| In China,primary liver cancer is the second leading cause of cancer death,and its incidence is on the rise.The 5-year relative survival rate of liver cancer after age-standardization was only 14.1%.The onset of primary liver cancer is insidious,and most patients have been in the advanced stage of diagnosis and have lost the opportunity for surgical treatment.For patients who cannot be operated,in addition to topical treatment,systemic therapy is often needed.Because liver cancer is not sensitive to chemotherapeutic drugs,the systemic drugs currently available to patients are very limited.Therefore,it is of great clinical significance to find targets that can affect the biological behavior of liver cancer cells and their corresponding targeted drugs.APOBEC3 is a family of cytosine deaminases with editing RNA or DNA activity unique to a newly discovered type of mammal in recent years.Many members of this family can affect the stability of the genome to varying degrees.Current studies have revealed that APOBEC3 can induce gene mutation and promote tumor development by affecting DNA damage repair ability.In the previous clinical data analysis and preliminary experiments,we found that high expression of APOBEC3F promoted the invasion and metastasis of liver cancer cells.Up to now,the biological function of APOBEC3F in tumors has not been reported.The dried Toad Skin water extract has a complex chemical composition and contains a variety of active ingredients,among which bufalin is currently recognized as the strongest anti-tumor effect.In vitro and in vivo,many experiments have demonstrated that bufalin has the effects of promoting apoptosis,inhibiting proliferation,invasion and metastasis,angiogenesis,and reversing multidrug resistance in various tumor cells.The distribution of APOBEC3F in hepatocarcinoma tissues,and the mechanism by which it affects the biological behavior of hepatocellular carcinoma cells and its sensitivity to bufalin have still not been well clarified.It is of great importance to solve these problems in appropriate patient selection to use bufalin as the main active ingredient of anti-tumor treatment strategies.This study contains three parts as follows:Part 1.APOBEC3F can be used as a potential target for the treatment of hepatocellular carcinomaObjective:To clarify the expression and distribution of APOBEC3F in hepatocarcinoma tissues and its influence on the biological behavior of hepatocarcinoma cells,and evaluate its value as a therapeutic target for HCC.Methods:The expression levels of APOBEC3F m RNA in HCC tumor tissues and adjacent tissues were analyzed in Gene Expression Omnibus（GEO）and The Cancer Genome Atlas（TCGA）databases.The expression levels of APOBEC3F m RNA and APOBEC3F protein were validated in 8 pairs of HCC tumor tissues and corresponding adjacent tissues.The relationships between APOBEC3F m RNA expression and clinicopathological features and survival status of HCC patients were analyzed in GSE14520 and GSE36376 profiles.In SK-Hep1 and Bel-7404 cell lines,APOBEC3F si RNA interference was conducted to observe the effects of APOBEC3F on the proliferation and metastasis abilities in HCC cells.Results:The results of GSE25097,GSE36376 and TCGA databases showed that APOBEC3F m RNA was significantly higher in tumor tissues than that in adjacent tissues in HCC patients（P<0.001 or P<0.01）.Compared with healthy individuals,APOBEC3F m RNA was significantly increased in peripheral blood from HCC patients（P<0.05）.We analyzed the expression levels of APOBEC3F m RNA in tumor tissues and paracancerous tissues of 8 pairs of HCC patients.The results showed that APOBEC3F m RNA was highly expressed in 6 pairs（75%,6/8）.In addition,immunohistochemistry and western blot assay showed that APOBEC3F protein was highly expressed in tumor tissues than that in adjacent tissues from HCC patients.We analyzed the clinical information of 240 HCC patients after surgical resection in the GSE36376 dataset.The results demonstrated that high expression of APOBEC3F m RNA is a risk factor of vascular invasion,intrahepatic metastasis and elevated alpha-fetoprotein（AFP）in HCC patients（P=0.037,P=0.038 and P=0.015）.The proportion of patients with vascular invasion（59.2%vs 45.8%,P=0.039）,intrahepatic metastasis（28.3%vs 17.5%,P=0.046）and AFP increase（39.2%vs 26.7%,P=0.039）in the APOBEC3F m RNA high expression group was significantly higher than that of HCC patients with low expression.In the two data sets of GSE14520 and GSE36376,Cox regression analysis showed that elevated APOBEC3F m RNA was a risk factor for disease-free survival（DFS）in HCC patients（HR=1.32,95%CI=1.01–1.27,P=0.028 and HR=3.38,95%CI=1.25–9.72,P=0.017,respectively）.In the