Investigation Of TGIF's Role In Gastric Carcinoma And Its Mechanism

Gastric carcinoma (GC) is a frequent neoplasm that severely threatens people's health. Although its mechanism is still unknown, many studies have shown that TGF-βparticipates in the processes of its initiation, invasion and metastasis. Both TGF-βand retinoid signaling pathways can inhibit cell growth and induce cell apoptosis and differentiation. TG-interacting factor (TGIF) is an inhibitor of both TGF-βand retinoid signaling pathways; moreover, the activation of MAPK pathway can lengthen its half-life time. However, its role in carcinogenesis is still unknown, particularly in gastroenteric tumors. Thus we attempt to investigate the effect of TGIF on biologic behaviors of GC cells and its mechanism. GC cell line, SGC-7901, is stablely transfected with plasmid PcDNA3.1-TGIF, with Western blot and immunocytochemistry screening for high expressing clone for TGIF. The proliferation of transfectant cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) and transmission electron microscopy (TEM). Then, Tumorigenicity of the transfectant cells was analyzed in nude mice compared with that of vector control and parental cells. MMP2, MMP9 and VEGF proteins were examined using immunohistochemistry in tumor tissues of nude mice. Lastly, VEGF, active MMP2 and MMP9 were determined by ELISA and gelatin zymogram respectively. TGIF can partially resist the growth inhibition induced by TGF-β1. TGIF has no effect on the proliferation, cell cycle and apoptosis of SGC-7901 cells without exogenous TGF-β1. Compared with two control cells, TGIF transfectant cells have more cell organellae under TEM, and the forming cell clones are smaller in plate forming efficiency. In vivo experiment, tumors originated from TGIF transfectant cells have no cancerous embolism formed inside whereas tumors originated from vector control or parental cells have obvious embolisms. The expression levels of MMP9 and VEGF in tumors from TGIF transfectant cells are much lower than that from the two controls, whereas MMP2 has no detectable difference among the three cells. TGIF was further confirmed to inhibit the expression level of VEGF in SGC-7901 cell's supernatants and also could inhibit the secretion of VEGF induced by TGF-β1. The experiments of gelatin zymogram show that SGC-7901 cells can secrete active MMP9 but not MMP2; moreover, TGF-β1 is capable of inducing the expression of active MMP9. The vector control cells get the similar results, whereas TGIF transfectant cells have no expression of active MMP9. Although TGIF can partially resist TGF-βmediated growth inhibition, it cannot worsen the biologic behaviors of GC cells. On the contrary, it can induce at least partially the differentiation of GC cells, and inhibit the metastasis of GC through the downregulation of VEGF and MMP9. To confirm the negative role of TGIF in growth inhibition and cell cycle arrest TGF-β1 mediated. GC cells line, SGC-7901, was transfected with TGIF antisense oligodeoxynucleotide (ASDON). Then we analyzed the transfectant efficiency by RT-PCR, its proliferation by MTT method, cell cycle and apoptosis rate via FCM. After transfection with TGIF ASDON, the proliferation of SGC-7901 cells was partially inhibited, and its inhibition rate was increased to 20.4% at 72h, whereas it has no effect on cell cycle and apoptosis in SGC-7901 cells. Followed by thetreatment of TGF-β1, the growth rate of cells transfected with TGIF ASDON was distinctly reduced by 30%, and the content of G1 phase cells increased more obviously compared with the cells transfected with mutated oligonucleotides or parental cells. TGIF can resist the negative regulation of TGF-β1 over proliferation and cell cycle. To further systemically investigate its mechanism by which TGIF acts on GC cell line, SGC-7901, we use the classic high throughput proteomic technology, two dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and bioinformatics, to analyze the differential profiles between vector control and TGIF transfectant cells. The total proteins from TGIF transfectant and vector control cells were separated by 2D electrophorosis. 760±41 and 680±38 spots were detected in TGIF transfectant and vector control cells respectively. Locus repeat analysis of protein spots showed that the mean deviation in IEF direction in TGIF transfectant and vector control cells were 0.864±0.123 mm and 0.843±0.115 mm respectively whereas the mean deviation in SDS-PAGE direction in these two cells were 0.812±0.109 mm and 1.125±0.123 mm respectively. So the well-resolved and reproducible 2DE patterns from TGIF transfectant and vector control cells were established. Analysis of 2DE patterns revealed that there were 592±18 spots matched between TGIF transfectant and vector control cells. One spot was upregulated and 19 downregulated in TGIF transfectant cells. Subsequently 4 interesting protein spots were excised from gels and then digested in-gel by trypsin respectively. After that, the digestions weredetermined by MALDI-TOF mass spectrometry. Lastly, the resulting pepide mass fingerprints (PMF) were used to search the MS Database with the software ---Mascot peptide mass fingerprint. Fascin 1 and alpha enolase were successively identified. TGIF may inhibit the metastatic capability of GC through downregulation of Fascin 1 and alpha enolase. Meanwhile, the downregulation of alpha enolase may contribute to the differentiation of GC cells. Malignant tumor cells and their surrounding cells can secret TGF-β1, which can upregulate the expressions of VEGF and MMP9, and promote the invasion and metastasis of neoplasms. TGIF is an inhibitor of TGF-β1 signaling pathway. We have shown that the metastatic capability of TGIF transfecting cells was reduced in nude mice tumorigeneity, and TGIF can inhibit the invasion and metastasis of tumor cells by downregulating the expressions and secretions of MMP9 and VEGF. To further examine the roles of TGIF, MMP9 and VEGF proteins in the metastasis of GC, we analyzed the expressions of these proteins and their correlation with clinical features on formalin-fixed, paraffin-embedded tissue sections from 76 patients with GC between 2000 to 2002 in Xiangya Hospital using immunostaining. The positive rate of TGIF protein in cancerous tissue was 46.1%(35/76), obviously lower than that in surgical marginal mucosa (P<0.05), and its downregulation was associated with the lymph node metastasis(P<0.05). The positive rates of MMP9 and VEGF proteins in cancerous tissue were 59.2%(45/76)and 56.6%(43/76)respectively, with higher expressions than that in surgical marginal mucosa (P<0.05). The overexpression of MMP9 was correlated with the lymph node metastasis,while the overexpression of VEGF was associated with invasion and the lymph node metastasis (P<0.05). The expressions of VEGF and MMP9 were inversely correlated with that of TGIF (P<0.05). TGIF may overcome the invasion and metastasis of GS by inhibiting the expressions of MMP9 and VEGF.