| Objective:Sirtuin 5(SIRT5)is an NAD+-dependent class III protein deacetylase,and its role in prostate cancer has not been reported.Therefore,in order to explore the diagnosis and treatment of prostate cancer,we studied the effect of SIRT5 on prostate cancer.SIRT5 was evaluated in 57 prostate cancer tissues by immunohistochemistry.We found that the tissue expression level of SIRT5 in patients with Gleason score ≥ 7 was significantly different from that of patients with Gleason score <7(P <0.05,R>0).In addition,related experiments through pathway screening showed that SIRT5 regulates the activity of the mitogen-activated protein kinase(MAPK)pathway,which in turn regulates the expression of MMP9 and cyclin Cyclin D1.In mass spectrometry analysis,we found the acetyl-Coenzyme A acetyltransferase 1(ACAT1)protein.As a substrate of SIRT5,ACAT1 is regulated by SIRT5,and SIRT5 regulates the activity of the MAPK pathway through ACAT1.These results indicate that SIRT5 promotes the activity of the MAPK pathway through ACAT1 and increases the proliferation,migration and invasion of prostate cancer cells.All in all,the above results indicate that SIRT5 expression is closely related to the progression of prostate cancer.Understanding the underlying mechanisms may provide new goals and methods for the diagnosis and treatment of diseases.Results: 1.In order to study the expression of SIRT5 in human prostate cancer tissues,we performed immunohistochemical experiments on 57 randomly selected prostate tissue sections.SIRT5 is not expressed in normal prostate tissue,but it is highly expressed in tumor tissues.According to the Gleason scoring standard,57 tissue sections were scored and counted.When the Gleason score is equal to 7,prostate cancer tissue can be divided into two groups:(3 + 4)and(4 + 3).The difference between the two groups was not statistically significant(P = 0.797).Similarly,there was no significant difference in the expression of SIRT5 in tissues with Gleason scores of 6 and 7(P = 0.396).In addition,the expression differences between the two groups with scores of 8 and 9 were not statistically significant(P = 0.262),so the scores of 8 and 9 were grouped into one group.However,when the scores 8 and 9 were combined into one group,the SIRT5 protein expression was significantly different from the group with a score of 7(P = 0.028)and a score of 6(P = 0.001).Therefore,we conclude that SIRT5 expression is related to the Gleason score of prostate cancer.In the cBio Portal database,we found that the SIRT5 gene has mutations in prostate cancer,and most of these mutations are deep deletions and missense mutations.These mutations may be related to the prognosis of prostate cancer patients.However,there is no obvious statistical significance in our data.Therefore,we ignored the impact of SIRT5 gene mutation on prostate cancer.62 different tumor cell lines with increased SIRT5 expression have been identified in the Human Protein Atlas database.In PC-3 cells(a representative prostate cancer cell line),this increase was more pronounced compared with other tumor cell lines.Compared with other tumor cell lines,the expression of SIRT5 in PC3 cells is also higher in the Shankavaram Cell Line(Oncomine)database.2.Based on the above findings,we next studied the role of SIRT5 in prostate cancer.The LNCa P and PC-3 cell lines were selected for these studies.First,it was observed that the expression of SIRT5 remained low at 72 hours after transfection until the end of the experiment.Cell proliferation and colony formation experiments were performed to evaluate the influence of SIRT5 expression on the proliferation ability of prostate cancer cells.The results of these measurements in two cell lines indicate that up-regulation of SIRT5 expression can promote cell growth.In addition,transfection of either cell line with si RNA-SIRT5 resulted in decreased cell proliferation and decreased colony formation,indicating that SIRT5 can promote the growth of prostate cancer cells.In the Transwell migration experiment,after the SIRT5 protein was overexpressed in LNCa P cells,cell migration was enhanced.In addition,after interference with SIRT5 protein expression,LNCa P cell migration