GOLPH3促进前列腺癌细胞增殖和转移的机制研究

Mechanisms of GOLPH3 Promoting Proliferation and Metastasis of Prostate Cancer Cells

目的研究前列腺癌中新型高尔基相关蛋白GOLPH3的表达特性,并探索其在前列腺癌细胞中的功能作用和分子机制。方法采用定量PCR和Western blot方法检测前列腺癌细胞中GOLPH3表达情况。运用免疫组化法检测前列腺组织中GOLPH3的表达情况。采用CCK-8方法检测敲减GOLPH3后PC-3细胞增殖生长情况;使用Transwell方法检测敲减GOLPH3后PC-3细胞侵袭和迁移能力。Western blot方法检测敲减GOLPH3后PC-3细胞周期蛋白表达的变化;Western blot方法检测EGFRSrc和EGFR-Akt-mTOR信号通路中关键蛋白及其磷酸化在GOLPH3 RNAi及其阴性对照组细胞中的表达变化。结果 GOLPH3在前列腺癌细胞和组织不同程度表达。GOLPH3敲减后,PC-3细胞增殖和生长受到明显抑制作用(P<0.05),PC-3细胞穿过滤膜的细胞数明显减少(P<0.05)。GOLPH3基因沉默后PC-3细胞增殖和转移能力明显下降。GOLPH3敲减后,PC-3细胞在G2/M期出现了非常显著的阻滞,提示GOLPH3基因在G2/M期参与了PC-3细胞增殖周期过程;PC-3细胞中p21的表达明显上调,CDK1/2、cyclin B1表达明显下调,Akt和mTOR表达上调,p-Akt、p-mTOR、p-p70S6K则下调;pEGFR、p-Src蛋白表达量显著降低,而EGFR、Akt、mTOR、Src、FAK、p-FAK、p70S6K蛋白表达量无明显变化。揭示阻断GOLPH3能抑制EGFR-Src信号通路的活化。结论 GOLPH3在前列腺癌细胞和组织中过量表达。GOLPH3在前列腺癌细胞增殖和转移过程中发挥重要作用。GOLPH3通过抑制p21,激活CDK1/2、cyclin B1等细胞周期蛋白和Akt-mTOR-p70S6K磷酸化来促进前列腺癌细胞增殖。GOLPH3能调控EGFR蛋白磷酸化及其下游Src蛋白磷酸化的表达。GOLPH3可能通过调控EGFR-Src信号通路及其底物MMP9影响前列腺癌的侵袭和转移。

Objective To study the expression of GOLPH3,a novel Golgi-related protein,in prostate cancer,and to explore its functional and molecular mechanisms in prostate cancer cells. Methods The expression of GOLPH3 in prostate cancer cells was detected by quantitative PCR and Western blot. The expression of GOLPH3 in prostate tissue was detected by immunohistochemistry. The proliferation of PC-3 cells was detected by CCK-8 method,and transwell was used to detect the invasion and migration of PC-3 cells after knocking down GOLPH3. Western blot was used to detect the expression of PC-3 cell cycle protein after knockdown of GOLPH3. Western blot was used to detect the expression of EGFR-Src and EGFR-Akt-mTOR signaling pathway in GOLPH3 RNAi and its negative control group. Results GOLPH3 was expressed in prostate cancer cells and tissues to varying degrees. The proliferation and growth of PC-3 cells were significantly inhibited by GOLPH3 knockdown（ P〈0. 05）. The cell number of PC-3 cells was significantly decreased（ P〈0. 05）. The proliferation and metastasis of PC-3 cells were significantly decreased after GOLPH3 gene silencing. After GOLPH3 knockdown,PC-3 cells in the G2/M period were a very significant block,suggesting that GOLPH3 gene in G2/M phase was involved in PC-3 cell proliferation cycle process; p21 expression in PC-3 cells were significantly increased,The expression of p-EGFR and p-Src protein were down-regulated,while the expression of p-AKT,p-mTOR and p-p70S6K were down-regulated. The expression of p-EGFR and p-Src protein was significantly decreased,while EGFR,Akt,mTOR,Src,FAK,p-FAK,p70S6K protein expression did not change significantly. Suggesting that blocking GOLPH3 can inhibit the activation of EGFR-Src signaling pathway. Conclusions GOLPH3 is over expressed in prostate cancer cells and tissues. GOLPH3 plays important roles in the proliferation and metastasis of prostate cancer cells. GOLPH3 promotes the proliferation of prostate cancer cells by inhibiting p21,activating CDK1/2,cyclin B1 and cyclin and Akt-mTOR-p70S6K phosphorylation. GOLPH3 regulates the phosphorylation of EGFR protein and the expression of Src protein phosphorylation downstream. GOLPH3 may affect the invasion and metastasis of prostate cancer by regulating EGFR-Src signaling pathway and its substrate MMP9.