Study On The Correlation Between The Content Of DNA Cytosine-modified Bases And Small Hepatocellular Carcinoma And Targeting IRAK1 For Human Hepatocellular Carcinoma

Part ? Study on the Correlation between Changes in Content of DNA Cytosine-Modified Bases and Clinical Prognosis of Small Hepatocellular CarcinomaObjectives: The investigation of new molecular markers for diagnosis of hepatocellular carcinoma was critical for early detection,standardized treatment and improvement of prognosis of hepatocellular carcinoma.This study investigated the correlation between changes in content of DNA cytosine-modified bases and the recurrence,metastasis and prognosis of small hepatocellular carcinoma(SHCC),thereby providing a basis for evaluation of the treatment and survival prognosis of SHCC.Methods: All the patients in this study met the inclusion criteria.The content of DNA cytosine-modified bases 5m C,5hm C and 5f C in 63 patients with SHCC was detected by mass spectrometry;the content of 5hm C and expression levels of CD133,CD44,OV6,CK19,Ep CAM and other HCC stem cell markers in 63 patients with SHCC were detected by immunohistochemical assay;the correlation between the content of DNA cytosine-modified bases and clinicopathologic factors and survival prognosis of SHCC was analyzed.Results:1.Mass spectrometry revealed that the content of genomic DNA 5m C,5hm C,5f C in cancer tissue was significantly lower than that in adjacent tissue(P<0.05).Correlation analysis showed that the 5m C content in cancer tissue and the 5hm C content in cancer tissue were positively correlated,so were the 5hm C content in cancer tissue and the 5f C content in cancer tissue(P<0.05).Correlation analysis on pathological factors showed that 5m C content in cancer tissue was correlated with serum HBV-DNA content(P<0.05);5m C content in adjacent tissue was correlated with AFP,microvascular tumor thrombus and tumor metastasis(P<0.05);5hm C content in cancer tissue was correlated with survival time and serum HBV-DNA content(P<0.05);5hm C content in adjacent tissue was correlated with tumor metastasis,AFP and survival time(P<0.05);5hm C content in cancer tissue/5hm C content in adjacent tissue was correlated with T stage,microvascular tumor thrombus and serum HBV-DNA content(P<0.05);5f C content in cancer tissue was correlated with tumor size,AFP,survival time(P<0.05);5f C content in adjacent tissue was correlated with liver cirrhosis(P<0.05);5f C content in cancer tissue/5f C content in adjacent tissue was correlated with microvascular tumor thrombus and T stage(P<0.05);HBV-DNA content in cancer tissue was correlated with liver cirrhosis(P<0.05),but not with other pathologic factors.Univariate survival analysis demonstrated that 5hm C content in cancer tissue,5hm C content in cancer tissue/5hm C content in adjacent tissue,5f C content in cancer tissue,postoperative recurrence,tumor capsule,microvascular tumor thrombus,Child-Pugh classification and T stage were correlated with the disease-free survival and overall survival of patients with SHCC;besides,serum HBV-DNA content was correlated with disease-free survival(P<0.05);COX multivariate analysis showed that 5hm C content in cancer tissue,postoperative recurrence and tumor metastasis were independent prognostic factors for disease-free survival and overall survival of patients with SHCC;microvascular tumor thrombus was also an independent prognostic factor for overall survival(P<0.05).2.Immunohistochemical assay revealed that the 5hm C content and expression levels of CK19 in adjacent tissue were significantly higher than those in cancer tissue(P<0.05);the overall expression levels of HCC stem cell markers CD133,OV6,CD44 and Ep CAM in cancer and adjacent tissue were low without statistical difference(P>0.05).A small sample analysis showed that a larger ratio of 5hm C-positive cells to CK19-positive cells near adjacent tissue was associated with a higher incidence of metastasis,a higher mortality(P<0.05),a shorter disease-free survival,a shorter overall survival(P<0.01),and therefore a poorer prognosis.However,a large sample analysis showed no such correlation with disease-free survival or overall survival.Conclusions:In patients with SHCC,the 5hm C and 5f C content in cancer tissue