Effects Of RNAi On EGFR Gene In Human Osteosarcoma Cells

Osteosarcoma is the most predominant type of malignant bone tumor in childrenand adolescents between 15-25 years old. The prevalance of osteosarcoma in male istwo times than female. The prognosis of osteosarcoma is worse. 15-20％of patientshave clinically detectable metastases espesilly in lung at initial diagnosis. The 3-5year survival rate is only 5-20％even after amputation. Lesions deteted in the distalfemur and proximal tibia are account for 75％of total cases in osteosarcoma. Thepathology changes in osteosarcoma are mainly osteolytic. Despite treatment advances,chemotherapy is only marginally effective in most advanced cases. For those patientsrefractory to or intolerant of the current chemotherapy, treatment options are limited.Hence, more effective therapy with fewer side effects is needed.At present, the machanisiam of osteosarcoma is still unclear. The abnormalexpression of oncogene, suppressor gene, and apoptosis related gene, growth factorreceptor and disorder of intracellar signal pathway were regarded as to have key rolewith metastasis occur in osteosarcoma. Epidermal growth factor receptor (EGFR) is aglycoprotein with a molecular weight of 170,000 to 180,000. It is an intrinsictyrosine-specific protein kinase, which is stimulated upon epidermal growth factor(EGF) binding. EGFR signaling involved in cell growth, angiogenesis, DNA repair,and autocrine growth regulation in MG63 as well as in a wide spectrum of human cancer cells. Thus, it has recently emerged as an innovative target for thedevelopment of new cancer therapy.RNA interference (RNAi) is an evolutionarily conserved process in whichrecognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptionalsuppression of gene expression. This suppression is mediated by short hair RNA(shRNA), which induces specific degradation of mRNA through complementary basepairing. In several model systems, ie: mostly in lower order animals, this naturalresponse has been developed into a powerful tool for the investigation of genefunction. Recently it was discovered that introducing synthetic 21-23 nucleotidedsRNA duplexes into mammalian cells could efficiently silence gene expression. Inthis study, we investigated the possibility whether RNAi could silence EGFR gene incommonly used MG63 cancer cell lines. We also assessed the degree of EGFR genesilencing and its functional outcome in terms of effects on cell proliferation andgrowth inhibition in vitro.ObjectiveTo investigate whether RNA interference (RNAi) could induce gene silencing inosteosarcoma (MG63) cells; to evaluate the degree of epidermal growth factor (EGFR)receptor gene silencing and its effect on functional outcome of MG63 cells.Meterials and methodsTo determine whether EGFR is overexpressed and the effect of RNAi on EGFRgene in MG63 cells, EGFR gene RNAi lentivirus vector was constructed, MG63 celllines were transfected with target sequence-specific shRNA as well as variouscontrols. IHC and Western Blot were used to measure the expression of EGFR proteinin MG63 cells and reduction the EGFR protein in EGFR gene knocked down MG63cells. Quantitative reverse-transcriptase PCR was used to detect the expression andsilencing of the EGFR gene level. MTT assay, DAPI, TUNEL, transmission electronmicroscope (TEM) and Annexin V/P I double stain analysis were used to assess thefunction effects of EGFR gene science. ResultsWe have demonstrated here EGFR gene was overexpressed in MG63 cells. InMG63 cell line, we displayed sequence specific silencing of the EGF receptor with16.2％,27.3％,47.7％,52.3％and 54.5％(P＜0.01)in 12h,24h,48h,60h and72h respectivly of down-regulation of EGF receptor gene mRNA. The Quantitativereverse-transcriptase PCR technique was built.The expression of EGFR gene proteinis also downregulated significantly. Compare with the control group, the cellproliferation suppresion rate in the EGFR gene silence group in MG63 is 12.6％,30.0％(P＜0.01),39.1％(P＜0.01),47.1％(P＜0.01) and 62.1％(P＜0.01) in 12h,24h,48h,60h and 72h respectivly with MTT assay. The reduction in EGFR causedMG63 cells apoptosis, apoptotic MG63 cells with condensed or fragmented nucleistained by DAPI were observed and Brown stained apoptosis cells were seen byTUNEL and apoptosis bodies was shown by TEM also. The ratio of apoptosis werechanged from 1.9％to 12.6％, 31.6％and％36.2％respectively. Differencebetween control group and other groups had statistical significance (P＜0.05).ConclusionThese sequence specific shRNA-EGFR showed a blockbuster effect indownregulation of EGFR gene expression, inhibition of the cellular proliferation andmotility and arrestment of the cell cycle. The successful application of dsRNA-EGFRto reverse the neoplastic phenotype of EGFR overexpressing cells extends the list ofavailable therapeutic modalities in the treatment of human cancer. Our results suggestthat RNAi-mediated silencing of EGFR may provide an opportunity to develop a newtreatment strategy for osteosarcoma.