Studies On Inhibition Of Epidermal Growth Factor Receptor Expression And Cell Cycle Progression In Epithelial Cancer Cells By RNA Interference

PartⅠStudies on RNA Interference Mediated Inhibition of Epidermal Growth Factor Receptor Expression and Its Biologic Effects in Epithelial Cancer Cells[Objects] The epidermal growth factor receptor (EGFR) is commonly over-expressed in a variety of human epithelial cancers, and has important roles in cancer pathogenesis and progression. EGFR thus provides a rational target for cancer therapy. We studied RNA interference mediated inhibition of epidermal growth factor receptor expression and its biologic effects in different human epithelial cancer cell lines (A431, HeLa and SPC-A-1) .[Methods] Cells were transfected with chemically synthesized small interference RNA (siRNA) using Lipofectamine2000．The receptor was detected by immunofluorescence staining and flow cytometry and EGFR mRNA was quantified by real-time PCR. The biologic effects on cell growth were assessed by colony-formation assay and MTT assay.[Results] 1) EGFR is over-expressed in A431, HeLa and SPC-A-1 detected by immunofluorescence staining, flow cytometry and real-time PCR. 2) EGFR expression was downregulated on day 1 and disappeared on day 2 after transfection detected by immunofluorescence staining while nonspecific siRNA had no interference effect. 3) siRNA-EGFR significantly decreased mRNA level by 73．9%, 44．6% and 57．7%, receptor expression by 77．0%, 61．3% and 65．2% in A431, HeLa and SPC-A-1, respectively. 4) siRNA-EGFR reduced colony number by 27．2%, 53．9% and 59．1% in A431, HeLa and SPC-A-1, respectively. 5) siRNA-EGFR inhibited cell viability by 31．7% on day 1 after transfection in A431 and by 33．2% and 31．3% on day 2 in HeLa and SPC-A-1 detected by MTT assay. The inhibition rate reduced on day 3～4, to agree with the RNA interference efficacy.[Conclusions] Our data suggested RNA interference could downregulate EGFR and inhibit cell proliferation in human epithelial cancer cells with different pathological types and EGFR expression levels, promising an extensive application in future targeted cancer therapy. cancer; Targeted therapyPartⅡStudies on Downregulation of Epidermal Growth Factor Receptor Expression and Inhibition of Cell Cycle Progression by RNA Interference[Objects] To determine effects of plasmid-based short hairpin RNA (shRNA)targeting EGFR on cell cycle progression and the probable mechanisms in humanlung adenocarcinoma cell SPC-A-1．[Methods] Plasmid-based short hairpin RNA targeting EGFR was transfected intoSPC-A-1 by lipofectamine2000．The effect of shRNA targeting EGFR on cell cycleprogression was assessed by flow cytometry. The expression of EGFR, downstreamsignaling proteins PI3K, AKT, ERK1/2 and cell cycle proteins cyclin D1, p21, p16was detected by immunoblotting.[Results] 1) Expression of EGFR in SPC-A-1 was downregulated by 89．9% on day 6and by 77．5% even on day 10 after transfection. 2) Plasmid-based siRNA caused G1cell cycle arrest with G0/G1 cell percent increasing by 21．4%, S cell percent reducingby 23．1%. 3) Expression of downstream signaling proteins phosphorylated PI3K,AKT and ERK1/2 were significantly downregulated on day 6 after transfection, whilenonphosphorylated PI3K, AKT and ERK1/2 had no change. 4) Expression of cyclinD1 was downregulated and expression of p21 was upregulated after transfection,while the expression of p16 had not change.[Conclusion] Our data suggested that plasmid-based shRNA could inhibit EGFRexpression more effectively and cause G1 cell cycle arrest by blotting downstreamsignaling pathway, downregulating cyclin D1 and upregulating p21 in human lungadenocarcinoma cell SPC-A-1．...