RNAi-mediated Silencing Of Type 1 Insulin-like Growth Factor Receptor Inhibited Growth And Metastasis Of BGC823 Human Gastric Cancer Cel In Vitro

Chapter I Overexpression and clinical significance of Type 1 Insulin-like Growth Factor Receptor (IGF-IR) in human gastric cancerObjective: To detect the expression of IGF-IR in gastric cancer(GC), and to evaluate its clinical significance. Materials and Methods: IGF-IR expression in 50 fresh GC tissues as well as in 113 GC patients who had accepted resection were detected by RT-PCR , Western blot and Immunohistochemistry. Associations of IGF-1R with clinicopathological characteristics were subsequently assessed. Results: RT-PCR and Western blot showed positive expression level of IGF-1R were significantly higer than Para-cacinoma(PC) or Non-carcinoma(NC) tissues(P<0.05). IGF-IR expression was associated with depth of gastic wall invasion, regional lymph node metastasis, TNM stages(P<0.05). Immunohistochemistry showed IGF-IR was stainned mainly on cell memberance and in cytoplasm. Similarly, overexpression level of IGF-IR was significantly higher in GC than PC or NC(P<0.05). IGF-IR was associated with depth of gastic wall invasion, regional lymph node metastasis, TNM stages as well as differentiation status of GC(P<0.05). Furthermore overexpression level of IGF-IR was correlated with poor prognosis. Conclusions: IGF-IR was obviously up-regulated in GC, Overexpression of IGF-1R might be the feature of GC and benefit us in predicting the prognosis.Chapter II Down-regulate the Expression of (IGF-IR) by Small Interfering RNA (siRNA) in human gastric cancer cell line BGC823.Objective: To construct the siRNA expression vector targeting IGF-IR and evaluate its ability to downregulate the expression of IGF-IR in human gastric cancer cell line BGC823. Materials and Methods: Three siRNA expression vectors, pIGF -IR-siRNA1, pIGF-IR- siRNA2 targeting IGF-IR , were constructed using pSUPER vector, and adenovirus, pcontrol-siRNA was constructed as control, and it was identitied by PCR. The levels of mRNA and protein expressions were determined by RT-PCR and Western blot analysis after recombinant plasmid pIGF-1R-siRNA1, pIGF-IR-siRNA2 and pcontrol-siRNA were transfected into BGC823 cells 48h, respectively. Results: pSUPER-IGF-IR-siRNA1, pSUPER-IGF-IR-siRNA2 and pSUPER-Control-siRNA were validly transfected into gastric cancer cell line. After 48h transfection, level of IGF-IR mRNA and protein expression of IGF-IR in cell line BGC823 were significantly decreased by pSUPER-IGF-IR-siRNAl as well as pSUPER-IGF-IR-siRNA2 comparing with either pSUPER-Control-siRNA or PBS-negative-group(P<0.05). Conclusions: We successfully contructed the siRNA for down-regulating the expression of IGF-IR in gastric cancer cell line.Chapter III Down-regulation of IGF-IR expression by Small Interfering RNA Inhibited Human Gastric Cancer Cell Proliferation,invasion and Increased Apoptotic Response in vitroObjective: To evaluate the effect of IGF-IR silence on the proliferation, apoptosis and invasion of Human Gastric Cancer Cell line BGC823. Materials and Methods: The cell cultures were measured for cell proliferation levels at different time points (at 0h, 24h,48h and 72h after transfection) using MTT assay; cell cycle of gastric cancer was observed by FCM; both PI stainning and Apoptotic DNA ladder detection were used to examine the apoptosis of tumor cells. Transwell test and cell "scratch" experiment were used to evaluate the invasion and metastasis of BGC823 cell after transfection. Results: MTT assay indicated that the proliferation level of BGC823 cells were no different at 24h, but obviously decreased at 48h and were maintained at 72h after transfection with pSUPER-IGF-IR-siRNA2. The inhibition ratio of viable cell at 48h and 72h were 62.15±0.98% and 59.82±0.68%, obviously higher than negative control (P<0.05). FCM showed at 48h pSUPER-IGF-IR-siRNA2 transfection induced an increase in G₀/G₁ phase cells (59.42±4.01%) as compared with pSUPER-Control-siRNA or PBS-negative-group (19.72±1.61 % or 14.33±2.15%) (P<0.05). It was shown that down-regulation of IGF-IR obviously enhanced apoptotic response to 3 % ethanol of BGC823 cells by using apoptotic DNA ladder detection. In the transwell test, BGC823 cells transfected with pSUPER-IGF-IR-siRNA2 significantly reduced invasion. And the results of cell "scratch" experiment indicated that there exsisted significant difference of the scratch distance at beginning of 12h. The scratchs were cured in the pSUPER-Control-siRNA transfection group and PBS-negtive group at 36h, but only about 40% was observed in group transfected by pSUPER-IGF-IR-siRNA2. Conclusions: IGF-IR silence greatly inhibited proliferation and invasion of human gastric cancer and obviously increased apoptotic response.Chapter IV The study of the role of IGF-IR in gastric carcinoma based on co-immunoprecipitation -Western blot -mass Spectrometry technologyObjective: To investigate the dysfunction molecular mechanism of IGF-IR protein in gastric cancer cells. Methods: Based on the co-immunoprecipitation analysis and mass Spectrometry technology. first, co-immunoprecipitation techniques from gastric cancer cells BGC823 enrichment IGF-IR-binding protein, to SDS-PAGE electrophoresis technology combined with IGF-IR protein complexes were separated, the second cut differences in protein bands through the bleaching, reduction, alkylation, in situ enzyme, sample extraction, desalination and sample, and then analyzed by ESI-MS/MS, peaklist document analysis system for the importation of Mascot retrieval. IGF-IR for identification of binding proteins. Results: 24 protein spots outcome data were identified in cluding Sorcin protein. Conclusion: It is feasible to analysis of gastric cancer cells in IGF-IR molecular interaction based on co-immunoprecipitation combination to mass Spectrometry , the mechanism of IGF-IR and Sorcin protein interaction is maybe play important role in regulation development and the drug resistance in gastric tumor cells.