LHPP Inhibits The Proliferation,Invasion And Metastasis Of Hepatocellular Carcinoma Cells By Down-regulation The Phosphorylation Level Of EGFR

Objective:To study the correlation between LHPP gene and survival and prognosis of patients with hepatocellular carcinoma,and its function and mechanism in malignant biological behaviors such as proliferation,invasion and metastasis of hepatocellular carcinoma,so as to provide a theoretical basis for finding effective molecular therapeutic targets for hepatocellular carcinoma.Methods:Western blot was used to detect the expression of LHPP in cancer tissues and adjacent tissues of 8 patients with hepatocellular carcinoma.q PCR and Western blot were used to detect the expression of LHPP in 5 hepatocellular carcinoma cells and normal hepatocytes cell.IHC was used to detect the expression of LHPP in 94 cases of hepatocellular carcinoma and 86 cases of adjacent tissues,The correlation between the expression of LHPP and prognosis was analyzed.Hep G2 and Huh-7 cells were infected with lentiviral vectors.LHPP overexpression cells(LHPP),LHPP overexpression control cells(vector),LHPP low expression cells(KD1,KD2)and LHPP low expression control cells(NC).The results were verified by q PCR and Western blot.CCK-8,cloned plates,subcutaneous tumor formation in nude mice,flow cytometry,Western blot and IHC were used to verify the effect of LHPP on proliferation,apoptosis and cycle distribution of hepatoma cells.The effect of LHPP on invasion and metastasis of HCC cells was verified by Wound healing assay,Transwell migration and invasion assay and Western blot.CO-IP,IF,IHC and Western blot were used to verify whether LHPP interacts with EGFR,whether it controls the phosphorylation level of EGFR and its downstream pathways.The overexpression of LHPP cells were treated with the activator of EGFR(NSC228155)and the low expression of LHPP cells were treated with the inhibitor of EGFR(PD153035)Blot and other experiments confirmed the effect of LHPP on the proliferation and invasion and metastasis of hepatoma cells,and verified that LHPP was involved in regulating EGFR phosphorylation and the expression of key proteins in downstream STAT3/AKT/ERK signaling pathway.Results:1.The results of q PCR,Western blot and IHC showed that the expression of LHPP in liver cancer tissues and cells decreased,and patients with low LHPP expression had poor prognosis.2.The results of CCK8,clone formation,flow cytometry,Western blot,subcutaneous tumor formation in nude mice and IHC showed that the expression of Bcl-2 protein decreased,BAX protein expression increased and cell apoptosis increased in overexpressing LHPP cells.The ratio of cell cycle distribution to G1 phase increased,and the ratio of S phase to(or)G2/M phase decreased in overexpressing LHPP cells.The expression of cyclin A2,cyclin B1,cyclin D1,cyclin E1 and CDK2 decreased,while the expression of p27 and p21 increased in overexpressing LHPP cells.Cell proliferation and the ability of subcutaneous tumorigenesis of hepatoma cells were inhibited,and the expression of proliferation related proteins PCNA and Ki-67 decreased in overexpressing LHPP cells.The apoptosis were decreased in cells with low expression of LHPP,and Bcl-2expression were increased and BAX expression were decreased.The percentage of G1 distribution decreased,the ratio of S phase and / or G2/M phase increased,the expression of cyclin Cyclin A2,Cyclin B1,Cyclin D1,Cyclin E1,and D1 increased,and the expression of m RNA was decreased in cells with low expression of LHPP.Cell proliferation and subcutaneous tumorigenicity were enhanced,and the expression of PCNA and Ki-67 was upregulated in cells with low expression of LHPP.3.The results of Wound healing assay,Transwell invasion and migration assay and Western blot showed that the invasion and migration ability of cells were significantly decreased after overexpression of LHPP,the expression of E-cadherin protein related to metastasis was increased,and the expression of vimentin protein was decreased;while the invasion and migration ability of cells were enhanced after low expression of LHPP,the expression of E-cadherin protein was decreased,and the expression of vimentin protein was up-regulated.4.Western blot、CO-IP、IF、IHC results showed that LHPP interacted with EGFR,and overexpression of LHPP was involved in down regulating the phosphorylation of EGFR in hepatoma cells,and could inhibit the phosphorylation of key proteins in STAT3 / AKT / ERK signaling pathway downstream of EGFR.5.The results of CCK8,Transwell invasion and migration assay and Western blot showed that NSC228155 enhanced the proliferation,invasion and metastasis of HCC cells,increased the phosphorylation level of EGFR,and activated the downstream STAT3 / AKT / ERK signaling pathway.5.The results of CCK8,Transwell invasion and migration assay and Western blot showed that NSC228155 enhanced the proliferation,invasion and metastasis of HCC cells,increased the phosphorylation level of EGFR,and activated the downstream STAT3 /AKT / ERK signaling pathway.Conclusions:1.The expression of LHPP decreased in HCC and HCC cells,and was closely related to the prognosis of patients.2.Overexpression of LHPP inhibits the proliferation and invasion and metastasis of hepatoma cells in vivo and in vitro,while low expression of LHPP promotes the malignant biological behavior of hepatoma cell proliferation,invasion and metastasis.3.LHPP and EGER interact with each other,and overexpression of LHPP is involved in down regulating the phosphorylation level of EGFR in hepatocellular carcinoma cells and inhibiting the activation of downstream STAT3/AKT/ERK signaling pathway,thereby mediating the proliferation and invasion and metastasis ability of hepatoma cells.