Plasmid-mediated Rna Interference Of Human Telomerase Reverse Transcriptase In Lung Cancer

Background and objectiveLung cancer has become the one of the most common primary malignant cancer. In China, the mortality of lung cancer is in the first place. Although the current prefered treatment for lung cancer is surgical treatment, meanwhile taken radiotherapy, chemotherapy and other traditional therapies at the same time, but these traditional therapies do not have the breakthrough, so people are still working hard to develop new effective treatments.Gene therapy change people's genetic material-based bio-medical treatment in cancer treatment, which has become a new hot spot in recent years. RNA interference (RNA interference, RNAi) is a small interfering RNA (small interference RNA, siRNA) to guide the specific degradation of mRNA targeting post-transcriptional gene silencing. By RNA interference silencing of target gene inhibition, thus inhibiting disease-related RNA, abnormal protein synthesis, and ultimately achieving the purpose of regulating cell growth.The relationship between telomerase activity and tumor development is very close links. Telomerase activation in lung cancer occurrence and development plays an important role as its catalytic subunit human telomerase reverse transcriptase (hTERT), which is the activity of telomerase play a limiting factor of telomerase, thus treatment of lung cancer is expected to become an ideal target for RNA interference. In this study, it was tconstructed that plasmid vector-mediated siRNA interference targeted hTERT gene, and stably transfected into lung cancer cell line H1299. By real time quantitative PCR technology, western blot technique, the cell cycle by flow cytometry techniques, the role of hTERT gene silencing in lung cancer cell proliferation was explored which induced by plasmid-mediated targeting hTERT siRNA interference vector.Methods1. Construction of interference vectors, according to the experimental group study, select a pair of siRNA, designed and synthesized DNA template primers, the template primer was cloned into pGenesil-1.1 plasmid vector promoter followed by restriction enzyme digestion and gene sequencing constructed interference vector .2. Reorganization interference vectors and empty vector transfected by cationic liposomes to lung cancer cell H1299. After selection and monoclonal culture, stable transfected cell was obtained. Protein expression level of hTERT was detected by real Time RT-PCR technology and western blot.3. Cell growth and proliferation capacity were measured by MTT and colony formation count.4. Cell cycle of lung cancer cells was measured by Flow cytometry.Results1. The interference of vector pGenesil.1-hTERT was successfully constructed. Restriction enzyme digestion and gene sequencing confirmed that the insertion sequence met the design requirements.2. PGenesil.1-hTERT transfected cells H1299 were obtained after stable transfection of monoclonal cell line H1299-pGenesil.1-hTERT and H1299-pGenesil.1. Compared with stable blank plasmid transfected cells group, expression of hTERT mRNA cells decreased significantly in stable transfection of siRNA plasmid, inhibition rate was 93.97±0.83%, P <0.01.3. The expression of hTERT protein was suppressed Lung cancer H1299- pGenesil. 1-hTERT cells, compared with H1299-pGenesil.1 cells. 4. Compared with H1299-pGenesil.1, cell proliferation ability Lung cancer H1299-pGenesil.1-hTERT cells was decreased.5. Compared with the control group, H1299-pGenesil.1, G1 phase cells increased and S/G2 phase cells were decreased in H1299-pGenesil.1 group , Respectively 18.3% (P <0.05), 10.4%( P <0.05), 7.9% ( P <0.05).Conclusions1. The vector of pGenesil.1-hTERT has been constructed successfully.2. The expression of hTERT mRNA and protein was significantly suppressed by transfection siRNA interference plasmid. It could regulate the activity of telomerase , proliferation and cell cylce of lung cancer cells...