Effects Of EGFR Downregulation Via RNAi On Downstream Signal Transduction Pathways And Multi-drug Resistance Genes In Human Lung Adenocarcinoma Cell

BackgroundBecause of the dysregulation of epidermal growth factor receptor (EGFR) and its downstream cellular signal transduction pathways, EGFR over expression was observed in a large portion of different typesof tumor cells, which was closely related to the cells' malignant behaviors. Lung cancer has become the leading cause of death worldwide in recent years, which is a great threat to the health care system. EGFR expression or over expression could be detected in approximately 40-85% of non-small cell lung cancer (NSCLC) patients' tissues. Patients with EGFR over expression and/or activation usually progress faster and have poorer prognosis with lower sensitivity to radiotherapy and chemotherapy. Because almost 60-80% of lung caner patients were diagnosed in the advanced stage, which means the deprivation of surgical treatment option, the chemotherapy oriented multidiscipline treatment was the most feasible way to these patients. But primary and/or second drug resist nce remains the most common and important reason in lung cancer chemotherapy failure and post-chemotherapy relapse.RNA interference (RNAi) is a post-transcriptional gene silencing (PTGS) process mediated by double-strand RNA (dsRNA), which can efficiently inhibit the expression of target gene. Previously, our laboratory has already optimized the best target sequence of EGFR gene by chemical synthesed siRNA, which can efficiently suppress the growth of lung adenocarcinoma cells in vitro. Then we constructed a short hairpin RNA (shRNA) expression vector targeting EGFR. gene. We also observed that after downregulating EGFR in SPC-A1 cell by siRNA or shRNA, the SPC-A1 cell showed a 4 to 7 folds increase in sensitivity to cytotoxic agents like cisplatin, adriamycin and paclitaxel, as well as increase in sensitivity to radiotherapy. Based on these results, we observed the dosage effect and time-effect of shRNA expression vector targeting EGFR gene on survival rate of SPC-A1 cell and the expression of EGFR mRNA and protein. We further investigate the alterations of two main cellular signal transduction pathways downstream to EGFR, the Raf/MEK/ERK and PI3K/AKT pathway, and the mRNA expression level of three main genes that play important roles in tumor multidrug resistance (mdr1, lrp1 and gst-π), which may reveal the potential molecular mechanisms involved in the chemo-sensitive and radio-sensitive effects of shRNA that down regulated the EGFR expression of SPC-A1 cell.CharpterⅠ. Effects on EGFR expression and lung adenoma cell biology by siRNA expression vector targeting EGFRObjective: The present study aims at determining gene-silencing effects of plasmid-based short hairpin RNA (shRNA) targeting EGFR on the dosage effect and time-effect of EGFR expression and SPC-A1 cell biology such as survival rate and colony forming capacity.Methods: The shRNA plasmid was extracted and purified before transfection experiment. SPC-A1 cell was conventionally defrosted and cultured before transfection experiment, pShEGFR and pShNEG was transfected into SPC-A1 cell by Lipofectamine 2000 in each group, respectively. The SPC-A1 cell survival rates of different groups were detected by methyl thiazolyl tetrazolium chromatometry assay (MTT assay). Colony forming capacity of different groups was detected by colony formation assay. EGFR mRNA and protein of SPC-A1 cell at different time was quantified by real-time PCR and western blot after pShEGFR and pShNEG transfection respectively.Results: After transfection with pShEGFR, the survival rate and colony forming capacity of SPC-A1 were decreased by 78.7% and 68.8 (P＜0.01) with certain dosage and time effects. EGFR mRNA expression reached its lowest level (decreased by 84.3%) in day 2 after transfection with pShEGFR, and stepped up slightly in day 4 to day 10 while still significantly lower than the control group (P＜0.01). EGFR protein decreased significantly in day 2 after transfection with pShEGFR and reached the lowest point in day 6 with the inhibition rate about 82.6% (P＜0.01), and then slightly stepped up while still significantly.lower than the control group (P＜0.01).Conclusion: Our data suggested that shRNA expression plasmid-based siRNA could effectively inhibit EGFR mRNA and protein in SPC-A1 with certain dosage and time effects. The shRNA expression plasmid-based siRNA could also significantly influencethe SPC-A1 tumor cell biology behaviors such as survival rate and colony forming capacity, which provided a prospect for being developed as a novel cancer gene therapy in the future.CharpterⅡ. Effects on main cellular signal transduction pathways downstream to EGFR