| Epstein-Barr virus (EBV) is a gamma herpes virus closely associated with a variety of human malignancies, including nasopharyngeal carconima (NPS), Burkitt's lymphoma(BL) and gastric carcinoma (GC). Among the EBV latent genes, latent membrane protein 1(LMP1) is considered as the principal oncoprotein. LMP1 could promote cell proliferation and inhibit cell apoptosis, LMP1 inhibits the neoplastic cell differentiation, LMP1 may be implicated in the carcinogenesis, invasion and metastasis of carcinoma.Small or short inhibiting RNAs are siRNAs which act as mediators of sequence-specific mRNA degradation. RNA-induced silencing complex (RISC) guides hybridization of the siRNA antisense strand to its complementary target sequence and initiates cleavage of the target mRNA. Recently, siRNA has gained widespread use as a tool to achieve sequence-specific inhibition of target gene expression invitro.In this study, small interfering RNAs (siRNA) targeting LMP1 was employed to investigate the effect of LMP1 on cell proliferation and apoptosis in EBV-positive gastric epithelium cell lines. Our findings also support the possibility for using siRNA targeting LMP1 as therapeutics in those EBV-associated tumors expressing the LMP1 gene.Objective To synthesize siRNA that targets LMP1 coding gene and transfect EBV positive GT38 cells. To investigate the specific silencing effect of siRNA to the expression of LMP1 and the influence on target cell due to LMP1-specific silence.Methods①The fluorescently-labeled siRNA (FAM-siRNA) was transfected into target cells using 20, 30, 50, 80 and 100nM final concentration by lipofectamine 2000 protecting from light. Fluorescence microscope was used to detect transfection efficiency which is equal to the ratio of fluorescent cell numbers and total cellular score in the same field of vision.②Three chemically synthetic siRNA(siRNA649, siRNA979 and siRNA1348) targeting LMP1 were transfected into target cells by lipofectamine 2000 at 50nM final concentration, and then the LMP1 expressions were tested by RT-PCR.③The best one targeting LMP1 was transfected into target cells by lipofectamine 2000 as suitable final concentration, then the expression of LMP1 and NFκB were tested by Western blotting. Bcl-2, Bax, MMP9 and ICAM-1 mRNA were tested by RT-PCR. Hoechst 33258 staining, transmission electron microscope and flow cytometry were used to detect apoptosis, variation of ultramicrostructure and cell cycle of target cells, respectively.Results①Punctiform green fluorescence appeared in all groups of GT38 cells transfected FAM-siRNA at 12h. We counted the numbers in random 5 fields of vision and calculated transfection efficiency. 50nM final concentration was adopted to transfect into target cells in our experiment.②RT-PCR results showed that siRNA649, siRNA979 and siRNA 1348 markedly inhibited the expression of LMP1 in target cells compared with the non-specific control (F=235.99, P<0.05), and the effect of siRNA649 was most obvious. RT-PCR results showed obviously discrepancy 24h, 48h and 72h post-transfection with siRNA649 compared with cell control group (F=89.93. P<0.05), and the effect of 48h and 72h post-transfection with siRNA649 were most obvious. Western blotting results also showed obviously discrepancy 48h, 72h and 96h post-transfection with siRNA649 compared with cell control group.③Apoptosis was observed in target cells transfected by siRNA649 with Hochest33258 staining. Chondriosome vacuolization and chromatin pyknosis appeared in target cells transfected by siRNA649 with transmission electron microscope. Flow cytometryanalysis showed no obviously discrepancy 24h, 72h and 120h post-transfection with siRNA649 compared with non-specific control group and cell control group.④Western blotting results showed obviously discrepancy of NFκB in cellular nucleus and cytoplasm post-transfection with siRNA649 compared with cell control group.⑤RT-PCR results showed that siRNA649 markedly inhibited the expression of Bcl-2(F=39.83, P<0.05),MMP9(F= 177.47, P<0.05),ICAM-1(F=467.69, P<0.05) and Bcl-2/Bax(F=5.45, P<0.05) in target cells compared with the untransfected cell control. No obviously discrepancy was found in Bax.Conclusion Chemically synthetic siRNA targeted LMP1 could effectively silence the expression of LMP1 in EBV positive cell GT38. The inhibition effect of LMP1 mRNA is associated with the transfection time and is proportioned to siRNA concentration. The effect of inhibition is specific and relates to the siRNA sequence. The NFκB expression decreased when LMP1 was silenced. Then it may down regulate Bcl-2/Bax,MMP9,ICAM-1 though NFκB pathway and lead target cells apoptosis. This cell line can be used as a good model cell to explore LMP1 bioactivity and its effect in oncogenesis of EBV correlated tumors.
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