The Mechanism Study Of NME2 Regulating 4EBP1 Phosphorylation To Promote The Proliferation Of HCC Cells By Inhibiting Autophagy

Background: Liver cancer remains a global health challenge.Hepatocellular Carcinoma(HCC)is the fourth leading cause of cancer-related mortality worldwide,accounting for about 90% of liver cancer.These include aflatoxin B1 exposure,HBV and HCV infections,AALD,obesity,NAFLD,and hemochromatosis,as well as genetic disorders such as tumor suppressor or cancer gene mutations,which promote HCC development.In China,although the treatment of HCC is more diversified and individualized,it is still difficult to improve the 5-year survival rate.Most HCC is diagnosed at an advanced stage.Therefore,it is necessary to clarify the important molecular mechanisms of HCC occurrence and development and to judge the prognosis of HCC,to develop new strategies for HCC prevention and treatment.NME2(non-metastatic cells2),also known as NM23-H2 or NDPKB,is located on chromosome 17q21 and encodes the nucleoside diphosphate kinase B subunit.NME2 is a metastasis-associated protein involved in the regulation of gene transcription.It is differentially expressed in malignant tumors and may be involved in the pathological process of cancer.However,there are few reports of NME2 in HCC.We previously found that NME2 is highly expressed in HCC through bioinformatics mining,and the expression of NME2 is related to the regulation of autophagy.To further elucidate the effect and mechanism of NME2 on the malignant behavior of HCC,the present study used NME2 as the starting point to explore the role and mechanism of NME2 in HCC.Objective:(1)To clarify the expression of NME2 in HCC patients and its relationship with clinical prognosis(2)To verify the role of NME2 in HCC in vivo and in vitro.(3)To explore the effect and mechanism of NME2 on autophagy in HCC.Methods:(1)Immunohistochemical staining was used to evaluate the expression of NME2 in cancer and para cancer in 96 HCC patients.Follow-up data of 96 HCC patients were completed.(2)NME2 knockout hepatoma cell lines Huh7 and Hep G2 were constructed by transfection of small interfering RNA and lentivirus.After successful NME2 knockout was verified by Western blot,the effect of NME2 knockout on HCC cell proliferation was verified by cell cloning and MTT proliferation.(3)Construct NME2-knockout and overexpressed Huh7 cell lines for subcutaneous tumor formation in nude mice.Fifteen healthy nude mice were randomly divided into 3 groups: control group,NME2 knockout group,and NME2 overexpression group,with 5 mice in each group.To observe the effect of NME2 on hepatocellular carcinoma proliferation in vivo.(4)The changes of LC3 B,Beclin-1,P62 and ATG5 in Huh7 cells after NME2 knockout were detected by Western blot.(5)The effects of NME2 knockout on the autophagosome and autolysosome of HCC cells were observed by laser confocal microscopy and transmission electron microscopy.(6)Immunohistochemical method was used to verify the correlation between NME2 and LC3 B protein in tumor tissue of nude mice.(7)The effect of NME2 on autophagy-related pathway proteins was verified in vitro and reversed.Results:(1)The relatively high expression of NME2 in HCC patients was negatively correlated with the 5-year survival rate of patients.(2)Knockdown of NME2 in Huh7 and Hep G2 inhibited the proliferation and colony formation of HCC cells.(3)NME2could promote the proliferation of Huh7 cells in vivo.(4)NME2 knockdown could up-regulate the expression of LC3B(Ⅰ and Ⅱ)protein in Huh7 cells.Chloroquine(CQ)combined with NME2 knockdown induced more LC3B(Ⅰ and Ⅱ)protein expression than NME2 knockdown alone.(5)Compared with the control group,NME2 knockout increased the number of autophagosomes and autolysosomes in Huh7 cells under laser confocal microscopy and transmission electron microscopy.(6)Chloroquine reversed the effect of NME2 knockdown on the proliferation of Huh7 cells.(7)The expression of LC3 B protein was decreased in NME2 overexpression cells and tumor tissues of nude mice.(8)The phosphorylation of 4EBP1 was regulated by NME2,and4EBP1 mutation at Thr37/46 counteracted the ability of NME2 knockout to activate autophagy and inhibit proliferation in HCC cellsConclusion:(1)NME2 has high expression in HCC,and negatively correlated with the prognosis of patients,and has the function of promoting tumor proliferation.(2)NME2 promoted the proliferation of HCC cells by activating autophagy.(3)P-4EBP1 mediates the regulation of autophagy and proliferation by NME2 in HCC.