Gene Therapy Experimental Study Of Recombined Ientivirus-mediatal SiRNA Technology Targeting Pleiotrophin In Small Cell Lung Cancer

Objective To construct of siRNA lentiviral expressing vector targeting PTN(pleiotrophin)gene in human small cell lung cancer H446 cell and to study the RNAi effect on tumor growth and apoptosis of small cell lung cancer. At the same time, to investigate the changes in gene expression profiles of angiomotin (Amot), schlafen5 (Slfn5), metalloproteinase 9 (MMP-9) and vascular endothelial cell growth factor (VEGF) which are associated with the angiogenesis, tumor growth and invasion after gene silencing of pleiotrophin (PTN) in H446 cell of human small cell lung cancer. To construct of human small cell lung cancer H446 cells nude mice xenograft tumor models, and then observe the growth inhibition role of interference PTN virus towards xenograft tumor.Methods PTN expression in H446 cell was determined by RT-PCR and Western Blotting. Four pairs of small hairpin RNA specific for PTN were designed, synthesized and cloned into the Plvthm vector. The resulting lentiviral vector containing shPTN were confirmed by DNA sequencing named LV-shPTN. 293T cells were co-transfected with LV-shPTN, pRsv-REV, pMDlg-pRRE and pMD2G, then packed and purified slightly to produce lentivirus. After infecting H446 cells with recombinant lentivirus, PTN expression were determined by real-time RT-PCR and Western Blotting. Finally we packed and purified the selected lentiviral vector with best interference efficiency in large scale and determined the titer of virus. Use the packed virus to infect H446 cell, the experiment was divided to normal cell group, negative control group, PTN interference group, chemotherapy group and combined group with RNAi and chemotherapy. The effect on cell growth and apoptosis of H446 cell infected with high titer virus were analyzed using MTT and FCM. The expression of Amot, Slfn5, MMP-9 and VEGF were detected using RT-PCR method in nomal non-interference group, negative control group, PTN interference group and combined group with PTN interference and chemotherapy. Use direct subcutaneous inoculation method with H446 cells to construct of human small cell lung cancer H446 cells nude mice xenograft tumor models, the experiment was divided to nomal non-interference group, chemotherapy group, PTN interference virus group, combined group with RNAi and chemotherapy and negative control virus group. After multi-point subcutaneous inject virus or cisplatin near the tumor, the tumor volume were measured separately at No.1,2,7,15 days. At No. 15 day PTN expression in nomal non-interference group, negative control virus group and PTN interference virus group was determined separately by RT-PCR and Western Blotting.Results The results of RT-PCR and Western blot test showed that PTN expression in H446 cell was higher then in H460. DNA sequencing analysis confirmed that shPTN lentiviral vector was successfully established as expected .The interference efficiency of constructed ShRNA sequence(sGCAGCTGTGGATACTGCTGAA) targeting PTN was as high as 72.1% and 59.2% at the mRNA and protein level respectively in H446 cell line. The titer of concentrated virus was 1×108TU/mL. Compared to normal cells and control group, cell viability of PTN interference group was decreased. Compared to RNAi group and chemotherapy group alone, the combined group with RNAi and chemotherapy showed less cell viability and a higher apoptotic rate in a concentration independent manner of the virus. Compared to negative control group, the expressions of Slfn5 and MMP-9 in H446 cell were increased 165.1% and 47.3% while the ones of Amot and VEGF were down-regulated 33.1% and 26.6% respectively after gene silencing of PTN. The changes of the gene expression profile became more evident when chemotherapy was superimposed on PTN interference. For animial experiments, compared to nomal non-interference group and negative virus control group, the expressions of PTNmRNA and PTN protein in PTN interference virus group were decreased significantly in tumor tissue, the interference efficiency was as high as 39% and 28% respectively. Compared to nomal non-interference group and negative virus control group, the tumor volume of PTN interference virus group was reduced significantly. Reduced tumor volume became more evident when chemotherapy was superimposed on PTN interference.Conclusion By constructing PTN RNAi vector and tranfecting H446 cell, we can effectively reduce the PTN transcription and expression, inhibit the growth and promote the apoptosis of tumor cells, and then can further influence the expression of proliferation and metastasis-related genes in H446 cell. Animial experiments also showed that targeted therapy for PTN could significantly inhibit the growth of xenograft tumor in vivo.This method may become a useful therapeutic strategy for SCLC overexpressing PTN.