The Role And Mechanism Of SPOCK2 In Prostate Cancer

Objective:Prostate cancer is the most common cancer among men in developed countries and the second leading cause of death.In China,the incidence of prostate cancer is also increasing year by year.Prostate cancer presents as a chronic asymptomatic course in the early stage,and with a good prognosis.Most patients with local or regional prostate have a 5-year survival rate of more than 90%.Meanwhile,with the use of prostate specific antigen(PSA)screening,the detection rate of early prostate cancer has been greatly improved.However,mortality rates for advanced prostate cancer remain high,with a 5-year survival rate of only 30%.Therefore,the study of the causes of the occurrence and development of prostate cancer is of great significance to improve the prognosis of prostate cancer.SPARC(osteonectin),cwcv and kazal like domains proteoglycan 2(SPOCK2),is a member of the Spock family,encodes a protein that binds to glycosaminoglycan and form a component of the extracellular matrix,SPOCK2 is also known as testincan-2.The abnormal expression of this gene is associated with the occurrence and development of a variety of diseases.Studies have found that abnormal methylation in the promoter region of SPOK2 gene is closely related to prostate cancer,breast cancer,and colon cancer,etc.,and it is believed that the apparent inactivation of this gene can be used for the early diagnosis of the above three tumors.This is the first time to elaborate the correlation between SPOCK2 gene and the occurrence and development of prostate cancer.Meanwhile,in the study of the related mechanism of glioma,it was found that SPOCK1and SPOCK3 could inhibit the MT1-MMP or MT3-MMP mediated MMP2 activation by binding with MT1-MMP or MT3-MMP to form a complex,and then inhibit the invasion of brain glioma cells.However,the highly expressed SPOCK2 in glioma can bind to SPOCK3 to relieve the inhibitory effect of SPOCK3 on MMP2,thus increasing the aggressiveness of glioma.However,the expression of this gene in prostate cancer tissues and its role and mechanism in the occurrence and development of prostate cancer have not been reported in the literature before.This study aims to explore the role and mechanism of SPOCK2 gene in the occurrence and development of prostate cancer by detecting the expression of SPOCK2 gene in prostate cancer tissues,and also find the effect of biological and molecular behavior and the effect of its expression on MT1-MMP/MMP2 by up-regulation of SPOCK2 gene on the prostate cancer cells.Methods:1.Real-time PCR was used to detect the expression of SPOCK2 gene mRNA in prostate cancer tissues.2.To detect the effect on molecular biological behavior of prostate cancer cells by up-regulation of SPOCK2 expression.2.1 Human prostate cancer cell line DU145 and LNCaP were transfected with SPOCK2 overexpression vector.2.2 Real-time PCR was used to detect the expression of SPOCK2 gene in cells before and after transfection.2.3 The protein expression of SPOCK2 in cells before and after transfection was detected by Western Blot.2.4 CCK8 kit was used to detect cell proliferation before and after transfection.2.5 Cell cycle was detected by flow cytometry before and after transfection2.6 Cell apoptosis was detected by flow cytometry combined with Annexin V-FITC/PI double staining assay before and after transfection.2.7 Transwell invasion chambers were used to detect changes in cell invasion ability before and after transfection2.8 Cell migration ability before and after transfection was detected by wound healing assay.2.9Cell adhesion assay was used to measure cell adhesion ability.3.Detect the effect on the expression and activity of MT1-MMP/MMP2 in prostate cancer cells after up-regulation of SPOCK2 expression.3.1 The protein expressions of Spock2,MT1-MMP and MMP2 in each group were detected by Western Blot.3.2 Detection of the expression of actived MMP2 and MMP9 in prostate cancer cells by zymography gel assayResults:1.Expression of SPOCK2 mRNA in prostate cancer tissues.Detection the level of SPOCK2 mRNA in prostate cancer tissues by real-time PCR.The results showed that SPOCK2 mRNA expression in prostate cancer group(2.14±1.26)was significantly lower than that in normal control group(6.58± 1.97),the difference was statistically significant(P <0.05).2.To detect the effect of SPOCK2 upregulation on molecular biological behavior of prostate cancer cells.2.1 Expression of SPOCK2 mRNA in prostate cancer cells after transfection.The results showed that the mRNA expression of SPOCK2 gene in DU145 and LNCap cells was significantly increased after transfection,and the difference was statistically significant(P <0.05),indicating that the transfection of recombinant SPOCK2 fragment effectively increased the mRNA expression of SPOCK2 gene in DU145 and LNCaPcells.2.2 Expression of SPOCK2 gene protein in prostate cancer cells after transfection The protein expression of SPOCK2 gene in prostate cancer cells before and after transfection was detected by Western Blot.The results showed that the protein expression of SPOCK2 in DU145 and LNCap cells was significantly increased after transfection,and the difference was statistically significant(P <0.05),indicating that the transfection of recombinant SPOCK2 fragment effectively increased the protein expression of SPOCK2 in DU145 and LNCap cells.2.3 Effect of upregulation of SPOCK2 expression on proliferation of prostate cancer cells Cell proliferation after up-regulation of SPOCK2 