was closely correlated with tumor recurrence,metastasis and survival prognosis.The content of DNA cytosine-modified bases in cancer tissue could be a potential biomarker for the recurrence and prognosis of SHCC.It had important potential value in clinical application and was worthy of further in-depth study.Part ? Research on the Effects of the IRAK1 on the Proliferation,Migration and Cycle of Hepatoma CellObjective: Based on previous studies,we found that IRAK1 significantly expressed in hepatocellular carcinoma and hepatocellular carcinoma cell lines,and there were differences in the phosphorylation status and levels at different loci,suggesting that IRAK1 played an important role in the development of hepatocellular carcinoma.In this study,we used a pharmaceutical IRAK1/4 inhibitor to study the effects and mechanisms of inhibiting IRAK1 activity on hepatocellular carcinoma cell migration,proliferation,apoptosis,cell cycle in vitro,HCC xenograft in vivo and other biological functions,thereby revealing the important role of targeting IRAK1 in hepatocellular carcinoma treatment.Methods: 1.Using immunohistochemical method to detect the expression p-IRAK1(T209)level in liver tissues;and to evaluate the expression p-IRAK1(T209)level in hepatocellular carcinoma cell lines by Western Blot technique.2.Western Blot method was used to study the effect of IRAK1/4 small-molecule inhibitor on the expression levels of IRAK1 and p-IRAK1 in kinds of hepatocellular carcinoma cell lines;the effects of different doses of IRAK1/4 small-molecule inhibitor on hepatocellular carcinoma cell line IRAK1 and p-IRAK1(T209)were investigated.3.Cell Counting Kit-8(CCK-8)proliferation assay and cell Colony formation analysis were used to study the effect of IRAK1/4 small-molecule inhibitor on the proliferative capability of hepatocellular carcinoma cells after inhibiting the phosphorylation level of p-IRAK1(T209).4.Transwell migration assay and wound healing assay were used to study the effect of inhibiting p-IRAK1(T209)level on the adhesion and migration capacity of hepatocellular carcinoma cells.5.Flow cytometry with Annexin V/PI staining was used to study the changes in hepatocellular carcinoma cell apoptosis and cell cycle when the phosphorylation level of p-IRAK1(T209)is down-regulated with IRAK1/4 small-molecule inhibitor.6.HCC xenograft were used to study the effect of IRAK1/4 small-molecule inhibitor on subcutaneous liver cancer growth in mice,and immunohistochemical method was used to detect the change in proliferation factor of subcutaneous hepatocellular carcinoma.Results:1.p-IRAK1(T209)were highly expressed in the HCC tissues comparing with adjacent normal tissues by immunohistochemical staining and highly expressed in hepatocellular carcinoma cell lines by Western Blot technique.2.Western Blot assay results showed that,IRAK1/4 inhibitor was able to significantly down-regulate the p-IRAK1(T209)levels in several hepatocellular carcinoma cell lines,and the down-regulation amplitude was positively correlated with inhibitor concentration;20?m IRAK1/4 inhibitor was an ideal inhibitor dose,while the changes in the overall levels of p-IRAK1(S376)and IRAK1 were not significant.3.IRAK1/4 inhibitor was able to down-regulate p-IRAK1(T209),thereby effectively inhibiting the proliferation capacity,migration capacity and cloning level of hepatocellular carcinoma cells.4.After down-regulating the p-IRAK1(T209)level in hepatocellular carcinoma cell,IRAK1/4 inhibitor was able to induce hepatocellular carcinoma cell apoptosis and cycle arrest.5.IRAK1/4 inhibitor was able to significantly inhibit the growth of subcutaneous hepatocellular carcinoma;compared with the control group,the difference was significant(P<0.05).Conclusions:The study revealed the important role of IRAK1 in promoting HCC growth.The level and status of p-IRAK1(T209)was inhibited by the pharmaceutical IRAK1/4 inhibitor.Theeffects and mechanisms of inhibiting IRAK1 activity on hepatocellular carcinoma cell migration,proliferation,apoptosis,cell cycle and other biological functions in vitro,which led to lessened HCC xenograft tumor growth in vivo.In summary,IRAK1 promotes cell proliferation and resistance to apoptosis in HCC,and can be a novel target for HCC treatment.