GSE14520dataset,Kaplan-Meier survival analysis showed that the mean disease-free survival months of patients with high expression of APOBEC3F m RNA was significantly shorter than that of patients with low expression（32.25±3.81 months vs.42.68±2.06months,log rank P=0.012）.In the GSE36376 dataset,Kaplan-Meier survival analysis revealed that the HCC patients with high expression of APOBEC3F m RNA was had shorter mean DFS months than those with low APOBEC3F m RNA expression（42.38±6.37 months vs.54.74±3.86 months）,but no statistical difference was observed（Log rank P=0.065）.We performed APOBEC3F si RNA interference on SK-Hep1 and Bel-7404 HCC cell lines.The cell count assay showed that the cell number of SK-Hep1and Bel-7404 cells was significantly lower than that of the control group after APOBEC3F si RNA interference for 24h（P<0.05 and P<0.01）,CCK8 assay demonstrated that the proliferation ability of SK-Hep1 and Bel-7404 cells was significantly decreased after APOBEC3F si RNA interference for 24 h,48 h and 72 h（P<0.05,P<0.01 or P<0.001）.Cell wound healing assay showed that the cell migration ability of SK-Hep1 and Bel-7404 cell lines was significantly decreased after APOBEC3F si RNA interference,and the morphology of SK-Hep1 cells was transformed from fusiform to round-like after APOBEC3F si RNA interference.Conclusion:APOBEC3F m RNA and protein are highly expressed in tumor tissues of HCC patients.APOBEC3F overexpression is closely associated with vascular invasion,intrahepatic metastasis and AFP elevation,and is a risk factor for DFS in HCC patients.Downregulation of APOBEC3F expression could inhibit the proliferation and migration ability of HCC cancer cells.Therefore,APOBEC3F can be a potential target for the treatment of HCC.Part 2.Roles of APOBEC3F-mediated Intestinal immune network for Ig A production in hepatocellular carcinomaObjective:To clarify the roles of APOBEC3F-mediated Intestinal immune network for Ig A production signaling pathway in the tumorigenesis and development of HCC.Methods:The APOBEC3F co-expressed genes was analyzed by Oncomine database,and the KEGG（Kyoto Encyclopedia of Genes and Genomes）signaling pathway and GO（Gene Ontology）biological process enrichment analysis were performed on the APOBEC3F co-expressed genes.Bioinformatic analysis on the intestinal immune network for Ig A production signaling pathway with most APOBEC3F-coexpressed genes enriched was conducted.APOBEC3F si RNA interference assay was performed in SK-Hep1 and Bel-7404 cell lines and key moleculars in intestinal immune network for Ig A production signaling pathway including CCR9,CCR10,CXCR4 and p Ig R were detected by Western blot.Additionally,we evaluated the associations between p Ig R expression and clinico-pathological features and survival in HCC patients.Results:Totally 128 genes were detected as coexpressed genes of APOBEC3F in Oncomine database.Enrichment analysis of APOBEC3F co-expressed genes showed that most of the genes were enriched in intestinal immune network for Ig A production,antigen processing and presentation and cell adhesion moleculars signaling pathways,while APOBEC3F co-expressed genes were mainly involved in immune response.Antigen processing and presentation and cell adhesion moleculars signaling pathways are important components of intestinal immune network for Ig A production signaling pathway in KEGG.In SK-Hep1 and Bel-7404 cell lines,APOBEC3F si RNA interference could significantly decreased key moleculars in intestinal immune network for Ig A production signaling pathway including CCR9,CCR10,CXCR4 and p Ig R protein expression.In TCGA database,p Ig R m RNA was significantly upregulated in tumor tissues than that in nontumor tissues（P=0.003）.Meta-analysis of 8 studies in the Oncomine database showed that p Ig R m RNA was significantly overexpressed in tumor tissues of HCC patients compared to normal tissues（P=1.18E-15）.We detected the p Ig R protein levels in 4 paired HCC patients.The results showed that p Ig R protein was highly expressed in tumor tissues of HCC patients.In the TCGA database,p Ig R high expression group had more male patients（74.0%vs 61.1%,P=0.009）.Patients in p Ig R high expression group were significantly older than p Ig R low expression group（61.30±12.68yrs vs 57.55±14.29yrs,P=0.009）.The proportion of risk factors for hepatocarcinogenesis in p Ig R high expression group was significantly higher（80.7%vs71.7%,P=0.045）.Patients in p Ig R high expression group had higher risk of developing cirrhosis（24.9%vs 13.9%,P=0.008）.The disease-free survival（DFS）of patients with high p Ig R expression was significantly lower than that of patients with low p Ig R level（median survival months:15.74 vs 21.62,log rank