Role And Mechanism Of Prostaglandin E Receptor 2 And Its Signaling Pathway In The Development Of Hepatocellular Carcinoma

Objective In order to identify the relationship between EP2 receptor and the development of hepatocellular carcinoma,the lentiviral vector was used to construct the stable HCC cell line with EP2 interfered,and the effects of EP2 on proliferation,migration and invasion abilities of HCC cells were verified by cell function experiment in vitro.The second generation sequencing technique was used to screen and verify the downstream signaling pathway regulated by EP2 in HCC.Methods 1 The EP2 shRNA plasmid expression vector targeting EP2 was designed and constructed,and the recombinant plasmid and lentiviral vector packaging auxiliary plasmids p Helper 1.0 and p Helper2.0 transfected 293 T cells,then produced lentiviral vectors.2 The cultured hepatocellular carcinoma cells(SK-Hep-1,SMMC-7721,MHCC97-H,HepG2,Huh7)were detected by Western Blot technique,and the cell lines with high-expression of EP2 were selected for subsequent experiments.3 The lentiviral vector constructs a stable HCC cell line which interferes with EP2 expression,detects its jamming efficiency by Western blot and qPCR,and detects the effect of EP2 on the proliferation,migration and invasion abilities of HCC by using cell function experiments such as Transwell,cell scratch and CCK8.4 The differentially expressed gene between MHCC97-H cell line and its control group was screened by RNA digital expression spectrum(RNA-Seq)sequencing and the function of gene was explored through bioinformatics analysis,and the related targets and molecular signaling pathways of EP2 regulating HCC were screened by qPCR and Western blot.Results 1 The results of DNA sequencing and identification showed that plasmid with target silencing EP2 gene shRNA fragment was constructed correctly,packaging and producing lentiviral vector LV-shEP2.2 Western Blot results showed that EP2 were expressed in five cell lines.The EP2 expression of MHCC97-H and HepG2 cells was higher than that of other three strains,and MHCC97-H and HepG2 cells were selected for follow-up experiments.3 The results of Western blot and qPCR showed that EP2 expression of MHCC97-H and HepG2 infected by LV-shEP2 was significantly lower than LV-ctrl and blank control groups in mRNA and protein.4 In the CCK8 experiment,the OD value of MHCC97-H and HepG2 cells infected by LV-shEP2 was significantly lower than that of the blank control and LV-ctrl Groups in 96h(P<0.01).5 In the cell scratch experiment,the ability of MHCC97-H and HepG2 cells infected by LV-shEP2 to migrate to the central zone was weakened.In the Transwell experiment,the number of cells in LV-shEP2 infected groups(MHCC97-H and HepG2 cells)was significantly less than that of the blank control group and LV-ctrl group,and the difference was statistically significant(P<0.01).6 The results of RNA-SEQ and qPCR showed that after interfering with the EP2 receptor of MHCC97-H,Wnt6,FZD6,CCND2 expression were lower,and Wnt signaling pathway was enriched by KEGG.7 Western Blot results showed that there was no significant change in the total expression of ?-catenin receptor in MHCC97-H and HepG2 cells infected by LV-sh EP2,but the ?-catenin expression of phosphorylation increased significantly.Conclusion 1 The successful construction of recombinant lentiviral vector LV-shEP2 has high infection efficiency and can stabilize the inhibition of EP2 expression.2 EP2 receptor can enhance the migration,invasion and metastasis ability of HCC cells.3 EP2 receptor may promote the development of HCC through the Wnt signaling pathway.