The Role And Mechanism Of NAP1L5 In Proliferation,migration And Invasion Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is one of the leading causes of cancer death worldwide.Although the comprehensive treatment based on surgical resection is widely used in HCC,the overall therapeutic effect is not good.In recent years,some new immunotherapy and targeted drugs have emerged,which have improved the therapeutic effect of HCC to a certain extent.This also highlights the necessity of exploring the mechanism of the occurrence and development of liver cancer and finding effective molecular targets.Nucleosomeassemblyprotein1-like5(NAP1L5)is a protein coding gene,which encodes a protein similar to Nucleosomeassemblyprotein1(NAP1).It belongs to the NAP1 family and is a histone chaperone that plays an important role in biological gene transcription.However,the role of NAP1L5 in the pathogenesis of hepatocellular carcinoma remains to be studied.Therefore,this study makes a preliminary exploration on the function and mechanism of differentially expressed NAP1L5 in hepatocellular carcinoma,which provides a theoretical basis for NAP1L5 to become an effective therapeutic target for HCC.Method:1.The differential expression of NAP1L5 in cancer tissues and paracancerous tissues was searched and analyzed in Cancer Genome Map Project(TCGA)database.Real-time quantitative PCR(RT-q PCR)and western bolt were used to verify the expression of NAP1L5 in tissues(tissue samples from 8 pairs of liver cancer tissues and corresponding paracancerous tissues from patients with liver cancer in affiliated Hospital of Guizhou Medical University)and cell lines.The expression of NAP1L5 was verified again by immunohistochemical(IHC)staining on the tissue microarray.According to the difference of NAP1L5 expression,the patients were divided into two groups.The clinical correlation analysis,Cox regression analysis and Kaplan-Meier survival analysis were performed to analyze the relationship between NAP1L5 expression and clinicopathological features and prognosis.2.Three kinds of short interference RNA(si RNA)were designed to transfer si RNA into MHCC97 H and Hep G2 cell lines.After verifying the efficiency of RT-q PCR and western bolt,si RNA1# and si RNA2#,which had the most significant interference effect were selected to down-regulate NAP1L5,and then cell function experiments such as EDU,colony formation assay,flow cytometry,Transwell assay and wound healing assay were carried out to verify their regulation on HCC cells.The overexpressed plasmid of NAP1L5 was designed and transfected into MHCC97 H and Hep G2 cell lines.After the efficiency of RT-q PCR and western bolt was verified,the cell function experiments such as EDU,clone formation assay,flow cytometry,Transwell assay and wound healing assay were carried out to verify the effect of NAP1L5 on the proliferation,apoptosis,invasion and metastasis of hepatocellular carcinoma cells.MHCC97 H cell line with stable overexpression of NAP1L5 was constructed by lentivirus,and the nude mice model of in situ subcutaneous tumorigenesis was constructed(control group MHCC97 H cells were transfected into control vector).The transplanted tumor tissues of mice were stained with HE,IHC,Ki-67 and TUNEL to evaluate the effect of NAP1L5 overexpression on tumor proliferation and apoptosis.The mouse model of pulmonary metastasis was established by tail vein injection of the stable transductive cell lines mentioned above.The incidence and number of pulmonary metastases in the experimental group and the control group were compared to verify the effect of NAP1L5 overexpression on the metastatic ability of hepatocellular carcinoma cells in vivo.3.Two MHCC97 H cell lines with stable overexpression of NAP1L5 and control vector were constructed by lentivirus.Magnetic beads with Flag tags were used to adsorb the proteins that NAP1L5 could bind and retain the protein lysate.The possible binding target proteins were found by mass spectrometry analysis,and the downstream mechanisms that may be related to the occurrence and development of hepatocellular carcinoma,such as WNT,RSA,PI3 K signaling pathways,were found by enrichment analysis of the above proteins.After transfection of plasmids,short hairpin RNA(sh RNA)and control vectors in MHCC97 H and Hep G2 cell lines,western bolt verified the regulatory relationship between NAP1L5 on downstream signaling pathways and the effects of NAP1L5 on downstream apoptosis,cell cycle,epithelial-mesenchymal transformation(EMT)related proteins.After adding agonists of downstream signaling pathway,the proliferation,migration and invasion of hepatoma were regulated by NAP1L5 in stable overexpression MHCC97 H cells through colony formation,EDU,Transwell assay,western bolt to verify whether NAP1L5 regulated the proliferation,migration and invasion of HCC through this signaling pathway.The binding relationship between NAP1L5 and the target protein was verified by co-immunoprecipitation(Co-ip),and the regulatory relationship between NAP1L5 and its interaction protein was verified by western bolt in the above MHCC97 H and Hep G2 cell lines.After clarifying the regulatory relationship between them,si RNA was used to down-regulate NAP1L5 and its capture protein in MHCC97 H cells at the same time.Clone formation,EDU,Transwell,westernbolt and other experiments