The Role Of Circadian Clock Gene NR1D1 In Urothelial Carcinoma Of The Bladder

Objective:To study the expression level of circadian clock gene NR1D1 in urothelial carcinoma of the bladder(bladder cancer)and the relationship between NR1D1 level and clinical characteristics,as well as to explore the effect of NR1D1 on the biological behaviors of bladder cancer cells and its potential mechanism.Materials and Methods:Using the data downloaded form The Cancer Genome Atlas(TCGA)database,the level of NR1D1 m RNA in bladder cancer and normal tissues was compared.The association between NR1D1 level and clinical characteristics was also analyzed.Moreover,we compared the immune cells infiltration and immune checkpoint(ICP)in subgroups with high and low NR1D1 level.The differentially expressed genes(DEGs)related to NR1D1 and the level of NR1D1 between subgroups of oncogenes with wild and mutation type were determined.GO and KEGG analysis were employed to identify potential biological function and signal pathways of NR1D1 in bladder cancer.Paraffin-embedded tissues of patients who were diagnosed with bladder cancer in our center were obtained.Immunohistochemistry(IHC)of NR1D1 was performed and the association of NR1D1 expression level with clinical characteristics and prognosis was investigated.Human bladder cancer cell lines T24,5637 and RT4 were used for in vitro experiments.Firstly,bladder cancer cells were stimulated by different concentration of Rev-erbα(the protein coded by NR1D1)agonist,SR9009,which could activate Rev-erbα.CCK-8 experiment was carried out after 24 h and 48 h,respevtively.Furthermore,cells were treated with SR9009 with an optimal concentration to conduct transwell and colony formation experiments.Thirdly,lentivirus and si RNA targeting NR1D1 were constructed to over-express(OE)and knock-down(KD)NR1D1 in bladder cancer cells,respectively.The biological functions were repeatedly detected using CCK-8,transwell and colony formation experiments.Cell cycle and apoptosis were tested by flowcytometry.PI3K/AKT/m TOR pathway proteins in OE-NR1D1 bladder cancer cells were determined by Western blot.Additionally,the effect of Rapamycin(Rapa)on KDNR1D1 and KD-Control cells were detected by CCK-8 and colony formation experiment.Finally,OE-NR1D1 and OE-Control bladder cancer cells were subcutaneously implanted in BALB/c nude mice.After treating those mice with Rapa or vehicle for 14 d,mice were sacrificed and tumor tissues were removed.Tumors were compared among all groups and then were used for Western blot to verify the mechanism that we identified in vitro.All results were based on the level of α = 0.05,a P < 0.05 was considered as statistically significant.Results:408 bladder cancer cases and 19 normal cases were obtained from TCGA database.It showed that the level of NR1D1 m RNA was not significantly different between bladder cancer tissue and normal bladder tissues either by independent samples t test or paired t test,as was the same between male and female patients(all P > 0.05).It presented with the fact that NR1D1 was defferent among subgroups of age,including≤ 50 y vs.> 80 y、≤ 50 y vs.51-60 y and ≤ 50 y vs.61-70 y(all P < 0.05),as that was observed in patients diagnosed with different stages.NR1D1 level in stage III was higher than that in stage II,but lower than stage IV(all P < 0.01).Patients with high grade also had a higher level of NR1D1 than those with low grade(P < 0.05).Seven types of immune cells,such as na(?)ve B cells,as well as seven m RNA of ICP genes,such as PD-1,were were significantly different between the high and low NR1D1 level groups(all P < 0.05).Additionally,we identified 704 DEGs related to NR1D1,including 379 up-regulated and 325 down-regulated genes.The level of NR1D1 was not significantly different between the subgroups of oncogenes with wild or mutation type,such as TP53,FGFR3 and ERBB2(all P > 0.05).GO enrichment analysis demonstrated that NR1D1 might serve as serine-type endopeptidase inhibitors in molecular function,and it could be observed in the the sites of cell junctions and be involed in multiple biological processes,such as epidermal development,keratinization,and