[Construction of siRNA lentiviral expressing vector targeting pleiotrophin gene and its impact on growth and apoptosis in H446 cells of human small cell lung cancer].
OBJECTIVE
To construct a siRNA lentiviral expressing vector targeting PTN (pleiotrophin) gene in human small cell lung cancer H446 cells and to study the RNAi effect on tumor growth and apoptosis.


METHODS
Four pairs of small hairpin RNA specific for PTN were designed, synthesized and cloned into the Plvthm vector. The resulting lentiviral vector containing shPTN was 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 was determined by real-time RT-PCR and Western blot. Finally the selected lentiviral vector was packed and purified with best interference efficiency in large scale and the titer of virus was determined. The packed virus was used to infect H446 cells, and then the experiment was divided to a normal cell group, a negative control group, a PTN interference group, a chemotherapy group and a combined group with RNAi and chemotherapy. The effect on cell growth and apoptosis of H446 cells infected with high titer virus was analyzed using MTT and FCM.


RESULTS
DNA sequencing analysis confirmed that shPTN lentiviral vector was successfully established as expected with interference efficiency as high as 72% and 59% at the mRNA and protein level. The titer of concentrated virus was 1 x 10(8) TU/ml. Compared to normal cells and control group, the 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.


CONCLUSIONS
PTN RNAi vector construction and H446 cell transfection effectively reduced the PTN transcription and expression, inhibited the growth and promoted the apoptosis of tumor cells. This method may become a useful therapeutic strategy for small cell lung cancer overexpressing PTN.
Yu
Yong
Y
Department of Respiratory Medicine, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
Shi
Min-hua
MH
Xu
Xun
X
Hu
Hua-cheng
HC
D017209:Apoptosis / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D055752:Small Cell Lung Carcinoma / Q000235:genetics; D014162:Transfection
D002352:Carrier Proteins; D016207:Cytokines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C066845:pleiotrophin