The Study Of AKR1B10 In Early Diagnosis Of Hepatocellular Carcinoma And Its Inhibitor In Regulation Of HepG2 Cell Proliferation

Objectives:Early diagnosis of hepatocellular carcinoma(HCC),which is closely related to the prognosis,is very important.Alpha-fetoprotein(AFP)is the most widely-used serum marker for early diagnosis of HCC in clinical practice,which is still lack of sensitivity and specificity.Therefore,we still go for seeking new and more sensitive serum markers for early diagnosis of HCC.By now,some published researches show that AKR1B10 is closely related to several cancers,which may be a serum biomarker for early diagnosis of cancers and the target for anti-tumor therapy.1.We detect serum AKR1B10 levels in HCC patients,and compare them with patients of chronic hepatitis and liver cirrhosis.We evaluate the value of AKR1B10 in early diagnosis of HCC,and aim to improve the sensitivity and specificity in HCC diagnosis by detection of AKR1B10 and AFP.2.We detect the expression of AKR1B10 in HCC tissues,compare differences of AKR1B10 levels in tissues of different differentiation,and explore the relationship between AKR1B10 levels and tumorigenesis.3.We aim to discover the role of AKR1B10 inhibitor in regulation of HepG2 cell proliferation and apoptosis by dealing HepG2 cell with Epalrestat,which is a kind of AKR1B10 inhibitors,and reducing AKR1B10 expression stably.Meanwhile,we discuss whether AKR1B10 inhibitor enhances the effancy of Sorafenib,which is the HCC molecule-targeting agent,or not.Methods:Part One: The role of serum AKR1B10 in early diagnosis of hepatocellular carcinoma.We enroll 84 cases with hepatocellular carcinoma,74 cases with liver cirrhosis,29 cases of chronic hepatitis as subjects,and 30 cases of healthy persons as control.We detect their serum AKR1B10 levels by sandwiches ELISA kit,compare them among different groups and do statistical analysis.We invent HCC early diagnosis model with AKR1B10 and AFP,and discuss the sensitivity and specificity of early diagnosis of HCC.Part Two: The expression of AKR1B10 in HCC tissues of different differentiation.We enroll 44 cases of liver cancer tissue,in which there are 16 cases in high differentiation,19 cases in moderate differentiation,and 9 cases in poorly differentiated,and 37 cases of liver cirrhosis tissue.We detect the expression of AKR1B10 by immunohistochemical,and compare the IOD among different groups.Part Three: The role of AKR1B10 inhibitor in regulation of HepG2 cell proliferation.We deal HepG2 cell with Epalrestat,which is a kind of AKR1B10 inhibitors,and Sorafenib,which is the HCC molecule-targeting agent.Meanwhile,we develop a cell line,which is stable to interfere AKR1B10 expression by shRNA.We discuss the role of AKR1B10 inhibitor in regulation of HepG2 cell proliferation,and make it clear whether Eplarestat can enhance the effancy of Sorafenib or not by detecting HepG2 cell proliferation and apoptosis.All data are statistically analysed by SPSS 17.0 software.The continuous variable make standard description statistics,statistics use mean ± standard deviation as statistical description,multiple sets of comparison use single factor analysis of variance,and the two comparisons use the LSD method.With P<0.05 for the difference is statistically significant.Results:Part One: The role of serum AKR1B10 in early diagnosis of hepatocellular carcinoma.(1)The levels of serum AKR1B10 in HCC,liver cirrhosis,chronic hepatitis groups are separately 4.02±3.53ng/ml,2.61±2.00ng/ml,1.12±1.01ng/ml.The highest level of AKR1B10 is in the HCC group,which is significantly higher than that in liver cirrhosis and chronic hepatitis group.The difference is statistically significant(P < 0.05).(2)Among patients in HCC,the serum AKR1B10 level of patients in BCLC early stage and intermediate stage are separately 5.94±4.90ng/ml,5.91±3.76ng/ml,which are significantly higher than that in advanced group and terminal group(2.61±1.54ng/ml,2.21±1.60ng/ml).The difference is statistically significant(P < 0.05).(3)The AUC of AKR1B10 and AFP in the diagnosis of HCC is 0.759,0.796,separately.The sensitivity of combined diagnosis of HCC is 92.86%(78/84),specificity is 52.63%(70/133),positive predictive value is 55.32%(78/141),and negative predictive value is 92.11%(70/76).The sensitivity and negative predictive value of the combined diagnosis is significantly higher than the above two measures separately.Part Two: The expression of AKR1B10 in HCC tissues of different differentiation(1)The expression of AKR1B10 in HCC tissue is much higher than that in liver cirrhosis tissues.(2)The expression of AKR1B10 among HCC tissues in well differentiation,moderate differentiation and poor differentiation are different.The expression of AKR1B10 in moderate differentiation tissue is the highest,while it is lower in the high differentiation tissue and rare in poor differentiated tissue.The difference is statistically significant(P < 0.05).(3)In Barcelona clinical liver cancer staging,the expression of AKR1B10 is the highest in the early stage,while the intermediate stage and advanced stage are lower.The difference is statistically significant(P < 0.05).Part Three: The role of AKR1B10 inhibitor in regulation of HepG2 cell proliferation.(1)The proliferation of HepG2 cell is inhibited and the apoptosis is increased by dealing HepG2 cell with Epalrestat.(2)After we interfere the expression of AKR1B10 gene,the proliferation of HepG2 cell is significantly inhibited,and the apoptosis is increased.(3)The proliferation of HepG2 cell is inhibited and the apoptosis is increased by dealing HepG2 cell with Epalrestat and Sorafenib together,which is significantly higher than using a single drug.Conclusions:(1)AKR1B10,which can be used as a biomarker in early diagnosis of HCC,can be detected from the serum of HCC patients(including those in the early stage of BCLC staging).(2)The combination of serum AKR1B10 and AFP can significantly improve the sensitivity and specificity in early diagnosis of HCC.(3)AKR1B10,which is associated with differentiation of the tumor,can be detected in HCC tissue and it is highly expressed in the moderately and well differentiated HCC tissue.(4)AKR1B10 is associated with the BCLC staging,the expression of which is the highest in HCC patients in the early stage of BCLC staging.(5)The AKR1B10 inhibitor can significantly inhibit the proliferation of HepG2 cell,and promote its apoptosis.(6)The combination of the AKR1B10 inhibitor,which enhances the effancy of Sorafenib,and Sorafenib can further inhibit the proliferation of HepG2 cell,and promote its apoptosis.They inhibite the proliferation of tumor cell synergistically.