Research Of The Effect Of RNAi-mediated Gene Silencing Of C-erbB-2 On Proliferation And CDDP Drug Sensitivity Of Lung Adenocarcinoma Calu-3 Cells

ObjectiveThe c -erbB -2 gene is a member of the epidermal growth factor (EGF) -receptor (EGFR) family and encodes a 185 - kDa protein with tyrosine ki-nase activity. The amplification or enhanced transcription of the c - erbB - 2 gene demonstrated in many different types of human cancers, including breast, ovarian, lung cancers are associated with enhanced malignancy and the meta-static phenotypes and intrinsic chemoresistance and poor prognosis. RNAi technology is a new genetic technique, and can suppress gene expression with high efficiency and specificity. In this study, we investigated the effect of synthesized c - erbB - 2 specific siRNA on c - erbB - 2 dysregulated human lung adenocar-cinoma cells.Methodscalu - 3 cells were transfected with siRNAs formulated IipofectAMINE 2000, The c - erbB - 2 and Cyclin D₁ mRNA level were detected by reverse transcription - polymerase chain reaction ( RT - PCR) , The biological morphology and growth inhibition of calu - 3 and the chemosensitivity of transfected cells to cisplatin (CDDP) were observed with light microscopy and MTT assay respectively. The c - erbB -2 protein level and the cell cycle and apopotosis rate were analyzed by FCM. VEGF level in culture supernatant was detected by ELISA. Caspase -3 activation was tested by fluorospectrophotometry.ResultsSiRNA targeting c - erbB - 2 down - regulated the transcription of c - erbB -2 oncogene and protein level. The cells infected with c - erbB -2 siRNA 48h after transfection showed markedly less c - erbB - 2 protein. The c - erbB - 2 protein positive expression rate in c - erbB - 2 siRNA group, in untreated control group, empty vector group or unrelated siRNA group was (25. 04 ± 1. 56)%,(98.24 ±2.23)%,(95. 67±1.98)% or (94.79 ±0. 87)% , respectively. There was significant difference between c - erbB - 2 siRNA group and other else three groups (P <0.01). Slowed cell proliferation and cell cycle arrested at Gq/Gj stage [ c - erbB -2 siRNA group (56.6±3.6)% vs. untreated control group (45.5±3.2) %, P <0.01] could also be observed, accompanied with enhanced cell apoptosis. The FCM results showed that the apoptosis rate of CDDP combined with c - erbB - 2 siRNA was elevated when compared with unrelated siRNA group, empty vector group and untreated control group.ConclusionSpecific siRNA targeting c - erbB - 2 could effectively inhibit c - erbB - 2 expression and cell proliferation in c — erbB - 2 positive calu - 3 cell lines, consequently led to the decline of Cyclin D, and VEGF level and activation of Caspase - 3 pathway, thus arrested cell cycle at Go/1 stage and enhanced cell apoptosis. Simultaneously sequence specific siRNA - c - erbB - 2 was capable of enhancing the chemosensitivity of calu - 3 cells to cisplatin. SiRNA mediated gene silencing may be a useful therapeutic strategy for cancer.