| Interferon-induced transmembrane proteins have anti-proliferation,anti-tumor and anti-viral effects.In this experiment,fluorescence quantitative PCR,prokaryotic expression and purification,immunohistochemistry,ELISA,Western Blot,immunofluorescence detection and other methods were used to investigate the expression and function of yak IFITM1/2/3 genes.The cDNA sequence of yak IFITM1 gene was 546 bp,of which the coding region was 375 bp,encoding 124 amino acids.The expression level of yak IFITM1 gene in liver tissue was significantly higher than that in spleen,lung and kidney tissues(P<0.05).Yak IFITM1 protein was expressed in the yak liver in three growth stages and was located in the cell membrane.Yak IFITM1 protein was successfully expressed with a molecular weight of about 22.593 k Da,and the activity of yak hepatocytes and HepG2 cells were significantly inhibited after 10 μg/m L yak IFITM1 protein treated(P<0.05).The m RNA levels of BCL2/BAX,Cdk1 and m OTR in yak hepatocytes were extremely significantly decreased(P<0.01),the BAX protein level was extremely significantly increased(P<0.01),and the m RNA levels of BCL2/BAX,Cdk1 and MMP9 genes in HepG2 cells were extremely significantly decreased(P<0.01).The cDNA sequence of yak IFITM2 gene was 602 bp,of which the CDS region was 381 bp,encoding 126 amino acids.Yak IFITM2 gene was expressed in the spleen of 1-day-old,15-month-old and 5-year-old yak.The yak IFITM2 protein was successfully expressed with a molecular weight of about 33.680 k Da.The proliferation and migration abilities of HepG2 cells were significantly inhibited after treatment with yak IFITM2 protein(0.1 and 1 μg/m L)(P<0.05).The m RNA levels of BCL2/BAX and m TOR genes were significantly decreased,but the m RNA levels of BAX gene were significantly increased after treatment with yak IFITM2 protein(0.1 and 1 μg/m L)(P<0.05).The BAX protein level was also significantly reduced after treatment with 1μg/m L yak IFITM2 protein(P<0.05).A mouse tumor model was constructed,and the results of clinical symptoms,histopathology and immune organ indices indicated that the yak IFITM2 protein could reduce the formation of lung tumor lesions,and the degree of lung pathological damage was less than that of the model group.Ki67 protein levels of lungs in the IFITM2 group were significantly lower than those in the model group,and Ki67 protein levels of lungs in the model group were significantly higher than those in the control group(P<0.05).The cDNA sequence of yak IFITM3 gene was 578 bp,of which the CDS region was 441 bp,encoding 147 amino acids.The expression of yak IFITM3 gene in liver was extremely significantly higher than that in heart,spleen,lung and kidney(P<0.01).Yak IFITM3 protein was localized on the yak hepatocytes plasma membrane.The yak IFITM3 protein was successfully expressed with a molecular weight of about 19.5 k Da.The proliferation,cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 μg/m L yak IFITM3 protein(P<0.05).The mitochondria of HepG2 cells were concentrated,cristae widened,and the double film density of mitochondria was increased after treating with 10 μg/m L yak IFITM3 protein.After 10 μg/m L yak IFITM3 protein treating,the m RNA levels of VDAC-2,VDAC-3and p53 genes were extremely significantly increased(P<0.01),but the m RNA level of GPX-4 gene was extremely significantly decreased(P<0.01).Finally,the activities of macrophages were significantly promoted after treating with yak IFITM3 protein(1,10 and 20 μg/m L)(P<0.05).The m RNA levels of IL-1β,IL-6 and TNF-α were significantly lower after treating with yak IFITM3 protein(10 and 20 μg/m L)(P<0.05),the m RNA levels of IL-10 gene were significantly higher after treating with yak IFITM3 protein(1,10 and 20 μg/m L)(P<0.05).Significant reduction of IL-6 and TNF-α secretion by macrophages after addition of yak IFITM3 protein(10 and 20 μg/m L)(P<0.05).Through the antibacterial experiment,it was found that the yak IFITM3 protein had the effect of inhibited Klebsiella pneumoniae,and the MIC value was 16 μg/m L.A mouse model of bacterial infection was constructed,and the results of clinical symptoms,histopathology,immune organ indices and serum biochemical indices showed that the injection of yak IFITM3 protein could prevent bacterial infection,and the degree of pathological damage was significantly reduced relative to the infected group.The m RNA levels of IL-1β,IL-6,TNF-α,TRAF6 and TLR4 genes in the infection group were significantly lower than that in the control group,but the m RNA levels of IL-1β,IL-6,TNF-α and TLR4 genes in the IFITM3 group were significantly higher than that in the infection group(P<0.05).In conclusion,the above results are beneficial to further study the function of IFITM proteins. |
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