gene expression in DU145 and LNCaP cells was detected by CCK8 kit.The results showed that the proliferation rates of the two kinds of cells were significantly decreased from 24 h after transfection,and the proliferation rates of the transfection group were significantly lower than those of the negative control group and the blank control group,with statistical significance(P <0.05).These results indicated that up-regulation of SPOCK2 expression could inhibit proliferation of DU145 and LNCaP in prostate cancer cells.2.4 Effect of up-regulation of SPOCK2 expression on cell cycle of prostate cancer cells Cell cycle changes were detected by flow cytometry after SPOCK2 up-regulation in DU145 and LNCap cells.The results showed that the percentage of G0/G1 phase cells in the transfected group was significantly higher than that in the negative control group and the blank control group,and the difference was statistically significant(P <0.05).The percentage of S phase in the transfection group was significantly lower than that of the negative control group and the blank control group,and the difference was statistically significant(P <0.05).These results indicated that SPOCK2 could change the cell cycle,make the cells stagnate in the G0/G1 phase,and inhibit cell proliferation.2.5 Effect of upregulation of SPOCK2 expression on apoptosis of prostate cancer cells Flow cytometry combined with Annexin V-FITC/PI double staining analysis was used to detect the apoptosis of DU145 and LNCaP cells after SPOCK2 upregulation.The results showed that the apoptosis rate of the DU145 transfected group was significantly higher than that of the negative control group and the blank control group,the difference was statistically significant(P <0.05),while the apoptosis rate of the LNCaP transfected group was not significantly different from that of the negative control group and the blank control group(P >0.05),indicating that the up-regulation of SPOCK2 expression could promote the apoptosis of DU145 cells only.2.6 Effect of up-regulation of SPOCK2 expression on invasion ability of prostate cancer cellsThe invasion ability of DU145 and LNCaP cells after SPOCK2 upregulation was detected by Transwell invasion chamber.The results showed that the invasion ability of transfected group was significantly lower than that of negative control group and blank control group,and the difference was statistically significant(P <0.05),indicating that the upregulated expression of SPOCK2 could reduce the invasion ability of prostate cancer cells.2.7 Effect of up-regulation of SPOCK2 expression on migration of prostate cancer cells The migration ability of DU145 and LNCap cells after transfection was detected by scratch test.The results showed that the migration ability of cells in transfected group was significantly lower than that of negative control group and blank control group,the difference was statistically significant(P <0.05).These results indicated that the up-regulated expression of SPOCK2 could reduce the migration ability of DU145 and LNCaP cells.2.8 Effect of SPOCK2 upregulation on adhesion ability of prostate cancer cells Cell adhesion assay was used to detect the changes of adhesion ability in DU145 and LNCaP cells after up-regulation of SPOCK2 gene expression.The adhesion ability of transfected group was significantly lower than that of negative control group and blank control group,the difference was statistically significant(P <0.05).These results indicated that the up-regulated expression of SPOCK2 could reduce the adhesion ability of DU145 and LNCaP cells.3 Detection of the effect of up-regulation of Sp COCK2 on the expression of MT1-MMP/MMP2 in prostate cancer cells.3.1 Expression of SPOCK2 protein in prostate cancer cells after transfection After transfection,the protein expression of SPOCK2 gene in DU145 and LNCaP cells were significantly increased,with statistical significance(P <0.05).3.2 Expression of MT1-MMP2 and MMP2 protein in prostate cancer cells after up-regulation of SPOCK2The protein expressions of MT1-MMP2 and MMP2 in prostate cancer cells after up-regulation of SPOCK2 were significantly lower than those in negative control group and blank control group,respectively,with statistical significance(P <0.05).These results indicated that up-regulation of SPOCK2 expression could inhibit the expression of MT1-MMP2 and MMP2 protein.3.3 Expression of activated proteins of MMP2 and MMP9 in prostate cancer cells after up-regulation of SPOCK2The expression of activated MMP2 and MMP9 were detected by gelatin zymography after transfection,and they were significantly lower than those in the negative control group and the blank control group,respectively,with statistically significant differences(P <0.05).These results indicated that the up-regulation of SPOCK2 can inhibit the activation of MMP2 protein.Conclusion:1.The expression of SPOCK2 gene is decreased in prostate cancer tissues,and the abnormal expression of SPOCK2 gene may be related to the occurrence and development of prostate cancer.2.The up-regulated expression of SPOCK2 can inhibit cell proliferation,adhesion,invasion,apoptosis and other biological behaviors,and play an important role in prostate cancer through these above effects,which is expected to become a new target for the treatment of prostate cancer.3.The role of SPOCK2 in the development and progression of prostate cancer may be realized by regulating the expression of MT1-MMP and MMP2 protein,and regulating the activation of MMP2 and MMP9.