| Chemotherapy is one of the main treatments of cancer. However, dispite of the emergence of new reagents and schemes of chemotherapy, the clinical outcomes of chemotherapy have not made much progress. The main reason is that the cancer cells have developed multidrug resistance (MDR) to chemotherapy drugs. Thus, it has great significance to clarify the cause of MDR and explore the effective ways to reverse MDR . Ss-A/Ro ribonucleoprotein 60KD subunit (Ro 60) has been found highly expressed in MDR cell SGC7901/VCR by the modified differential-display polymerase chain reaction through SGC7901/VCR versus SGC7901, as was confirmed by reverse northern blot. Thereby, to elucidate the function and mechanism of Ro 60 in multidrug resistance of gastric cancer may help us to understand the mechanisms of MDR in gastric cancer.Aims: To investigate the function and mechanism of Ro 60 in multidrug resistance of gastric cancer.Methods: ?Clone Ro 60 encoding cDNA using RT-PCR method. ?Detect expression levels of Ro 60 in gastric cancer cell lines bysemi-quantitative RT-PCR. (3) Construct Ro 60 cDNA sense and anti-sense expression vector using DNA recombination technique, then transfect them into SGC7901 and SGC7901/VCR respectively, and detect expression levels of Ro 60 in transfected cells by semi-quantitative RT-PCR. (4) Draw life curves of transfected cells and perform in vitro drug sensitivity assay using MTT assay, calculate 1C50 values of gastric cancer cells to chemotherapy drugs. (5) Measure the intracellular accumulation and retention of adriamycin in gastric cancer cells using FACS. (6) Investigate the mechanism of Ro 60 in MDR of gastric cancer by drug block test and western blot.Results: (D Full length of Ro 60 cDNA was cloned, two transcription variants of Ro 60 were found, and were named Ro 60 variant 1(V1) and Ro 60 variant 2 (V2) respectively. The two variants were submitted to GenBank (Accession number: AY205315 : AY205314) . (2) The results of semi-quantitative RT-PCR show that the expression level of Ro 60 in SGC7901/VCR was higher than that in SGC7901 and in Hela cell line, so were the results of two variants. (3) The result of semi-quantitative RT-PCR showed that the expression level of Ro 60, VI or V2 in SGC7901 was increased after transfection with sense genes; and the expression level of Ro 60 in SGC7901 /VCR was decreased after transfection with anti-sense gene. (D In vitro drug sensitivity assay showed that SGC7901 cells transfected with Ro 60 sense genes showed significantly decreased sensitivity to VCR, 5-FU, MMC , CCDP and ADR , when comparing with SGC7901 and SGC7901-pcDNA3.1 cells; and SGC7901/VCR cells transfected with Ro 60 anti-sense gene showed significantly increased sensitivity to VCR, MMC, CCDP and ADR , when comparing with SGC7901/VCR andSGC7901/VCR-pcDNA3.1 cells. (5) As showed by flow cytometry, SGC7901 cells transfected with Ro 60 sense gene showed decreased accumulation and retention of adriamycin; SGC7901/VCR cells transfected with Ro 60 anti-sense gene showed increased accumulation and retention of adriamycin. (6)Results of verapamil block assay showed that the decreased sensitivity to chemotherapy drugs of SGC7901 cells transfected with sense genes could be reversed by verapamil partially.(7) As revealed by western blot, the expression level of P-gp and bcl-2 in SGC7901/VCR were decreased after transfection of Ro 60 anti-sense gene.Conclusions: (1) Two transcription valiants of Ro 60 were discovered in SGC7901/VCR, Ro 60 and it's two transcription variants were found to be highly expressed in SGC7901/VCR cells in comparison with SGC7901 cells and Hela cells. (2) The the SGC7901 cells transfected with sense genes show some competence of MDR, and SGC7901/VCR cells transfected with Ro 60 anti-sense gene show decreased competence of MDR. (3) Ro 60 might interfere with MDR through influencing drug accumulation in rumor cells. The function of Ro 60 in MDR of gastric cancer might be associated with P-gp, bcl-2.
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