P=0.0494）.Conclusion:APOBEC3F can induce the activation of intestinal immune network for Ig A production signaling pathway,resulting in p Ig R overexpression.PIg R upregulation could promote the progression of liver disease and was significantly associated with DFS in HCC patients.Part 3.New mechanism of bufalin inhibiting the proliferation and metastasis of hepatocarcinoma cells:regulation of APOBEC3F-mediated Intestinal immune network for Ig A productionObjective:To identify a new molecular mechanism of anti-proliferation and anti-metastasis in hepatocellular carcinoma.Methods:We verified the effect of bufalin on APOBEC3F protein expression in SK-Hep1 and Bel-7404 cell lines by western blot assay.The expression changes of key molecules in Intestinal immune network for Ig A production pathway treated by bufalin were also identified using western blot.The p Ig R overespression and knockdown cell lines were constructed using Bel-7404.Subcutaneous tumor model of p Ig R overexpression and p Ig R knockdown were constructed in C57BL/6 mice,in order to evaluate the effect of p Ig R expression on tumor development in C57BL/5 mice.The antitumor effects of bufalin on C57BL/6 tumor model were also observed.The Ki67,E cadherin,N cadherin and Vimentin expression levels in tumors were identified using immunohistochemical assay in C57BL/6 tumor model.Results:CCK8 assay showed that IC50 values of SK-Hep1 and Bel-7404 cells treated with bufalin were 10n M and 80n M,respectively.The proliferation ability of SK-Hep1and Bel-7404 cell lines was significantly decreased after treated with bufalin for 24hours.Bufalin combined with APOBEC3F si RNA could inhibit the proliferation of these two cell lines synergistically.Wound healing test and transwell assasy indicated that bufalin could significantly inhibit the migration of hepatoma cells SK-Hep1 and Bel-7404.Western blot results showed that bufalin could significantly inhibit the expression of APOBEC3F in SK-Hep1 and Bel-7404 cells.Key molecules of Ig A-related intestinal immune network signaling pathway including CCR9,CCR10,CXCR4 and p Ig R were significantly decreased after bufalin treatment in SK-Hep1 and Bel-7404 cells.The volume of subcutaneous tumors in p Ig R knockdown C57BL/6 mice was significantly lower than that in control group（P<0.05）,while tumor volume in p Ig R overexpression group was significantly higher than that in control group（P<0.05）.Bufalin can effectively inhibit the tumor growth of p Ig R overexpression mice（P<0.05）.Similarly,bufalin could significantly inhibit the growth of subcutaneous transplanted tumors in sh p Ig R mice（P<0.05）.The tumor weight and tumor body ratio of C57BL/6 mice in p Ig R overexpression group were significantly higher than those in control group（P<0.001）,and the tumor weight and tumor body ratio of mice in p Ig R knockdown group were significantly lower than those in control group（P<0.001）.The tumor weight of C57BL/6 mice in the p Ig R overexpression treated with bufalin was significantly lower than that in the p Ig R overexpression group（P<0.0001）.Bufalin could significantly reduce the tumor weight of in sh p Ig R mice treated with bufalin（P<0.05）.The expression of Ki67 in subcutaneous tumors of C57BL/6 mice was detected by immunohistochemical assay and semi-quantified by H-scores.The results showed that the expression level of Ki67 in tumor tissue of p Ig R overexpression mice was significantly higher than that of control group（P<0.05）,and the expression level of Ki67 in p Ig R knockout mice was significantly lower than that of control group（P<0.05）.Bufalin could effectively reduce the expression of Ki67 in p Ig R overexpression mice,but there is no significant difference.However,bufalin could significantly reduce the expression of Ki67 in sh p Ig R mice（P<0.05）.Moreover,the expression levels of E cadherin,N cadherin and Vimentin in tumors of C57BL/6 mice were detected by immunohistochemistry assay.The results revealed that the change of p Ig R expression level had no significant effect on the expression of E cadherin（P>0.05）.Overexpression of p Ig R can promote the expression of N cadherin and Vimentin,while knockdown of p Ig R can reduce the expression of N cadherin and Vimentin（P<0.05）.Bufalin could inhibit the expression levels of N cadherin and Vimentin both in p Ig R overexpression and p Ig R knockdown groups,but no significances were found.Conclusion:Bufalin could inhibit the proliferation and metastasis of hepatocellular carcinoma by inhibiting the activation of intestinal immune network for Ig A production mediated by APOBEC3F,which could be a new mechanism to promote the development of hepatocellular carcinoma. |
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