| Objective:We investigated the expression of Grb2-associated binding protein-2(Gab2) in colorectal cancer(CRC) tissues, and analyzed the correlation between Gab2 expression and clinicopathological characteristics of CRC patients. We researched the expression of Gab2 in human different CRC cell lines on the levels both m RNA and protein. Recombinant lentivirus vector bearing Gab2 gene and Gab2-sh RNA were transfected respectively into CRC cell lines, and Gab2 gene overexpression and silencingcell lines were successfully obtained. Then we exploredthe effects of Gab2 on biological characteristics including cellproliferation, migration, invasion and metastasis both in vitro and in vivo. We detected the protein phosphorylation levels of its downstream key signaling effectors in every group cells, and exploredthe possible signaling pathway that Gab2 participated in the proliferation, invasion and metastasis of CRC cells.Methods:1. The correlation between the expression of Gab2 and the clinical pathological parameters of CRC patients.In this study, 109 cases of specimens including 27 paired cancerous and matched adjacent normal frozen tissue samples, and 82 paraffin-embedded tissues were collected form CRC patients undergoing colorectal surgery. The m RNA and protein expression of Gab2 in frozen tissue samples were detected by q RT-PCR and Western blot, respectively. Immunohistochemistry was used to detect the expression of Gab2 protein in 82 cases of paraffin-embedded tissues, and to analyze the association of Gab2 expression with the clinicopathologic features of CRC.2. The expression of Gab2 in different CRC cell lines and its relationship with cell proliferation, migration and invasion.1) The expression of Gab2 in different CRC cell lines.We detected the expression of Gab2 in different CRC cell lines by q RT-PCR and Western blot, and screened out low and high Gab2 expressed CRC cell lines from the level of gene and protein.2) Lentiviral vector transfected CRC cell lines and its validation experiments.Lentiviral vector bearing Gab2 gene and its corresponding control empty vector were introduced into SW480 cells, and named LV-Gab2 and LV-Control; Lentiviral vector Gab2 gene silencing and its corresponding control empty vector were introduced into SW620 cells, and named LV-sh Gab2 and sh RNA-Control. After 72 h, we analyzed the m RNA and protein expression of Gab2 in different transfected cells by q RT-PCR and Western blot, respectively.3) The research about Gab2 regulating CRC cell proliferation, migration and invasion. The cell proliferation and colonigenic ability were detected by CCK-8 and colony formation assays, respectively; cell migration and invasion capabilities were evaluated with Transwell migrated and invasive assays, respectively. And then observe the roles of Gab2 in CRC cell proliferation, migration and invasion in vitro.3. The effect of Gab2 on growth and metastasis of CRC validated in vivo.1) Tumorigenicity assay: 24 BALB/c nude female mice(5-6 weeks) were randomly divided into four groups, six mice each group, the above four groups of transfected cells were hypodermic inoculation on back. Tumor formation was observed in these groups, including tumor volume and weight, and thus further validated the effects of Gab2 on the growth and proliferation of CRC cells in vivo.2) In vivo metastasis development assay: 24 BALB/c nude female mice(5-6 weeks) were immediately four groups, each group was six, and the above four groups of transfected cells were injected into the lateral tail veins. Tumor metastasis development was observed in these groups, including liver and lung metastasis rate and the number of metastatic nodules, and further validated the effects of Gab2 on the metastasis of CRC cells in vivo.4. To research the signaling pathway of Gab2 participating in CRC cell proliferation, invasion and metastasis.We first observed the effects of Gab2 on morphological alterations of CRC cells after lentiviral vector infection by inverted phase contrast microscopy. The m RNA and protein expression of E-cadherin and Vimentin in different transfected cells were detected by q RT-PCR, Western blot and immunofluorescence, the metastasis related factors including MMP7 and MMP9 were also detected by Western blot, and evaluated whether Gab2 regulated the processes of epithelial-mesenchymal transition(EMT)and in turn affected CRC cell migration and invasion. Then protein phosphorylation levels of its key downstream signaling effectors, including ERKl/2and AKT on Gab2 upregulated and interfered cells were detected by Western blot. Through these studies, we explore whether ERK and/or AKT signaling pathways participated in the proliferation, invasion and metastasis of CRC cells mediated by Gab2. At last, pharmacological inhibitors of ERK and/or AKT were applicated, and to definitude Gab2-mediated signaling pathway in the proliferation, invasion and metastasis of CRC cells.Results:1. Elevated m RNA(P=0.014) expression and protein(P=0.003) expression of Gab2 were found in most CRC tissues compared with the matched adjacent non-tumor tissues. Immunohistochemical analyses showed that Gab2 protein was upregulated in CRC tissues relative to adjacent normal tissues(P<0.05), and this overexpression was significantly correlated with lymph node metastasis, distant metastasis and TNM stage(all P<0.05).2. The expression levels on m RNA and protein of Gab2 expression were obviously increased in SW620 and LOVO cells, which have highly metastatic abilities, compared with either the poorly metastatic cell lines HT29 and SW480 or the normal human intestinal epithelial cell line FHC. A significant increase in the levels on m RNA and protein of Gab2 were observed in LV-Gab2 cells compared with the control cells(P<0.05), whereas a marked decrease in the levels on m RNA and protein of Gab2 were observed in LV-sh Gab2 cells compared with the control cells(P<0.05), which suggested that Gab2 gene overexpression and silencing cell lines were successfully constructed. Upregulation of Gab2 in SW480 cells markedly enhanced cellular proliferation activity and clone formation ability, and promoted cell migration and invasion(P<0.05). Conversely, downregulation of Gab2 in SW620 cells markedly reduced cellular proliferation activity and clone formation ability, and inhibited cell migration and invasion(P<0.05).3. Tumorigenicity analyses suggested that upregulation of Gab2 in SW480 cells induced tumorigenesis, and the tumor volume and weight was enhanced significantly when compared the control group(P<0.05).Conversely, silencing of Gab2 in SW620 cells reduced tumorigenesis, and the tumor volume and weight was decreased significantly when compared the control group(P<0.05). In vivo metastasis development analyses confirmed that the incidence of lung and liver metastases and the number of metastatic nodules were obviously enhanced in mice injected with SW480-Gab2 cells, compared with the control group(P<0.05). By contrast, silencing of Gab2 expression in SW620 cells inhibited the incidence of lung and liver metastases and the number of metastatic nodules compared with the control group(P<0.05).4. Elevated expression of Gab2 in SW480 cells, the cellular morphology changed from an epithelial phenotype to a mesenchymal phenotype with a decreased protein and m RNA expression of E-cadherin(P<0.05), and an increased protein and m RNA expression of vimentin(P<0.05). Conversely, silencing of Gab2 in SW620 cells, the cellular morphology changed from a mesenchymal phenotype to an epithelial phenotype with an increased protein and m RNA expression of E-cadherin(P<0.05), and a decreased protein and m RNA expression of vimentin(P<0.05). Overexpression of Gab2 in SW480 cells significantly increased phosphorylation of ERK1/2, MMP7 and MMP9(P<0.05), whereas knockdownof Gab2 in SW620 cells obviously reduced the activationof ERK1/2, and reduced the expression of MMP7 and MMP9(P<0.05).Conclusion:1. Gab2 was overexpressed in CRC tissues and cell lines.2. Gab2 promoted cells proliferation, invasion and metastasis of CRC both in vivo and in vivo.3. Gab2 may enhance cells invasion and metastasis of CRC through the regulation of EMT.4. Gab2 was involved in cells proliferation, invasion and metastasis of CRC mainly through the MEK/ERK1/2 signaling pathway. |
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