The Role And Molecular Mechanism Of PHD3-regulated Desmin In The Bone Metastasis Of Prostate Cancer

Background Prostate cancer is a common malignant tumor in urinary surgery.The morbidity of prostate cancer accounts for 7.8% of global cancer and its fatality rate ranks only second to lung cancer,breast cancer and colorectal cancer.In China,with the prolongation of human life,improved attention of health and continuous progress of prostate cancer diagnosis,the incidence of prostate cancer keeps growing continously.What is more,it is expected that the incidence is likely to largely grow in the future for a long period.Early diagnosis and treatment of prostate cancer has great theoretical significance and practical value for prognosis.Contribute to the progress of medical technology,prostate cancer could be detected by various means.However,a large proportion of patients suffer from bone metastasis when they are diagnosed.The bone has become the primary metastasis target organ of prostate cancer.The prostate cancer patients with bone metastasis are hardly to be cured.The patients are often accompanied by whole body bone pain,fracture and paralysis,which seriously threatens the life quality of patients.However,the mechanism of prostate cancer bone metastasis has not been cleared.Identifying the mechanism of bone metastasis is of great significance to prolong the patients' survival time and improve their life quality.Objectives 1 To screen and identify the differentially expressed proteins in prostate cancer and bone metastasis;2 To detect the expression and correlation of PHD3 and Desmin in prostate cancer;3 To explore the role and molecular mechanism of PHD3-regulated Desmin in the bone metastasis of prostate cancer.To provide a new target for the treatment of prostate cancer.Methods 1 Technical determination and identification of prostate cancer and bone metastasis associated differentially expressed proteins 1.1 Screening of prostate cancer and bone metastasis associated differentially expressed proteins by SELDI-TOF-MS The m/z values of differentially expressed proteins were screened in prostate cancer para-carcinoma tissues(para-carcinoma group),prostate cancer patients without bone metastasis(non-bone metastasis)and prostate cancer bone metastasis group(bone metastasis group)through the SELDI-TOF-MS technology.1.2 Isolation and identification of prostate cancer and bone metastasis associated differentially expressed proteins The differentially expressed proteins in prostate cancer tissue were isolated,purified and identified using the TRICINE-polyacrylamide gel electrophoresis(TRICINE-SDS-PAGE)and MALDI-TOF/TOF-MS technologies.2 Expression and correlation of PHD3 and Desmin in prostate cancer 2.1 PHD3 and Desmin protein expressions and correlation detected by the immunohistochemical method The PHD3 and Desmin expressions of the specimens in the para-carcinoma group,non-bone metastasis group and bone metastasis group were detected by the immunohistochemical method.The correlation between PHD3 and Desmin was statistically analyzed.2.2 PHD3 and Desmin expressions of the specimen by the Western blot method The PHD3 and Desmin expressions of the specimens in different prostate cancer tissues were detected by the Western blot method.2.3 The PHD3 and Desmin m RNA expressions of specimens were detected by real-time fluorescence quantitative PCR The PHD3 and Desmin m RNA levels of prostate cancer tissues in different groups were detected by real-time fluorescence quantitative PCR technique,suggesting that Desmin may have post-transcriptional levels of regulation.3 Effect and molecular mechanism of PHD3 and Desmin in prostate cancer metastasis 3.1 Effect of PHD3 on the expressions of Desmin protein and m RNA The expression of Desmin protein was interfered using si RNA.The expressions of Desmin protein ad m RNA were detected by the q RT-PCR and Western blot.After the over-expression plasmid transfection of LNCa P cells,the expressions of Desmin protein ad m RNA were detected by the above methods again.The expression of Desmin was observed by the immunofluorescence.The regulatory effect of PHD3 on Desmin expression was investigated.3.2 PHD3 inhibited the binding of NEDD4 with Desmin by combining NEDD4 protein The co-immunoprecipitation method was used to detect the binding of PHD3 with NEDD4.After the PHD3 expression was interfered,the expression of NEDD4 protein was detected.The effect of PHD3 on the binding of NEDD4 with Desmin was detected by the co-immunoprecipitation method.The interaction and influence among PHD3,NEDD4 and Desmin were explored.3.3 Effects of overexpressed Desmin on the LNCa P proliferation and metastasis with PHD3 deficiency by the tumor formation experiment in nude mice Cells groups: normal culture LNCa P group,PHD3 group after si RNA interference,PHD3 after si RNA interference + overexpressed Desmin group.The expressions of PHD3 and Desmin proteins were detected in all groups.The effects of PHD3 and Desmin on the metastasis and invasion of tumor cells were determined by the tumor formation experiment in nude mice.Results 1 Screening and identification of prostate cancer progression associated differentially expressed proteins 1.1 Screening of differentially expressed proteins in prostate cancer tissues by SELDI-TOF-MS The SELDI-TOF-MS technique was used to screen the