The Biological Function And Molecular Mechanism Of ILF2 In Hepatocellular Carcinoma

Background:In our country,hepatocellular carcinoma(HCC)is a common malignant tumor,90%patients had the history of hepatitis B.About 110 thousand people died of liver cancer each year in china,accounting for about 45%of the world's liver cancer deaths.Liver cancer has no obvious early symptoms,but once the symptoms appeared,most of patients have entered the advanced progress,the prognosis will be worse if the malignant degree is high.Although liver cancer resection,radiation therapy,chemical therapy and liver transplantation therapy has made much progress in recent years,but the long-term prognosis of patients with HCC is still not ideal.The main cause of poor prognosis is that effective therapeutic targets of HCC was not found and the molecular mechanism is unknown in the HCC progression.Therefore,searching for more sensitive and specific markers of HCC and exploring its underlying mechanism in the development of HCC,will be the key to further improve the survival rate of liver cancer and have a profound impact on early diagnosis and early treatment of HCC in future.Aim:The study was designed to test the mRNA and protein levels of ILF2 in HCC tissues and adjacent normal tissues or HCC cell lines.Further,the relationship between ILF2 levels and clinicopathological parameters were also analyzed.Moreover,we also unravel the correlation of ILF2 with the prognosis of HCC patients in TCGA database.To verify whether the high expression of ILF2 in liver cancer tissues and liver cancer cells is caused by transcription,HCC cell lines was treated with proteasome inhibitor MG132 at 0,2,4,8,24h,respectively.To elucidate the biological functions of ILF2 in HCC cell lines,we performed cytological experiments and in vivo tumorigenicity.We further uncover its possible underlying signal pathway in the progression of HCC.By interfering the expression of ILF2 on liver cancer cells and addding 0?M,5?M,10?M,20?M human epidermal growth factor receptor(EGFR)inhibitor erlotinib to culture medium,we studied the synergistic effect of two treatments on cell activity and proliferation.Based on microarray expression profiling,the data of GO analysis and KEGG database were used to analysed the differentially expressed genes,thus to further study ILF2's downstream molecules or signal pathways.Methods:1.HCC and matched adjacent paraffin embedded tissues from HCC patients with liver resection were extracted to further verify the mRNA and protein expression of ILF2 by real-time quantitative PCR(qRT-PCR),western blot and inmmunohistochemical staining(IHC).Meanwhile,The expression levels of ILF2 in L02,Huh7,Bel-7402,MHCC-LM3,SMCC-7721 and SK-HEP-1 cells were analyzed by qRT-PCR.2.Based on the IHC data,we studied the connection between the expression of ILF2 and clinical pathologic characteristics.3.Based on the information of TCGA database,we studied the relationship between ILF2 expression and prognosis of HCC patients with liver resection.4.To determine whether ILF2 upregulation occurs via transcription or not,the HCC cells were incubated with the proteasome inhibitor MG132 several times.5.We utilized small interfering RNA(siRNA)transfection and lentivirus overexpression methods to remove the endogenous ILF2 and enhance the expression of exogenous ILF2 in liver cancer cells,respectively.And then,the effect of ILF2 on cell proliferation and cell apoptosis were detected using cell counting kit-8(CCK8)assay,flow cytometry apoptosis assay,and colony formation assay.6.The effect of ILF2 on the tumor formation of HCC cells in vivo was observed by nude mouse tumorigenicity assay.7.Western blot was used to find the downstream molecules regulated by ILF2,exploring the molecular mechanism of ILF2 in progression of hepatocellular carcinoma.8.We upregulated the levels of ILF2 and added erlotinib in different concentrations to hepatocellular carcinoma cells.Cell Counting Kit-8(CCK-8)and clone formation experiment were used to detect the effects on the activity and proliferation of tumor cells after different treatment.9.Based on the microarray data,GO analysis of downstream significant differential gene in three aspects:Biological Process,Cellular Component and Molecular Function to understand the biological function of ILF2,which could help us get more comprehensive information.KEGG database was used for Pathway analysis of these differentially expressed genes,which is helpful for us to judge which signaling pathways are related to these differentially expressed genes,contributing to understanding the