotherapy based on cisplatin.Although neoadjuvant chemotherapy centered on cisplatin has shown good efficacy for advanced bladder cancer,patients' resistance to cisplatin has created limitations in its use.The clinical difficulty that needs to be overcome is to increase the sensitivity of bladder cancer patients to cisplatin drugs and reduce the recurrence rate of bladder cancer.Therefore,finding an effective method for the treatment of invasive bladder cancer has always been a major subject in the basic research field of bladder cancer.The research on treatment of invasive bladder cancer still needs to start from multiple angles of perspective of the molecular pathology of its occurrence and development.In this study,abnormal activation of the proto-oncogene netrin-1 is one of the molecular mechanisms that promote the malignant progression of bladder cancer tumors,which can promote tumor cell proliferation and reduce the sensitivity of cisplatin to chemotherapy.UNC5 B is one of netrin-1 dependent receptors,which can induce tumor cell apoptosis.The results show that netrin-1 / UNC5 B is expressed in bladder cancer and participates in apoptosis of cancer cells.And miR-214 overexpression can reverse tumor progression caused by netrin-1.By means of reporter gene analysis,in vitro functional experiments,we worked to confirm that netrin-1 / miR-214 regulates the invasion,metastasis and drug resistance of bladder cancer.Methods: Human bladder cancer cell lines 5637,T24,BIU-87,sv-huc-1,RT-4 and J82,through cell culture,cells are collected and total RNA and protein are extracted.At the same time,the surgical specimens of bladder cancer and its adjacent tissues excised from 2008 to 2015 in Urology department of the First Affiliated Hospital of China Medical University were collected.Total RNA and protein are extracted,the mRNA expression of netrin-1,UNC5 B and miR-214 was detected by RT-PCR,the protein expression of netrin-1 and UNC5 B was detected by Western-blot,the expression and localization of netrin-1/UNC5 B were detected by immunofluorescence and immunohistochemistry,and the relationship of clinical and pathological parameters between netrin-1/UNC5 B was analyzed.Cell proliferation and apoptosis are detected by shaking bacteria,plasmid extraction,cell transient and stable miR-214,cell proliferation test(CCK-8),clone formation test,Edu cell proliferation test and Flow cytometry cell apoptosis test,in order to analyze the expression level of netrin-1,UNC5 B and miR-214 in bladder cancer cell lines and tissues,moreover,their function on bladder cancer cells.It is further confirmed that mir-214 could promote the sensitivity of bladder cancer cells to cisplatin resistance by down-regulating netrin-1.Results: Real time PCR & Western-blot detected netrin-1 / UNC5 B expression levels from RNA and protein levels,respectively,and found that the expression of UNC5 B in adjacent tissues was significantly higher than that in bladder cancer tissues and higher in low-grade bladder cancer tissues.In high-grade cancer tissues,the opposite is true of netrin-1.The high / low expression of netrin-1 / UNC5 B is related to the clinical stage,pathological grade and metastasis of bladder cancer and Pearson correlation analysis shows that the expression of netrin-1 and UNC5 B is negatively correlated.Real time PCR & Western-blot detected the expression level of netrin-1 / UNC5 B in bladder cancer cell lines(T24,BIU-87,5637,and SV)from RNA and protein levels.UNC5 B expression was high in normal bladder cell line SV,and the malignant T24 cell line had low expression,while netrin-1 had the opposite expression.Immunofluorescence results showed that UNC5 B was expressed in bladder cancer cell plasm and netrin-1 was expressed in both cytoplasm and nucleus.RT-qPcR was used to detect the expression of miR-214 in 30 fresh bladder cancer tissues and corresponding normal tissues.Compared with normal tissues,miR-214 expression was significantly down-regulated in cancer tissues,and miR-214 was expressed in bladder cancer cell lines.Down-regulating and inhibiting cell proliferation and invasion,T24 and J82 cells were tested for CCK-8 with cisplatin(10 ?g / ml).The results showed that miR-214 reduced the drug resistance of bladder cancer cells.Significantly up-regulated the apoptotic rate of J82 and T24 cell lines after 24 hours of cisplatin treatment.MiR-214 transfection mimics the downregulation of total caspase-3 and total PARP,and up-regulates the expression of caspase-3 and cleaved PARP.Target Scan software was used to predict the potential targets of miR-214.The results showed that netrin-1 is one of the target genes of miR-214.After transfection of miR-214,the expression of netrin-1 protein and mRNA was significantly down-regulated.A reporter with netrin-1 wild type(ccU-GcU-G)and mutant(ccU-uu-G)3'-UTR binding sites was introduced into J82 cells together with miR-214 mimic,luciferase reporter.The analysis shows that miR-214 mimics significantly inhibited the luciferase intensity of the wild-type reporter compared to the control group.The above data shows that miR-214 binds to the 3'-UTR of netrin-1 and reduces its mRNA and protein expression.Compared with cells not treated with cisplatin,cisplatin(2 and 5 ?g / ml for 2 days)induced increased protein expression in J82 and T24 cells.There was a significant increase in mRNA in cells treated with 5 ?g / ml cisplatin.Annexin V / PI analysis showed that netrin-1 overexpression significantly inhibited cisplatin-induced apoptosis compared to the control group.Western-blot showed that netrin-1 gene transfection up-regulated AKT phosphorylation.Conclusion: The expression of miR-214 was down-regulated in bladder cancer tissues,and was significantly related to tumor stage lymph node status and tumor grade.Urine acellular miR-214 level can be used as an independent prognostic indicator of nonmuscle invasive bladder cancer recurrence.MiR-214 plays a tumor suppressive role in bladder cancer by down-regulating the expression of the oncogene PdRG1.In this study,the expression of miR-214 was down-regulated in bladder cancer tissues and cell lines.The inhibitory effect of miR-214 on the proliferation and invasion of bladder cancer cells was also verified in this study.However,its role in the development drug resistance to bladder cancer cells remains to be explored.Therefore,this study investigated the effect of miR-214 on chemotherapy resistance of J82 and T24 cells.The results show that miR-214 mimics significantly reduce drug resistance by up-regulating the apoptosis rate.A series of apoptosis-related proteins were tested,and the results showed that after transfection of miR-214 mimics,the expression levels of total caspase-3 and total PARP decreased,while splited caspase-3 and splited PARP were significantly up-regulated.These results indicate that miR-214 promotes apoptosis.In addition,AKT signaling is involved in the regulation of apoptosis by up-regulating Bcl-2 family proteins.Therefore,the effect of miR-214 on chemotherapy resistance may depend on its regulation of AKT / Bcl-2 signaling.This study further explored the mechanisms by which miR-214 regulates apoptosis and drug resistance.Target Scan software was used to predict the potential target gene of miR-214,and netrin-1 was identified as one of the target genes.Netrin-1 plasmid transfection significantly reduced bladder cancer cell apoptosis and significantly up-regulated AKT phosphorylation.In addition,miR-214 can significantly reduce netrin-1 mRNA and protein expression levels.There was a negative correlation between miR-214 and netrin-1 mRNA expression levels in bladder cancer tissues.In addition,luciferase report analysis showed that miR-214 can directly bind to 3'-UTR of netrin-1,indicating that netrin-1 is a direct target of miR-214.Together,these results indicate that miR-214 down-regulates netrin-1,which in turn inhibits p-AKT and reduces chemotherapy resistance.Netrin-1 plasmid transfection repaired miR-214 downregulated cisplatin resistance.To sum up,these data suggest that miR-214 inhibits chemotherapy resistance in bladder cancer by targeting netrin-1. |
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