V62 Gene Upregulated In Drug Resistance Stomach Cancer Cells

Stomach cancer is the most popular cancer in China with the highest mortality and multidurg resistance (MDR) is the main obstacle in chemotherapy. There have been many MDR related molecules found in the study of MDR, while only some of them are expressed in stomach cancer or they could only reverse the MDR in gastric cancer partly. So there is a question: is there any special MDR related molecule for stomach cancer? There are several MDR cell lines built in the institute of the PLA digestive diseases by laddering dose method with the gastric cancer cell line SGC7901 as the parental cell line. Furthermore, some upregulated or downregulated genes were displayed by subtractive hybridization or difference display reverse transcribe polymerase chain reaction(RT-PCR) between the MDR cell line SGC7901/VCR and its parental cell line. In this study we want to kown the relation between V62, one of the upregulated gene in MDR cell line SGC7901/VCR, and gastric cancer MDR and to identify if it's a gastric cancer MDR related molecule by gene transfection.Objection: the relation between upregulated gene V62 and stomach cancer MDRMethods:1. Searching for the homologues gene of V62 in genebank by gene blast.2. Emply the CDS of V62 gene by RT-PCR3. Cloning V62 gene into cloning vector pUCm-T4. Displaying V62 gene between MDR cell line SGC7901/VCR and its parental cell line by Northern blot5. Construction of sense/antisense eukaryotic expression vector of V62 gene with eukaryotic expression vector pcDNA3.1(+)6. Transcribing and translating V62 gene in vitro7. Construction of gene transfection cell lines by electroporation8. Checking the drug sensitivity of gene transfection cell lines by MTT assay9. Checking the drug accumulation and retention in gene transfection cell lines by flow cytometer10. Prediction of the stricture and function of V62 gene by bioinformatics Results:1. Blasting in the genebank with the 300bp v62 gene difference displayed between MDR cell line SGC7901/VCR and its parental cell line, we found that there were several genes showing high homology with it. These genes were classified into 4 type according to their gene squenceso They were allhypothetical protein having the same sequences to form a gene family. Because the V62 gene snippet from subtractive hybridization as long as 300bp was too short to identified which gene it answering for.2. Cloned the CDS of V62 gene by RT-PCR. Taking OligodT as the primer, mRNA of SGC7901 /VCR cells as the templet, catalyzed by AMV for 2h, the cDNA of V62 gene was reverse transcribed; with 5' and 3' primers designed according to the starting site and ending site of CDS of V62 gene, and templet from V62 gene, a 900bp and a 400bp PCR products were gotten catalyzed by high fidelity DNA polymerase Taq polymerase3. Screened by blue-white method, the targeted gene were cloned into cloning vector pUCm-T separately; targeted gene snippets could be released by double endoenzyme digesting; sequencing results showed that the targeted gene have the completely same sequences to gene AF24 and AF11 encoding hypothetical protein separately4. V62 gene was higher expressed in SGC7901/VCR cells than its parental cells according to the double primer northern blot, in which the first probe was templeted by targeted gene and the second probe was templeted by home-keeper gene β-actin as the control. But the result showed that the mRNA of targeted gene was as long as 2.4kb, which is different to any reported gene in this family, so it suggested that this was a special gene transcribing product5. Eukaryotic expression vectors of V62 gene, sV62-pcDNA3.1 (+) # asV62-pcDNA3.1 (+) , were constructed on the base ofthe cloning vector and confirmed by double digestive reaction6. V62-939 gene was transcribed and translated into a 42KD protein in the rabbit homocell transcribing and translating system in vitro, which supported that V62 was a gene that could encoding a protein7. The sense/anti...