mesenchymal cell apoptosis.Circadian rhythm,glycolysis/gluconeogenesis metabolism,pentose phosphate pathway and butyrate metabolism pathway were indentified by KEGG enrichment analysis.Based on the NR1D1 expression level by IHC of 63 bladder cancer cases.28(44.4%),26(41.3%)and 9(14.3%)cases were diagnosed with negative(score 0),weak positive(score 1)and strong positive(score 2)NR1D1 expression,respectively.No significant association was found between NR1D1 level and clinical characteristics(all P > 0.05).The univariate Kaplan-Meier analysis indicated that patients with NR1D1 positive expression(including score 1 and 2)had a longer disease-free survival(DFS)than those with score 0(30.59 m vs.53.91 m,P = 0.007).But there was no significant difference of overall survival(OS)between the two groups(69.71 m vs.64.69 m,P= 0.991).Besides,data showed that patients with muscle invasive bladder cancer(MIBC)had both shorter OS(85.29 m vs.54.16 m,P = 0.017)and shorter DFS(63.13 m vs.31.80 m,P = 0.019)than non-muscle invasive bladder cancer(NMIBC).The multivariate COX regression model revealed that NR1D1 positve expression was a favorable factor of DFS(Hazard ratio [HR]: 0.296,95% Confidence interval [CI]:0.108-0.810,P = 0.001),and muscle invasion was a poor factor of DFS(HR: 1.826,95%CI,1.277-2.610,P = 0.018).After treating three bladder cancer cell lines with 2μM～100 μM SR9009,we found that the cell viability significantly decreased as the concentration increased,and it almost reached a maximum effect at 32 μM.Then the cells were cultured with 32 μM SR9009,transwell and colony formation experiments were performed.Tt showed that both migration and colony formation were significantly suppressed(all P < 0.05).Furthermore,the OE-NR1D1 and KD-NR1D1 bladder cancer cells were established and verified by both Western blot and q RT-PCR.Compared with OE-Control groups,OE-NR1D1 bladder cancer cells had obviously suppressed cell viability,migration and colony formation(all P < 0.05),while those were found strengthened in KD-NR1D1 cells(all P < 0.05).Besides,KD-NR1D1 cells were observed with a lower proportion of dead cells and G0/G1 stage cells,but a higher ratio of G2/M stages(all P < 0.05).By perfoming Western blot with OENR1D1 cells,it showed a down-regulation of p-AKT,p-S6 and p-4EBP1,which were involved in PI3K/AKT/m TOR pathway.Next,we found Rapa could inhibit the cell viability and colony formation in both KD-NR1D1 and KD-Control cells(all P <0.01).Interestingly,the effect of Rapa on KD-NR1D1 cells was significantly greater than that of KD-Control cells with the same concentration(all P < 0.05).Finally,in vivo data demonstrated that the weight of tumor among the nude mice implanted with OE-NR1D1 T24 cells was significantly lighter than those with OE-Control cells(1.598 g vs.0.670 g,P < 0.001).For the nude mice implanted with either OE-NR1D1 or OE-Control T24 cells,treatment of Rapamycin could result in a smaller tumor(all P < 0.001).The p-S6 of removed sample was determined by Western blot and it was found that OE-Control group had a significantly higher level than the other three groups(all P <0.01).Conclusion:Though we did not observe a significant difference in the level of NR1D1 in bladder cancer tissues and normal tissues based on TCGA database,it should be noticed that NR1D1 might have potential relationship with immunity,which required to be further explored.IHC data showed that a lower level of NR1D1 might indicate earlier recurrence in bladder cancer.Cell viability,migration and colony formation of bladder cancer cells could be inhibited by Rev-erbα agonist SR9009 and over-expression of NR1D1.In contrary,down-regulation of NR1D1 significantly strengthened the biological function,including cell viability,migration,colony formation and cell cycle,while it reduced cell death.In vivo data also confirmed that over-expression of NR1D1 could significantly inhibit the tumorigenic ability of bladder cancer cells.Finally,PI3K/AKT/m TOR signal pathway might be one of the mechanisms by which NR1D1 affected bladder cancer.Thus,NR1D1 might become a novel target for the treatment of bladder cancer.