molecular mechanism of ILF2.Results:1.The result of western blot unraveled that ILF2 had a higher expression in 72 pairs of HCC tissues compared with matched adjacent tissues.qRT-PCR revealed that mRNA levels of ILF2 was higher in 27 pairs of HCC tissues than in matched adjacent tissues.The IHC results also revealed that expression of ILF2 was correlated with tumor size(p = 0.043).No correlation was observed in patients'age,gender,TNM stage,vascular invasion,histopathologic grading and cirrhosis.The levels of ILF2 in Huh7,Bel-7402,MHCC-LM3,SMCC-7721 and SK-HEP-1 cells were higher than the normal liver cells L02.According to analysis of ILF2 expression and prognosis in TCGA database,the results showed that the higher ILF2 expression was significantly associated with lower survival rate(p=0.013).2.The HCC cells were incubated with the proteasome inhibitor MG132 at 0,2,4,8,24h,after which cell lysates were subjected to western blot analysis.No significant changes in the level of ILF2 were observed in HCC cell lines(Huh7 and MHCC-LM3)treated with MG132.3.In vitro,our results showed that the clone formation and proliferation were suppressed in si-ILF2 group and the opposite effect was produced when the levels of ILF2 was increased.4.As expected,ILF2 overexpression significantly decreased apoptosis in cancer cells infected with recombinant ILF2 lentivirus compared with the control group.Consistent with these results,ILF2 downregulation increased the apoptotic cell count in the ILF2 siRNA group.TUNEL assay was employed to detect apoptosis in xenograft tumors,the reaults showed that the Lenti-ILF2 xenografts revealed less apoptotic rate compared with cells from control group.5.The results of nude mouse tumorigenicity assay were observed.Compared with the Lenti-NC group,the volume and weight of the tumors were significantly larger in mice bearing Lenti-ILF2.Consistently,the expression of Ki-67,a cell proliferation marker,was increased in nude mice after subcutaneous injection of Lenti-ILF2 cells6.These findings about molecular mechanism showed that ILF2 affected the expression of apoptotic proteins,such as BOK,BAX,BCL-2 and cIAP1.Bcl-2 and cIAP1 protein levels were significantly upregulated,and levels of Bax and Bok were downregulated in Lenti-ILF2 cells.Similar results were also validated in siILF2 cells,but there were no changes on Bak levels in cancer cells infected with Lenti-NC and Lenti-ILF2 or transfected with siNC and siILF2.Immunohistochemistry analysis from xenografts proved that Bcl-2 and cIAP1 levels were significantly increased in tumors from Lenti-ILF2-treated mice compared with the control group.7.Compared with si-NC control group,knockdown of ILF2 levels can enhance the inhibition of erlotinib on hepatocellular carcinoma cell activity,two kinds of treatment at the same time can exert a synergistic inhibitory effect.Colony formation assays in vitro were carried out in the same way,consistent with the above results,si-ILF2 combined with erlotinib treatment can significantly improve inhibition of erlotinib on liver cancer cell growth or proliferation.8.GO analysis in Biological Process,ILF2 was closely related to NF-KB,JNK and G1/S signaling pathways;in Cellular Component,ILF2 mRNA is involved in cell exosome processing and nuclear protein complexes;the results of Molecular Function information showed that ILF2 participated in the ion channel,combination of RNA and DNA.Pathway analysis revealed that ILF2 also was closely related to fatty acid metabolism,glucose metabolism and AMPK signaling pathway.Conclusion:1.The mRNA and protein levels of ILF2 in HCC tissues and HCC cells are significantly higher than the normal matched adjacent tissues and normal liver cells,respectively.The high expression of ILF2 is closely related to tumor size and low survival rate,and the expression of ILF2 is mainly regulated at transcriptional level.2.ILF2 could enhance proliferation abilities of HCC cells and inhibit the cell apoptosis,and promote proliferation of tumor cells in nude mice.3.At the cellular level,BOK,BAX,BCL-2 and cIAPl were proved to be regulated by ILF2.Furthermore,ILF2 and cIAP1 were significantly increased in tumors from Lenti-ILF2-treated mice compared with the control group.4.In vitro,colony formation experiments and CCK8 assays showed that,si-ILF2 combined with different concentrations of EGFR inhibitor erlotinib could exert joint or synergistic effect on inhibition of HCC cell proliferation and viability.5.Microarray results showed that ILF2 was closely related to mRNA processing,RNA/DNA binding,metabolism and AMPK signaling pathway.