Screening Based Proteomics And Clinical Verification Of Molecular Markers Of Hepatocellular Carcinoma Subtypes

ObjectivesTo analyze the molecular phenotype character of HCC subtypes with different clinical and pathological features, explore the molecular mechanisms of different biological characteristics, screen and verify potential molecular markers.1. To investigate the relevant clinicopathological characters of recurrence/metastasis of HCC in different period after radical resection.2. To compare the proteomics feature of HCC with different tumor number, find the molecular mechanism of different biological behavior of different HCC subtypes, search and verify the potential biomarkers.3. To compare the proteomics feature of HCC with different tumor size, find the molecular mechanism of different biological behavior of different HCC subtypes, search and verify the potential biomarkers.4. To explore the relationship between the expression of HK2 and recurrence/metastasis of HCC after radical resection. Methods1. Retrospectively analyzed the clinicopathological characters of recurrence/metastasis of HCC patients after radical resection. Univariate and multivariate logistic regression analysis were performed to analyze the risk factors for recurrence/metastasis of HCC in different period after radical resection.2. Compared the overall proteome feature between the HCC with single and multiple lesions by using iTRAQ-based quantitative proteomics approach. Screened the dysregulated proteins of HCC with different tumor number, and used bioinformatics technology to analyze the molecular function and signal pathways. Identified the potential biomarkers, and verified in mRNA level and protein level.3. Compared the overall proteome feature between the HCC with different tumor size by using iTRAQ-based quantitative proteomics approach. Screened the dysregulated proteins of HCC with different tumor size, and used bioinformatics technology to analyze the molecular function and signal pathways. Identified the potential biomarkers, and verified in mRNA level and protein level. The function and molecular mechanism of differential protein MT1 G in HCC was preliminarily studied.4. Detected the expression of HK2 in HCC tissue samples by using Western Blot method. Analyzed relationship between the expression of HK2 and the clinicopathological characters related to HCC recurrence/metastasis. Clinicopathological characters and the expression of HK2 were investigated to find the risk factors for recurrence/metastasis of HCC after radical resection by univariate and multivariate logistic regression analysis. Results1. The independent prognostic factors of recurrence/metastasis of HCC after curative hepatic resection in 12 months was find way, preoperative serum AFP value, tumor size and pathological differentiation of cancer cells; in 12 ~24 months was preoperative serum AFP value, tumors number; over 24 months was preoperative serum AFP value, tumor size, integrity of tumor capsule, pathological differentiation of cancer cells.2. There were 107 and 330 dysregulated proteins were identified in HCC tissue with multiple lesions(MC group) and HCC tissue with a single lesion(SC group), compared with their non-canceroustissue(MN and SN groups) respectively. The dysregulated proteins in MC group were concentrated in UBC and NFκB signal pathway, and the dysregulated proteins in SC group were concentrated in ERK and NFκB signal pathway. HSD17B13 was down-regulated only in MC group while HK2 was up-regulated only in SC group.3. There were 88 proteins in tiny HCC, 69 proteins in small HCC, 118 proteins in large HCC and 215 proteins in huge HCC, were identified respectively by comparing the proteome of cancerous tissues with its corresponding non-cancerous tissues. Analysis of dysregulated proteins involved signal pathway revealed that alternation of ERK1/2 and AKT signal pathways played important roles in the tumorigenesis or tumor growth in all HCC subtypes of different tumor size. Alternation of specific signal pathway was discovered in tiny HCC and huge HCC. A group of dysregulation proteins has been confirmed to be significantly correlated with the tumor size; these dysregulated proteins might be potential targets for study tumor growth of HCC.The expression of MT1 G in HCC was declined, and was negatively correlated with tumor size of HCC. The over-expression of MT1 G could inhibit cell proliferation and promote apoptosis.4. The expression of HK2 was related to the finding way, MVI, tumor size, tumor number of HCC. The postoperative cumulative survival rate of HCC in high HK2 expression group was significantly lower than that in low HK2 expression group. HK2 was an independent risk factor for recurrence/metastasis of HCC. Conclusions1. The independent prognostic factors for recurrence/metastasis of HCC after radical resection were different in different periods. Tumor number and tumor size were the independent prognostic factors for recurrence/metastasis of HCC in different periods after radical resection.2. There were different molecular characteristics and different involved signal pathways in HCC subtypes of different tumor number. HSD17B13 and HK2 might be potential biomarkers for HCC subtypes of different tumor number.3. There were different molecular characteristics and specific involved signal pathways in HCC subtypes of different tumor size. A group of dysregulated proteins were closely related to the tumor size of HCC. These dysregulated proteins might be potential biomarkers for HCC subtypes of different tumor size. Differential protein MT1 G may inhibit the proliferation of HCC through AKT signal pathway.4. The expression of HK2 was a predictor of the prognosis of HCC after radical resection. HK2 might become an effective molecular marker for recurrence/metastasis of HCC.