Transfection Of Rap2b Gene Into NIH3T3 Cells And Its Effect On P38MAPK Signaling Pathway

Lung cancer is one of the most common malignant tumors in terms of both incidence and mortality in the world. The occurrence and development of lung cancer is a complex biological process with multi-stage, multi-factor interaction and multi-gene participation. The expression levels of the related genes in lung caner tissues and normal tissues are significant different in this process. The discovery and study of these differentially expressed genes of lung cancer is not only conductive to reveal the molecular mechanism of tumor development, but also provides new markers and targets for diagnosis, prevention and cure of lung cancer.Rap2b gene is one of the genes screened from high expression library of human lung squamous carcinomas using suppression subtractive hybridization technique recently. Rap2b gene is one of the members of ras oncogene superfamily and has a high homology with ras. Previous research suggested that many tumors have mutation and over-expression of ras. Ras gene family members affect the downstream effectors mainly through the mitogen-activated protein kinase signal transduction system, P38MAPK is one of the members of the MAPK family. Therefore, rap2b gene might play an immportant role on the P38MAPK pathway.ObjectiveIn this study, in order to understand the role of rap2b on the P38MAPK pathway, we transfected the recombinant plasmid of pcDNA3.1-rap2b into NIH3T3 cells and applied of P38MAPK-specific inhibitors. This work will provide experimental data for elucidating the role of rap2b gene in human lung cancer.Experimental Methods1. Extraction of recombinant plasmid pcDNA3.1-rap2b and plasmid pcDNA3.1 Recombinant plasmids pcDNA3.1-rap2b and empty vector pcDNA3.1 were extracted using plasmid extraction kit and identified preliminary.2. Restriction endoenzyme digestion of recombinant plasmids Single-EcoRâ… digestion and EcoR I, Xho I double-enzyme digestion were carried out with rap2b recombinant plasmids, and results were observed by 1.0% agarose gel electrophoresis.3. Amplification of rap2b gene by PCRUsing rap2b recombinant plasmids as templates, PCR assays were performed with specific primers for rap2b genes, the amplified products were identified using 1.0% agarose gel electrophoresis, and purpose fragment of rap2b genes was observed.4. Sequencing of rap2b recombinant plasmidsRap2b gene was sequenced using universal primers of pcDNA3.1, and the sequence alignment was performed by BLAST in Genebank.5. Transfection of recombinant plasmids into NIH3T3 cellsRap2b recombinant plasmids without endotoxin were transfected into NIH3T3 cells,48 hours and 72 hours later identification were performed in nucleic acid and protein.6. Application of P38MAPK inhibitorsP38MAPK inhibitors were added 1 hour before transfection of recombinant plasmid and protein identification was performed 72 hours later.7. Western-blot assaysNormal cells were used as the control group, and the expression levels of phosphorylated ATF-2 protein and total ATF-2 protein were compared by Western-blot assays in the empty vector group, the recombinant plasmid group, the recombinant plasmid with inhibitor group.Results1. Extraction and identification of recombinant plasmid pcDNA3.1-rap2bSingle colonies were picked and cultured overnight, and plasmids were extracted and identified. EcoR I single digestion products were 5.5kb, EcoR I and Xho I double enzyme digestion products were 5.5kb and 0.629kb, which were consistent with the expected results. Using positive plasmids as templates, PCR assays were performed with specific primers for rap2b genes, and DNA fragments of 629bp were acquired. Recombinant plasmids pcDNA3.1-rap2b were confirmed by sequencing and alignment.2. The results of NIH3T3 cells transfection Endotoxin-free pcDNA3.1-rap2b plasmids were extracted and transfected into NIH3T3 cells using liposome. RNA were extracted and RT-PCR assays were performed, the objective band was consistent with expected result. RAP2B protein expression was detected in pcDNA3.1-rap2b group, and not detected in plasmid pcDNA3.1 group, blank control group.3. The results of Western-blot assaysNo difference was observed between the control group and the empty vector group. ATF-2 Phosphorylation protein expression level was higher in the recombinant plasmid group than the control group(P<0.05), and it was lower in the recombinant plasmid with inhibitors group than the recombinant plasmid group(P<0.05).ConclusionsRap2b gene activated the P38MAPK pathway and up-regulated the downstream ATF-2 phosphorylation, it played an important role on P38MAPK pathway. This study may provide experimental evidence to explore the role of rap2b gene in the occurrence of lung cancer.