Cloning, Identification And Functional Analysis Of The Gene Encoding A Gastric Cancer Multidrug Resistance Associated Protein MGr1-Ag

Multidrug resistance (MDR) is the main cause of the failure of chemotherapy on gastric cancer. Several molecules, such as P-gp, MRP, LRP, BCRP, GST, PKC and TopoII, have been confirmed associated with MDR of tumor cells. However, the expression levels of these MDR molecules aren't high in gastric cancer, which suggests other unknown molecules and mechanisms underlying gastric cancer MDR. MGrl-Ag is a MDR associated protein that was initially found in our lab by hybridoma technique on vincristine-resistant gastric cancer cell SGC7901/VCR. It has been confirmed that the expression level of MGrl-Ag in SGC7901/VCR cells is much higher than that in SGC7901 cells, and that monoclonal antibody MGrl could partly reverse MDR of SGC7901/VCR cells and enhance adriamycin accumulation and retention in SGC7901/VCR cells. It is believed that isolating the encoding gene and exploring the function and mechanisms of MGrl-Ag will help us to completely understand MDR mechanisms of gastric cancer cells.Aims: To clone and identify the encoding gene of MGrl-Ag, and to analyze the functions and mechanisms of MGrl-Ag gene.Methods: (1) Two dimensional electrophoresis, Western blot and immuno-enzyme assay were performed to characterize the physical, immunologicalcharacteristics of MGrl-Ag. (2) Monoclonal antibody MGrl was used to screen cDNA library. The positive cDNA fragments were amplified from positive phage clones using PCR. After sequencing, DMA homology analysis was performed according to GenBank. (3) The expressions of positive cDNA fragments in gastric cancer cells were determined using reverse RNA blot and Northern blot. (4) Candidate cDNA fragments were subcloned into prokaryotic expression vector and expressed in E coli. (5) Prokaryotic expressed protein products of candidate cDNAs were purified by SDS-PAGE and used as immunogens to prepare murine antisera. (6) The antisera were characterized by Western blot using MGrl antibody as positive control. (7) The antisense RNA expression vector according to MGrl-Ag gene was constructed using DNA recombinant technique and introduced into SGC7901/VCR cells by liposome. Western blot was performed to detect MGrl-Ag in transfectant cells and control cells. (8) Cell cycle analysis was performed using flow cytometry and proliferous index (PI) was calculated. (9) Drug sensitivity assay was performed using MTT assay. IC50 values and resistance index (RI) of gastric cancer cells to anticancer drugs were calculated. ODD The intracellular adriamycin of gastric cancer cells was determined using flow cytometry. GO The protein products of interesting gene were analyzed using in vitro transcription/translation and detected by murine antiserum using Western blot. 0$ The interesting gene was further analyzed using bioinformatics.Results: (1) Total proteins of SGC7901/VCR cells were separated by two dimensional electrophoresis, electro-transferred to nitrocellulose filter and detected by monoclonal antibody MGrl. The results revealed that MGrl-Ag has an apparent molecular weight of 42KDa and an isoelectric point of 4.8. (2) As immuno-enzyme assay showed, MGrl-Ag lost its binding activity to MGrl after treated by trypsin or pepsin, but retained binding activity to MGrl after treated by sodium periodate. (3)Among 2.1X106 phage clones in primary cDNA library, 22 phage clones were characterized as positive clones by MGrl antibody. (4) Homology analysis revealed that these 22 positive cDNA fragments (designated as R1-R22) belong to 12 genes. Given the apparent molecular weight of MGrl-Ag is about 42KDa, only 4 genes were selected for further identification. These 4 genes are human 67KDa laminin receptor precursor (LAMRP, R7/R9/R15/R16/R19/R20), R10/R12/R13 (novel gene), R18 (novel gene) and R22 (novel gene). (5) As reverse RNA blot suggested, the expression levels of R12 and R18 in SGC7901/VCR cells are lower than that in SGC7901 cells, but the expression levels of R9, R15 and R22 in SGC7901/VCR cells are higher than that in SGC7901 cells. Given that the expression level of MGrl-Ag in S...