The Role Of PRMT2 In Ang? Induced VSMC Proliferation And Its Mechanism Research

Protein arginine methyltransferase 2(PRMT2), one of the member of the protein arginine methyltransferase family, is involved in regulating cell function by methylating arginine residues. Recent studies have found that PRMT2 may play an important role in cardiovascular disease. Compared with normal mice, PRMT2 gene knock-out mice which giving pathological factors interference appeared obvious the vascular wall thickening, vascular intimal hyperplasia and vascular smooth muscle cells(VSMC) proliferation. Abnormal VSMC proliferation is often detected in the process of cardiovascular disease(CVD). Angiotensin?(Ang?) is one of the main bioactive factors which can induce VSMC proliferation by promoting intravascular inflammation. Many reports show that PRMT2 m RNA and protein expression are significant change in various kinds of inflammation models. Another studies confirm the NF-?B inhibit protein(I?B-?) is one of the PRMT2 substrate and PRMT2 can surpress inflammatory signaling pathway NF-?B. In this study, we tried to explore the role of PRMT2 in VSMC proliferation induced by Ang? and the possible mechanism.Part?: The effect of Ang? on PRMT2 expression.Objective: To indentify the effect of Ang? on PRMT2 expression.Methods: Human aortic vascular smooth muscle cells treated by Ang?with different concentrations(0, 0.01, 1, 100 n M) or different processing time(0, 6, 12, 24 h), we apply MTT to detect the cell proliferation rate and Western blot detects PRMT2 and PCNA protein expression,RT-PCR detect PRMT2 m RNA expression level.Results: VSMC proliferation rate, PCNA protein expression level tend to increase with increasing Ang?concentration(P<0.05), PRMT2 m RNA and protein expression level tend to decrease with increasing Ang?concentration(P<0.05); VSMC proliferation rate and PCNA protein expression level increased with increasing Ang? treatment time(P<0.05), PRMT2 m RNA and protein expression level decreased with increasing Ang? treatment time(P<0.05).Summary: Ang? down-regulate PRMT2 expression in a dose- and time- dependent manner.Part?: The role of PRMT2 in the process of VSMC proliferation induced by Ang?.Objective: To identify the role of PRMT2 in the process of VSMC proliferation induced by Ang?.Method: Building PRMT2 overe-xpression vector, and setting up empty plasmid(vector) group, Ang? treatment group, Ang?+PRMT2 group;Using si RNA technology to silence PRMT2 gene and setting up the negative control(NC) group, Ang? treatment group, the si PRMT2 group, Ang?+si PRMT2 group, MTT detects cell proliferation rate and Flow cytometry analyses cell cycle distribution, Western blot detect PRMT2 and PCNA expression level.Results: Ang?+PRMT2 group's S phase proportion?PCNA expression level and cell proliferation rate are decreased compared to Ang?treatment group(P<0.05), but PRMT2 expression and G0 / G1 phase proportion are increased(P<0.05), up-regulating PRMT2 expression can prevent VSMC proliferation induced by Ang?; si PRMT2 group cell proliferation rate, PCNA expression and cell cycle distribution have no obvious changes compared with NC group, it means only silence PRMT2 gene but no Ang? treatment haven't significant effect on VSMC proliferation, likely because PRMT2 prevent VSMC proliferation via blocking Ang? downstream signaling pathway;Ang?+si PRMT2 groups' s PRMT2 expression level is decreased but cell proliferation rate is increased compared with Ang? group(P<0.05),PCNA expression and cell cycle distribution( S phase proportion) also have increase, but without statistical significance, it indicates that down-regulating PRMT2 gene can promote VSMC proliferation induced by Ang II to a certain extent.Summary: PRMT2 surpress VSMC proliferation induced by Ang?.Part ?: The mechanism of PRMT2 surpress VSMC proliferationObjective: To explore the mechanism of PRMT2 surpress VSMC proliferationMethods: Setting up vector group, Ang? treatment group and Ang?+PRMT2 group, Immunofluorescence detect NF-?B subunit p65 distribution, Western blot detect inflammatory factor IL-1?, IL-6expression, I?B-? total protein and phosphorylation level,Immunecoprecipitation to validate the interaction between PRMT2 and I?B-?. We applay ELISA to detect each group's methylation product ADMA level.Results: Ang? group's p65 fluorescence signal intensity in the nuclei of is obviously enhanced compared with the vector group, IL-1?, IL-6expression and I?B-? phosphorylation level are increased(P<0.05), but the I?B-? total protein level is decreased(P<0.05), Ang?+PRMT2group's p65 fluorescence intensity within the nuclear is weaken compared with Ang? group, IL-1?, IL-6 expression and I?B-?phosphorylation level are decreased(P<0.05), but the I?B-? total protein level is increased(P<0.05), these results indicate that PRMT2 can effectively prevent p65 to transfer into nucleus?NF-?B activation and inflammatory factor expression; Immunecoprecipitation result shows I?B-? is the PRMT2 target protein; Ang? group's ADMA level is significant higher than vector group(P<0.05), Ang?+PRMT2group's ADMA level has obvious decrease compared with Ang? group(P<0.05). PRMT2 prevents VSMC proliferation induced by Ang? via methylate I?B-? to surpress I?B-? phosphorylation and NF-?B activation.Summary: PRMT2 prevent VSMC proliferation induced by Ang?likely through methylate I?B-? and block NF-?B signaling pathwayConclusion:1. PRMT2 may surpress VSMC proliferation induced by Ang?.3. PRMT2 prevent VSMC proliferation induced by Ang? likely through methylate I?B-? and block NF-?B signaling pathway.