in lung adenoma cell by siRNA expression vector targeting EGFRObjective: The present study aims at determining the mRNA, total protein and phosphorylated protein changes of extracellular signal-regulated protein kinase (ERK1/2), phosphatidylinositol 3-kinase (PI3K) and serine/threonine Kinase (AKT) after pShEGFR and pShNEG was transfected into SPC-A1 cell, in order to analyze their potential role in tumor cell biology behavior alteration, chemo-sensitization and radio-sensitization.Methods: pShEGFR and pShNEG was transfected into SPC-A1 cell by Lipofectamine 2000. The mRNA expression levels of erk1, erk2, pi3k and akt gene were quantified by real-time PCR in control group, Lipofectamine 2000 group, pShNEG transfection group and pShEGFR transfection group at different time spots. The protein expression levels of ERK1/2, PI3K, AKT, p-ERK1/2, p-PI3K and p-AKT were quantified by western blot in control group, Lipofectamine 2000 group, pShNEG transfection group and pShEGFR transfection group at different time spots.Results: The mRNA expression level of erk1 raised significantly in day 2, 8 and 10 after pShEGFR transfection (P＜0.05). The mRNA expression level of akt raised significantly in day 6 and 10 after pShEGFR transfection (P＜0.05). The mRNA expression level of erk2 and pi3k showed no significant change (P＞0.05). The ERK1 and ERK2 protein first,decreased by 39.3% and 44.8% respectively in day 4 after pShEGFR transfection (P＜0.05), then increased by 30.7%-38.9% in day 8 and day 10 after pShEGFR transfection (P＜0.05) compared to the control group. The p-ERK1 and p-ERK2 protein decreased significantly in the day of pShEGFR transfection by 49.8% and 38.5% respectively (P＜0.05) and decreased even further in day 4 to day 8 (P＜0.01), then they increased slightly in day 10 while still significantly lower than those of control group (P＜0.01). The PI3K protein decreased by 37.9% and 41.2% in day 4 and day 6 after pShEGFR transfection (P＜0.05) while other time spots showed no difference (P＞0.05). The AKT protein decreased by 32.4% in day 8 after pShEGFR transfection (P＜0.05) while other time spots showed no difference (P＞0.05). Compared to those in control group, the p-PI3K protein decreased by 29.4% (P＜0.05) in day 6, then decreased by 43.7% and 58.3% respectively in day8 and day 10 after pShEGFR transfection (P＜0.01). The p-AKT protein decreased by 61.2% in day 6 (P＜0.05), and decreased by 72.8% and 85.6% respectively in day8 and day 10 after pShEGFR transfection (P＜0.01).The expression levels genes and proteins mentioned above showed no significant change in pShNEG transfection group (P＞0.05).Conclusion: The mRNA level of erkl and akt of SPC-A1 cell changed significantly after pShEGFR transfection, the protein level of ERK1/2, PI3K and AKT also changed significantly. The protein level of p-ERK1/2, p-PI3K and p-AKT changed significantly alter pShEGFR transfection with different pattern compared to those of mRNA and total protein.CharpterⅢ. Effects on main multidrug resistance gene mRNA expression in lung adenoma cell by siRNA expression vector targeting EGFRObjective: The present study aims at determining the mRNA changes of three main multidrug resistance genes, mdr1 and lrp1 and gst-π, after pShEGFR and pShNEG was transfected into SPC-A1 cell, in order to analyze their potential role in the chemo-sensitization process. Methods: pShEGFR and pShNEG was transfected into SPC-A1 cell by Lipofectamine 2000. The mRNA expression levels of mdr1 and lrp1 and gst-πgene were quantified by real-time PCR in the control group, Lipofectamine 2000 group, pShNEG transfection group and pShEGFR transfection group at different time spots.Results: Compared with the mRNA level (0.000191±0.00003) in control group, the mRNA level of mdr1 (0.0000833±0.000014) decreased by 56.4% in day 2 after pShEGFR transfection (P＜0.05), then decreased by 78.1%, 84.9%, 71.5% and 76,2% in day 4, 6, 8 and 10 respectively (P＜0.01). Compared with the mRNA level (0.171±0.033) in control group, the mRNA level of lrp1 (0.088±0.024) decreased by 48.5% in day 2 after pShEGFR transfection (P＜0.05), then decreased by 68.3%, 77.7%, 73.1% and 41.8% in day 4, 6, 8 and 10 respectively (P＜0.05). The mRNA level of gst-πgene showed no significant change among the control group and pShEGFR transfection groups (P＞0.05). The mRNA level of the above three genes in pShNEG transfection group showed no significant difference (P＞0.05).Conclusion: The mRNA level of mdrl and lrp1 gene hadsignificant changes after pShEGFR transfection, which may be the results of upstream cellular signal transduction proteins' changes. The mRNA level changes of mdr1 and lrp1 gene may at least partially explain the mechanisms of chemo-sensitization after pShEGFR been transfected into SPC-A1 cell.