| Abstract: | EMP3 (Epithelial membrane protein-3)，基因位於染色體 19q13.3，屬PMP-22/ EMP/MP20 family成員之一，屬於跨膜四次的大家族 (TM4SF)，已知可能參與細胞凋亡和分化作用，但EMP3在人類肝癌扮演的角色仍不清楚。
為了釐清EMP3在人類肝癌細胞和組織的表現，利用西方墨點法與免疫螢光發現低?分化的(HA22T/VGH、SK-Hep-1)中EMP3表現?較高?分化(Huh-7、HepG2)明顯增加。利用人類肝癌組織和肝癌組織晶片證實EMP3會大量表現在肝癌組織，並與臨床病人資料比對證實EMP3與肝癌組織分化程度有正相關 (P < 0.02)。為了證實EMP3在肝癌的生物功能，我們利用shLuc和shEMP3分別感染SK-Hep-1與Huh-7細胞來抑制EMP3蛋白，分別以MTT、流式細胞儀以及群落生成實驗證實抑制EMP3會降低細胞生長、誘導細胞週期G0/G1停滯，同時也證實抑制EMP3會抑制 Cyclin D1、Cyclin E 和 Skp2，且增加 p21Cip1和p27Kip1 蛋白表現。另外我們也證實抑制EMP3會抑制肝癌細胞移動與侵襲，同時抑制EMP3也會降低MMP9與uPA蛋白和分泌量。之前文獻證實MAPK會參與腫瘤細胞生長和侵襲過程，我們證實抑制EMP3會降低ERK1/2和Akt磷酸化，為了證實ERK和Akt是否參與EMP3調控肝癌細胞生長和侵襲能力，因此我們分別加入PD98059或LY294002作用在shLuc和shEMP3的SK-Hep-1與Huh-7細胞，實驗結果發現只有抑制EMP3和處理LY294002更能顯著抑制肝癌細胞生長和侵襲，同時也抑制 MMP9 與 uPA蛋白表達。反之，過度表現Akt則是減緩抑制肝癌細胞爬行與侵襲。因Akt的上游是PI3K途徑，我們利用西方墨點法，證實抑制EMP3會抑制PI3K(p85)蛋白表現，但不影響p110蛋白表現。利用免疫共沈澱和免疫螢光法證實EMP3與PI3K (p85)蛋白交互作用和共位現象。進一步處理shEMP3也能降低EMP3與PI3K(p85)交互作用和共位現象。此外，利用化學免疫染色法證實人類肝癌組織晶片內EMP3與p85 (R=0.3851，P <0.01)、p-Akt (R=03789，P <0.01)、MMP-9 (R=0.3697，P<0.02)和uPA (R=0.4986，P<0.001)有正相關現象。
研究指出大量表達 EMP3會和P2X7R結合引起膜起泡作用與死亡。因此我們想要了解P2X7R在四株人類肝癌細胞的表現，西方墨點法證實低?分化肝癌細胞(SK-Hep-1和HA22T/VGH)內P2X7R高於中度和高度分化肝癌細胞(Huh-7和HepG2)。接下來，利用免疫共沈澱和免疫螢光法證實P2X7R與EMP3有共同結合現象。此外，抑制EMP3會減弱P2X7R蛋白表現。綜合以上結果，EMP3在肝癌中可能扮演著致癌基因的角色，未來研究可以針對分子標靶作為平台，應用在治療抗癌模式。
The epithelial membrane proteins-3 (EMP-3) comprise a subfamily of small hydrophobic membrane proteins. The putative four-transmembrane domain structure as well as the genomic structure is highly conserved among family members. EMP3 were mapped to chromosomes 19q13.3. EMP3 is expressed in many tissues, and functions in cell growth, differentiation, invasion and apoptosis have been reported. However, its involvement in human hepatocellular carcinoma (HCC) progression remains unclear.
In our study, we found that EMP3 protein expression is higher in poor differentiated HA22T/VGH and SK-Hep-1 cells than in moderate and well differentiated Huh-7 and HepG2 cells, and HCC cancer tissue correlated significantly with tumor differentiation (P < 0.02). To verify the biological function of EMP3 in HCC cells, we found that Lentivirus-mediated EMP3 interference inhibited growth as assessed by the MTT assay. Likewise, inhibition of EMP3 expression induced cell-cycle arrest in SK-Hep-1 and Huh-7 cells by up-regulating the expression of p21Cip1 and p27Kip1, down-regulating the expression of Cyclin D1、Cyclin E and Skp2 protein expressions. Knockdown of EMP3 decreased migration and invasion in SK-Hep-1 and Huh-7 cells, and down-regulation of MMP9 and uPA. We examined the effect of down-regulation of EMP3 on the activation of ERK1/2 and Akt signaling. Western blot analysis showed that the expression of phosphorylated Akt was significantly decreased in EMP3 knockdown cells. Moreover, overexpression of Akt abolished EMP3-mediated cell migration and invasion. Conversely, inhibition of Akt phosphorylation remarkably reduced the migration and invasion of HCC cells with decreased expression of EMP3. We also found that EMP3 interacted with p85 (regulatory subunit of phosphoinositide 3-kinase [PI3K]) by immunoprecipitation and immunofluorescence assay. Conversely, inhibition of endogenous EMP3 by shRNA significantly reduced the interaction of p85 in SK-Hep-1 cells. In agreement with these findings, Moreover, EMP3 expression positively correlates with p85 (R=0.3851, P <0.01)、p-Akt (R=0.3789, P< 0.01)、MMP9 (R=0.3697, P < 0.02) and uPA (R=0.4986, P < 0.001) in tumor tissues from patients with HCC.
From literature showed that the constitutive overexpression of EMP3 in HEK-293 cells led to cell blebbing and cell death, therefore, we found that P2X7R protein expression is higher in poor differentiated HA22T/VGH and SK-Hep-1 cells than in moderate and well differentiated Huh-7 and HepG2 cells. In addition, immunoprecipitation and immunofluorescence analysis revealed that EMP3 associated with P2X7R. Conversely, EMP3 depletion reduced the P2X7 expression. Collectively, our results show that overexpression of EMP3 promotes human HCC progression by enhancing cell proliferation and invasiveness. Therefore, EMP3 is a master regulator of human HCC growth and might be a useful molecular target for HCC prognosis and treatment. |