differentially expressed proteins of the specimens in the para-carcinoma group,non-bone metastasis group and bone metastasis group.The results showed that the m/z values of the differentially expressed proteins were located in 8452 Da and 13367 Da.1.2 Isolation and identification of prostate cancer occurrence and bone metastasis associated differentially expressed proteins Polyacrylamide gel electrophoresis(SDS-PAGE)was used to separate and purify the target protein.The m/z in 8452 Da and 13367 Da peptide fragment/protein sample was detected respectively.The peptide fragment was imported into the SEQUEST search program and searched in the Bioworks database.The peptide fragments/proteins of m/z at 8452 Da and 13367 Da were prolyl hydroxylase 3(PHD3)and Desmin.2 Expressions and correlation of PHD3 and Desmin in prostate cancer 2.1 Expressions and correlation of PHD3 and Desmin proteins detected by the immunohistochemical method The immunohistochemical method showed that the expressions of PHD3 and Desmin in prostate cancer tissues were significantly lower than those of paracarcinoma tissue.Moreover,the expressions of PHD3 and Desmin in prostate cancer and prostate cancer patients with bone metastasis were much lower.Statistical analysis showed that the expression of PHD3 was positively correlated with that of Desmin proteins.2.2 Expression of PHD3 and Desmin proteins in tissue by the Western blot method The above screened proteins were verified by the Western blot method.The result showed that the expressions of PHD3 and Desmin protein in the para-carcinoma group,non-bone metastasis group and bone metastasis group were gradually decreased.2.3 Expressions of PHD3 and Desmin m RNA in tissue were detected by the real-time fluorescence quantitative PCR Real-time fluorescence quantitative PCR(q RT-PCR)showed that the expression of PHD3 in prostate cancer tissue was significantly lower than that of para-carcinoma tissue.Moreover,the expression of PHD3 in prostate cancer tissue with bone metastasis was much lower.However,the expressions of Desmin m RNA showed no significant difference among all groups.3 Effect and molecular mechanism of PHD3 and Desmin in prostate cancer metastasis 3.1 Effects of PHD3 on the expressions of Desmin protein and m RNA The expression of PHD3 was intervened using si RNA and over-expressed plasmid.RT-PCR result suggested that the intervention efficiency was high and could be used in the following experiments.After the expression of PHD3 protein was intervened by sh RNA,the expressions of Desmin protein and m RNA were significantly decreased.After the LNCa P cells were transfected by PHD3 over-expression plasmid,the expression of Desmin protein was significantly increased,but the expressions of Desmin m RNA showed no significant change.Immunofluorescence staining showed that the expression of Desmin was increased after PHD3 over-expression,suggesting that PHD3 played a post-transcriptional regulation of Desmin.3.2 PHD3 inhibited the binding of NEDD4 with Desmin by combining NEDD4 protein The co-immunoprecipitation results showed that PHD3 could directly bind NEDD4.Although the expression of NEDD4 protein was not affected by interfering PHD3 expression,its binding with Desmin was significantly increased.While,the binding of NEDD4 with Desmin was inhibited after the purified PHD3 was added into the reaction system,suggesting that PHD3 did not affect the expression of NEDD4,but affected the NEDD4 on regulation of Desmin.3.3 Effects of the over-expression of Desmin on the LNCa P proliferation and metastasis with PHD3 deficiency by the tumor formation experiment in nude mice The tumor formation experiment in nude mice showed that the interference of PHD3 could significantly increase the metastasis of prostate cancer,but the over-expression of Desmin could inhibit the metastasis and invasion of tumor cells caused by PHD3 deletion.Conclusions 1 Isolation and identification of prostate cancer occurrence and bone metastasis related proteins PHD3 and Desmin by SELDI-TOF-MS.2 PHD3 and Desmin were correlated with the bone metastasis of human prostate cancer,their expressions were significantly decreased and positively correlated in tumor metastasis.3 PHD3 could competitively inhibit the Desmin binding and degradation of NEDD4 through combining with NEDD4.4 The recovery of PHD3 and Desmin expressions may be a new target for the treatment of prostate cancer bone metastasis.Innovation The prostate cancer associated differentially expressed proteins PHD3 and Desmin were screened and identified by the proteomics method in this research.The result showed that the PHD3 and Desmin were decreased in prostate cancer especially in the bone metastatic prostate tissues,and there was a positive correlation between them.As for the mechanism,PHD3 could competitively inhibit NEDD4 on the Desmin binding and regulation through combining NEDD4.Animal experiments further confirmed that the decreased expression of PHD3 could promote the bone metastasis of prostate cancer,and the recovery of Desmin expression had an important inhibitory effect on the bone metastasis of prostate cancer.There was no report about the effect of PHD3 regulating Desmin on prostate cancer metastasis.The intervention of Desmin PHD3 regulated pathway may provide a new therapeutic target for inhibiting the bone metastasis of prostate cancer.