Effect Of Gga-miR-148a-5p Target Regulation Of PDPK1 On Proliferation And Cycle Of Chicken Fibroblasts Infected With ALV-J

According to the results of previous research conducted by our research group,it was found that there were 7 differentially expressed miRNAs in the spleen tissue of 40-day-old SPF healthy chickens and chickens infected with J sub-type Avian leukemia(ALV-J).Therefore,this experiment was based on this,through bioinformatics analysis of 7differentially expressed miRNAs,gga-miR-148a-5p was finally selected as the research object.We predict that PDPK1 may be a target gene of gga-miR-148a-5p,and through interference,overexpression,CCK-8,Ed U,flow cytometry,dual luciferase reporter gene test and EMSA molecular and cell biology methods,it clarified the molecular mechanism of gga-miR-148a-5p targeting PDPK1 to regulate the pathogenicity of subgroup J avian leukemia.The test results were as follows:(1)The dual luciferase reporter gene experiment verified that PDPK1 was the target gene of gga-miR-148a-5p,and there is a target regulation relationship between the two.(2)Fluorescence quantitative results showed that after overexpression and inhibition of gga-miR-148a-5p,PDPK1 and gga-miR-148a-5p m RNA expression levels were negatively correlated.(3)CCK-8 test results showed that ALV-J can significantly promote the proliferation of CEF cells(P<0.05);after overexpression of gga-miR-148a-5p and interference with PDPK1,it can significantly promote the proliferation of ALV-J-infected CEF cells(P<0.05);on the contrary,the inhibition of gga-miR-148a-5p significantly inhibited the proliferation of ALV-J-infected CEF cells at 24h(P<0.01).(4)Ed U test results showed that the positive rate of Ed U in CEF cells infected with ALV-J was significantly higher compared with the control group(P<0.05);in CEF cells infected with ALV-J,after overexpression of gga-miR-148a-5p and interference with PDPK1,the positive rate of Ed U increased significantly compared with the control group(P<0.05).In contrast,after inhibiting gga-miR-148a-5p,the positive rate of Ed U in CEF cells infected with ALV-J was significantly reduced(P<0.05).The above results indicated that,in CEF infected with ALV-J,gga-miR-148a-5p can promote cell proliferation through targeted regulation of PDPK1.(5)The cell cycle results showed that the proportion of cells in the G2 and S phases of the ALV-J infected group increased significantly(P<0.05),and the proportion of cells in the G1 phase decreased compared with the control group,after overexpressing gga-miR-148a-5p and interfering with PDPK1,the proportion of cells in G2 and S phase of ALV-J infection group increased significantly(P<0.05),while the proportion of cells in G1 phase decreased;in contrast,after inhibiting gga-miR-148a-5p,the proportion of cells in G2 and S phases decreased significantly(P<0.05),while the proportion of cells in G1 phase increased.The above experimental results indicated that: in CEF infected with ALV-J,gga-miR-148a-5p can promote cell cycle progression through targeted PDPK1 regulation.(6)Double luciferase reporter gene test and EMSA test showed that,compared with the control group,ALV-J treatment and PDPK1 interference treatment had no significant effect on the activity of ALV-J infected CEF NF-κB.In summary,under the condition of CEF infected with ALV-J,gga-miR-148a-5p can promote ALV-J-induced cell proliferation and cell cycle progression by down-regulating PDPK1.At the same time,under the condition of CEF infected with ALV-J,ALV-J and PDPK1 interference treatment had no significant effect on the activity of NF-κB in CEF.