were used to verify whether NAP1L5 can regulate the downstream signaling pathway and affect the proliferation,migration and invasion of hepatocellular carcinoma by binding to the target protein.Result:1.In TCGA database,scatter plot analysis showed that the expression of NAP1L5 in HCC tissues was relatively low.Eight pairs of HCC tissue samples and corresponding paracancerous tissues from the affiliated Hospital of Guizhou Medical University were collected.The protein content of NAP1L5 was detected by western blot.It was found that the NAP1L5 protein content in cancerous tissues was lower than that in paracancerous tissues.RT-q PCR and western bolt showed that the m RNA and protein expression levels of NAP1L5 in HCC cell lines were lower than the non-cancer cell lines.Tissue microarray immunohistochemical staining showed that the expression of NAP1L5 was low in tumor tissues.According to the expression of NAP1L5,patients were divided into two groups: high expression of NAP1L5 and low expression of NAP1L5.Kaplan-Meier survival analysis showed that the OS and DFS of low NAP1L5 group significantly lower than that of high NAP1L5 group(p<0.05).Clinical correlation analysis showed that the expression of NAP1L5 was related to the survival and recurrence of the patients(p<0.05),and the low expression of NAP1L5 suggested a poor prognosis.Further univariate and multivariate COX regression analysis showed that the low expression of NAP1L5 was an independent prognostic factor for poor survival in patients with HCC(HR0.463,95%CI 0.222-0.96,p=0.040).2.After down-regulating the expression of NAP1L5,EDU and clone formation assay showed that the proliferation ability of HCC cells was significantly enhanced,flow cytometry showed that the apoptosis of HCC cells decreased significantly,and Transwell and wound healing assay showed that the invasion and migration ability of HCC cells were enhanced.After up-regulating the expression of NAP1L5,EDU and clone formation assay showed that the proliferation ability of HCC cells was significantly weakened,flow cytometry showed that the apoptosis of HCC cells increased significantly,and Transwell and wound healing test showed that the invasion and migration ability of HCC cells decreased.In the subcutaneous tumorigenesis experiment of mice,the average tumor weight and volume of the experimental group were significantly lower than those of the control group.The transplanted tumors of the two groups were stained with HE staining,IHC staining,Ki-67 staining and TUNEL staining,which further confirmed the inhibitory effect of NAP1L5 on the proliferation of hepatocellular carcinoma and the promotion of apoptosis.In the lung metastasis model,compared with the control group,the lung metastasis of nude mice in the experimental group was less than that in the control group,and the incidence of lung metastasis in the experimental group was also lower than that in the control group.3.Through mass spectrometry analysis,it was found that 199 proteins changed independently after NAP1L5 overexpression.Through functional enrichment analysis of these proteins,it was found that NAP1L5 may regulate the occurrence and development of hepatocellular carcinoma through PI3K/AKT/m TOR signaling pathway.After transfection of plasmid,sh RNA and control vector in MHCC97 H and Hep G2 cell lines,western bolt verified that the up-regulation of NAP1L5 decreased the phosphorylation level of AKT and m TOR,while the down-regulation of NAP1L5 increased the phosphorylation level of AKT and m TOR.In addition,the expression of cyclin D1,CDK4,CDK6,EMT-related proteins N-Cadherin,Vimentin,SNAIL and apoptosis-related proteins Bcl-2 decreased,while E-Cadherin and Bax levels up-regulated after up-regulation of NAP1L5.After NAP1L5 overexpression and control MHCC97 H cells were treated with SC79,an agonist of PI3K/AKT/m TOR pathway,the decrease of p-AKT and p-m TOR induced by NAP1L5 overexpression was restored,and the inhibitory effect of NAP1L5 overexpression on proliferation,migration and invasion of MHCC97 H cells was significantly weakened.Mass spectrometry analysis showed that there was a binding relationship between NAP1L5 and MYH9.Co-ip verified the binding relationship between NAP1L5 and MYH9,and western blot verified the negative regulation relationship between them.After down-regulation of NAP1L5 and MYH9 at the same time,through colony formation,EDU,Transwell,western bolt and other experiments,it was found that the down-regulation of MYH9 resulted in the enhancement of invasion,migration and proliferation of HCC mediated by the down-regulation of NAP1L5 was rescued,and the increased expression of p-AKT and p-m TOR was also restored.It is verified that NAP1L5 can affect the proliferation,migration and invasion of HCC by combining MYH9 to regulate downstream signaling pathways.Conclusion:1.NAP1L5 expression is relatively low in HCC tissues and HCC cell lines,and the low expression of NAP1L5 is related to the poor prognosis of HCC patients.2.As a tumor suppressor,NAP1L5 inhibits the proliferation,migration and invasion,promotes the apoptosis of hepatocellular carcinoma in vivo and in vitro.3.The targeting combination of NAP1L5 and MYH9 can inhibit the proliferation,migration and invasion of hepatocellular carcinoma by regulating the activity of PI3K/AKT/m TOR signaling pathway.