ABSTRACT
Objective@#To assess the effect of viral macrophage inflammatory protein (vMIP) -Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) , and to explore the mechanisms.@*Methods@#A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells, and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-Ⅱ gene on the APOBEC3G expression in 293T cells. Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN) -α for 36 hours, and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment. Some 293T cells transfected with vMIP-Ⅱ plasmids were treated with 75 μmol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μmol/L U0126 (an ERK signaling pathway inhibitor) separately; after 24 hours, total protein was extracted from 293T cells, and Western blot analysis was conducted to determine the expression of APOBEC3G. A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene, and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G, sequences with the lengths of 1 560, 960, 720, 480, 420, 360, 330 and 240 bp, and the regulatory element-free region (NEG) of APOBEC3G, separately. Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group), or the recombinant plasmid and empty plasmid (control group). Subsequently, the activity of the APOBEC3G promoter was evaluated, and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP-Ⅱ was analyzed. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference (LSD) -t test.@*Results@#The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013, 1.472 ± 0.013 respectively) than in the control group (1, 0.364 ± 0.030 respectively; t = 6.22, 6.54 respectively, both P < 0.05) . The APOBEC3G expression significantly differed among the empty plasmid group, vMIP-Ⅱ plasmid group, empty plasmid + IFN-α group and vMIP-Ⅱ plasmid + IFN-α group (1, 2.030 ± 0.108, 2.700 ± 0.081 and 2.600 ± 0.099 respectively; F = 67.026, P < 0.001) , but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t = 3.46, P > 0.05) . The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group, vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group (0.617 ± 0.025, 0.179 ± 0.061, 0.359 ± 0.012 respectively; F = 70.019, P < 0.001) , and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t = 9.66, 11.836 respectively, both P < 0.01) . Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS, 1 560-, 960-, 720-, 480-, 420-, 360-, 330-, 240-bp or NEG sequences (F = 81.092, P < 0.001) , and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group.@*Conclusion@#vMIP-Ⅱ upregulates the expression of APOBEC3G, likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.
ABSTRACT
Objective To assess the effect of viral macrophage inflammatory protein (vMIP)-Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G),and to explore the mechanisms.Methods A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells,and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-]Ⅱ gene on the APOBEC3G expression in 293T cells.Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN)-α for 36 hours,and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment.Some 293T cells transfected with vMIP-Ⅱplasmids were treated with 75 μ mol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μ mol/L U0126 (an ERK signaling pathway inhibitor) separately;after 24 hours,total protein was extracted from 293T cells,and Western blot analysis was conducted to determine the expression of APOBEC3G.A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene,and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G,sequences with the lengths of 1 560,960,720,480,420,360,330 and 240 bp,and the regulatory element-free region (NEG) of APOBEC3G,separately.Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group),or the recombinant plasmid and empty plasmid (control group).Subsequently,the activity of the APOBEC3G promoter was evaluated,and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP -Ⅱ was analyzed.Statistical analysis was carried out by using t test,one-way analysis of variance and least significant difference (LSD)-t test.Results The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013,1.472 ± 0.013 respectively) than in the control group (1,0.364 ± 0.030 respectively;t =6.22,6.54 respectively,both P ＜ 0.05).The APOBEC3G expression significantly differed among the empty plasmid group,vMIP-Ⅱ plasmid group,empty plasmid + IFN-oα group and vMIP-Ⅱ plasmid + IFN-α group (1,2.030 ± 0.108,2.700 ± 0.081 and 2.600 ± 0.099 respectively;F =67.026,P ＜ 0.001),but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t =3.46,P ＞ 0.05).The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group,vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱplasmid + U0126 group (0.617 ± 0.025,0.179 ± 0.061,0.359 ± 0.012 respectively;F =70.019,P ＜ 0.001),and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t =9.66,11.836 respectively,both P ＜ 0.01).Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS,1 560-,960-,720-,480-,420-,360-,330-,240-bp or NEG sequences (F =81.092,P ＜ 0.001),and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group.Conclusion vMIP-Ⅱ upregulates the expression of APOBEC3G,likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.