ability is reduced.Similar results were obtained in PC-3 cells.In addition,SIRT5 reduces the level of ROS.These results indicate that SIRT5 plays a role in promoting tumors in prostate cancer cells.3.To understand how SIRT5 protein affects prostate cancer cells,we performed Western blot analysis.Down-regulation of SIRT5 protein resulted in a significant decrease in cyclin D1 protein(related to the cell cycle)and MMP9 protein(related to migration and invasion).In addition,the activity of MAPK pathway proteins was significantly inhibited.When SIRT5 is overexpressed,the activity of MAPK signaling pathway proteins and the levels of downstream functional proteins increase.These changes are consistent with the results of in vitro proliferation and migration experiments,and prove that SIRT5 protein mainly regulates the MAPK signal transduction pathway in prostate cancer to play a tumor-promoting effect.After increasing the expression of SIRT5 in cells treated with si-SIRT5,the expression of related proteins also increased.In order to clarify whether it is the effect of SIRT5 changes,we added specific inhibitors of SIRT5 and found that the MAPK signaling pathway was inhibited,and the expression level of related functional proteins was significantly reduced.4.In order to study how SIRT5 affects the MAPK signal pathway,whether it directly affects the MAPK signal pathway,we conducted mass spectrometry analysis.The results showed that ACAT1 combined with SIRT5.To further investigate this finding and determine whether SIRT5 regulates ACAT1 at the gene or protein level,q RT-PCR was performed.Regardless of whether LNCa P cells were transfected or interfered with SIRT5,the level of ACAT1 m RNA was unchanged.The same results were obtained in PC-3 cells,indicating that SIRT5 does not regulate ACAT1 m RNA.Based on these findings,we shifted our focus to protein regulation and found that after interfering with the expression of SIRT5 in LNCa P cells,the protein level of ACAT1 decreased,while the increase of SIRT5 expression increased the protein level of ACAT1.On the contrary,after changing ACAT1 expression,SIRT5 protein level did not change.The same results were obtained in PC-3 cells.These results indicate that SIRT5 can regulate ACAT1 at the protein level,but ACAT1 cannot regulate the expression of SIRT5.After the expression of SIRT5 was reduced and MG-132 was added,the expression of ACAT1 protein did not increase significantly,indicating that SIRT5 does not regulate ACAT1 through the proteasome pathway.Co-IP experiments in LNCa P and PC-3 cells showed that SIRT5 and ACAT1 bind to each other in vitro.The results were confirmed by a confocal experiment,which revealed that the proteins SIRT5 and ACAT1 are co-localized in the mitochondria of the cell.5.In view of the above results,we understand the effect of SIRT5 protein on prostate cancer cells.Under normal expression of SIRT5,silencing ACAT1 protein can also inhibit the MAPK signaling pathway in LNCa P and PC-3 cells.Next,we explored the relationship between these proteins and MAPK signaling pathways in LNCa P and PC-3cells.Compared with the control group and cells transfected with flag-SIRT5 only,the co-transfection of si RNA-ACAT1 and flag-SIRT5 reduced the activity of the MAPK signaling pathway,thereby reducing the expression of downstream functional proteins Cyclin D1 and MMP9.After adding a specific inhibitor of ACAT1 protein,this inhibition is more obvious.Interference with ACAT1 also inhibited the function of SIRT5 in MTS,colony formation and Transwell migration experiments.In addition,when the expression of SIRT5 protein decreased,transfection of ACAT1 restored the expression of related proteins.Overall,these results indicate that SIRT5 regulates the MAPK signaling pathway through ACAT1 and has a tumor-promoting effect in prostate cancer.Conclusion: 1.High expression of SIRT5 in prostate cancer cells correlates with tumor Gleason score.2.SIRT5 promotes cell proliferation and migration.3.SIRT5 regulates the MAPK signaling pathway.4.SIRT5 combines with ACAT1 and regulates its expression.5.SIRT5 regulates the function of prostate cancer cells through ACAT1. |
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