| Background:Hepatocellular carcinoma(HCC)is one of the three most common malignant tumors with the highest morbidity and mortality that threaten human health worldwide.There are nearly 500,000 new cases and more than 400,000 deaths in China each year.The most effective means for clinical treatment of HCC is still the surgical resection and liver transplantation.However,because the concealed incidence rapid progress,and high degree of malignancy,of HCC,most patients have developed into advanced stage at the time of diagnosis,and lost the best chance for surgical eradication,resulting in a 5-year survival rate less than 25%.At present,there is still no effective treatment for HCC besides surgical treatment and liver transplantatio,which is closely related to the high complexity and uncertainty of HCC.So far,there is still lack of understanding on the major or key factor of HCC,which makes it impossible to specifically design and develop effective therapeutic drugs or methods of diagnosis.Therefore,searching for the molecular characteristics of HCC,including identification of key factors and diagnostic markers,has important theoretical significance and application prospects for development of effective treatment.In order to find new key factors or diagnostic markers in the process of HCC,we analyzed the differentially expressed genes in cancer tissues and normal liver tissues by expression microarray,and screened an obvious new gene LHX3.This study will analyze the expression profile of LHX3 in hepatocarcinoma,paracancerous tissues and normal liver tissues,evaluate its clinical features and prognostic significance in HCC patients,and finally determine the biological function of LHX3.This study found and identified a potential key factor in HCC,providing preliminary data for final elucidation of HCC pathogenesis and search for effective diagnostic markers.Objective:(1)Analysis of the expression changes of LHX3 gene in cancer cells,normal tissues,paraplastic tissues and cancer tissues;(2)Determine the relationship between LHX3 expression and clinical pathological information of patients;(3)To analyze the effect of LHX3 expression on the overall survival of patients,and to evaluate its significance in the prognosis of patients;(4)Analysis of the main biological functions of LHX3 in the development and progression of cancer.Methods:(1)Reverse transcription-polymerase chain reaction(RT-PCR)and real-time quantitative PCR were used to detect the expression of LHX3 mRNA in cancer cells,normal tissues and cancer tissues.(2)Immunohistochemistry(IHC)staining was used to detect the expression of LHX3 protein in hepatocellular carcinoma tissues and paraplastic tissues,and quantify its expression by intensity and positive rate of staining;(3)The t-test was used to analyze the difference of expression between two groups,chi-square test was used to analyze the correlation between LHX3 expression and clinical data of patients,Kaplan-Meier and Cox-regression methods was used to evaluate the effect of LHX3 expression on survival time of patients;(4)By constructing LHX3 expression and interference vectors,transfecting the cancer cell line with low expression and high expression,respectively,to analyze the exogenous overexpression of LHX3 or interfering with the endogenous expression of LHX3 on migration and invasion of cancer cells.Results:(1)RT-PCR and RT-qPCR analyses revealed that the expression of LHX3 in tumor tissues(4.511 ± 0.4351)was significantly higher than that in normal tissues(2.609 ± 0.4252).This result was further validated by the independent HCC cohort data in the TCGA(The Cancer Genome Atlas)database.The expression of LHX3 protein in 190 tumor tissues and 40 paracancerous tissues by IHC analyses showed that the expression of LHX3 protein in tumor tissues(8.279 ± 0.2025)was also significantly higher than that in adjacent tissues(5.100 ± 0.5838).(2)LHX3 protein expression is closely related to clinical stage(n=186,P=0.009)and tumor metastasis(n=184,P<0.001)in HCC patients,but not related to the age,pathological grade,gender and tumor size of patients(P>0.05).The expression of LHX3 in the patients at advanced stage was significantly higher than that in the patients at early stage(P=0.0019),and the expression of LHX3 in tumor metastasis patients was much higher than that in non-metastatic patients(P=0.0027).(3)LHX3 protein expression was associated with hepatitis C virus infection(HCV,P=0.002)and the clinical marker CD34 expression(P=0.043)of patients,but was not associated with the expression of other clinical markers HEP1,C-ERBB-2,KI67,P53,HBCAG,AFP,CK8,CK19 and HBSAG(P>0.05).(4)The overall survival of patients with high LHX3 expression was significantly worse(P=0.001),and LHX3 expression was an independent prognostic marker(HR=1.964,P=0.004);There is a difference of prognostic significance between the patients at early stage(I+II)and the patients at advanced stage(III+IV).LHX3 expression is an unfavorable prognostic factor(P=0.001)and an independent prognostic factor(HR=2.285,P=0.008)in the patients at advanced stage,whereas it is not associated with survival of the patients at early stage.(5)Exogenous overexpression of LHX3 in hepatocellular carcinoma cell lines can significantly enhance the migration and invasion of tumor cells.On the contrary,interference with endogenous LHX3 in hepatocellular carcinoma cell lines can significantly inhibit the migration and invasion of tumor cells.Conclusion:The expression of LHX3 was significantly increased in HCC,and its expression was closely related to clinical stage and tumor metastasis of HCC patients.LHX3 expression was an independent and unfavorable prognostic factor for the patients at advanced stage.Further functional analysis found that LHX3 can significantly promote tumor migration and invasion.These results indicate that LHX3 is a potential biomarker for prognosis assessment in the patients at advanced atage,and a candidate oncogene for HCC cancer metastasis. |
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