Expression Of SEMA3B Protein In Human Hepatocellular Carcinoma And Its Clinical Significance

Objective:Semaphorin3B (SEMA3B) is a secreted51kDa protein that is processed from a83kDa precursor. The gene encoding SEMA3B is located in the LUCA region of chromosome3p21.3and is highly methylated (～83%) in hepatocellular carcinoma cells (HCCs). It has been reported that SEMA3B plays an important role not only in the development of the neuron axons, but also in inducing apoptosis in lung and breast cancers. Recent studies showed that the expression of SEMA3B is down-regulated in many cancers including prostate and ovarian cancers. The down-regulation of SEMA3B mRNA expression was recently confirmed by semi-quantitative PCR. However, the expression of this gene at the protein level and its clinical significance has not been reported. In this study, we used both immunohistochemistry and Western blotting techniques to study the expression of SEMA3B at protein level. We also investigated the relation of the expression of SEMA3B with clinicopathologic factors and tumor angiogenesis in order to explore the importantance of SEMA3B in prognosis.Methods:1.Clinical Specimens and Pathological Materials The resections of the carcinoma and tumor-adjacent (≥1cm from the cancer tangent) tissues were collected from56patients with hepatocellular carcinoma. These patients (47males and9females, ages32to73) were hospitalized from July2008to June2009in the Department of General Surgery of Qilu Hospital of Shandong University. As controls,14normal liver tissues were also collected. All of these specimens were pathologically confirmed and the patients used in this study had not been treated by either radiofrequency ablation or intervention before surgery. Each of the collected specimens was divided into two parts, one was flash-frozen in liquid nitrogen and the other was paraffin-embedded.2.Immunohistochemistry To study the expression of SEMA3B, the sections were stained with SEMA3B polyclonal antibody (ABCAM, UK,1:200). Immunosignals were detected using streptavidin-biotin-fluorochrome (SABC) supplied within a kit (GeneTex, USA). To measure microvessel density (MVD), mouse anti-human CD34monoclonal antibody (Santa, USA,1:200in PBST) were used and the detection procedure was similar to that used for SEMA3B. As negative controls, PBS was used instead of primary antibody.For the purpose of quantification, semi-quantitative integration method was adopted. First, five fields on each slice were randomly selected under the magnification of100x, and500cells in each field were counted under a higher magnification (200x). The appearance of yellow to brown particles in the cytoplasm was considered a positive signal. For the purpose of statistical analyses, the staining intensity assigned as follows:no color=0point, yellow=1point, brown=2points and tawny=3points. The scores for SEMA3B-positive cells over total cells were defined as follows:≤25%=0point,25%to50%=1point,50%to75%=2points and>75%=3points. Overall negative and positive groups were sampled based on the sum of the above two scoring systems:total scores less than or equal to3was classified as negative group, while more than or equal to4, positive group. To measure MVD, the five highest vascular density areas were selected under100x light microscope, and CD34-positive cells or cell clusters in each area were counted under200x (0.708mm2). The average number of the five regions was used as the MVD value. The results were independently confirmed by two senior pathologists.3.Western blot200mg of each tissue were homogenized in0.6ml of lysis buffer (Bi Yun Tian, China). After incubation for30min at room temperature, the homogenates were centrifuged at16,000xg for10min at4℃and the supernatant was collected. Protein concentration was determined according to the instruction of the BCA Protein Concentration Quantification Kit (Bi Yun Tian, Beijing, China). For Western blotting,50℃for5min and separated on a10%polyacrylamide gel. The separated proteins were transferred onto a PVDF membrane, which was then blocked in TBST supplemented with5%of dry milk for2-3hours. The blocked PVDF membrane was incubated overnight at4℃with rabbit anti-human SEMA3B polyclonal antibody (ABCAM, UK,1:5000dilutions in TBST). The membrane was then washed3times with TBST, with each wash lasting for10min. After the last wash, the membrane was incubated for90min at room temperature with goat anti-rabbit secondary antibody (Bi Yun Tian Company, China,1:2000dilutions in TBST). The membrane was then washed3times with TBST and immunosignals were detected with the ECL Luminescent Reagent Kit (MILLIPORE, USA).4.Statistics For statistical comparison of counting data among the negative and positive groups,χ2test was used. For drawing the survival curve and comparing the differences among survival curves, Kaplan-Meier Survival Analysis and Log Rank methods were applied, respectively. All of the statistical analyses were performed with the SPSS18.0statistical software and P<0.05is considered to be a statistically significant difference.Results:1.Expression of SEMA3B was down-regulated in hepatocellular carcinoma cellsIn this study, we investigated the expression of SEMA3B protein in hepatocellular carcinoma (HCC) cells and studied its clinical significance. To compare the expression profiles of SEMA3B protein between normal and hepatocellular carcinoma cells, we carried out both immunohistochemical and Western blotting analyses. Immunostaining with the SEMA3B polyclonal antibody showed that this protein is located in the cytoplasm. The expression of this protein was significantly down-regulated (P<0.05) in the hepatocellular carcinoma cells when compared to the cells from either tumor-adjacent, or normal liver tissues. Statistically, the SEMA3B-positive cells in the normal liver and paraneoplastic tissues were78.6%and85.7%, respectively, while in hepatocellular carcinoma cells only42.9%expressed SEMA3B protein. In support of this result, our Western blotting analysis showed that the expression level of SEMA3B protein was much lower in the cancer tissues than that in the tumor-adjacent or normal tissues (85.7%and78.6%, respectively).2.Expression of MVD Microvessel density (MVD) can be measured by the number of CD34-positive endothelial cells. In this study, CD-34positive cells were immunohistochemically stained with the monoclonal anti-CD34antibody. The number of the CD43-positive cells in the area of0.708mm2(full field area under magnification of200) were recorded under light microscopy. To statistically study the MVD in different tissues, we defined92as a median value of MVD in this study. A value greater than92was considered up-regulated while a number less than or equal to92was considered down-regulated. Interestingly, we found that the expression profiles of MVD were in the opposite direction to those of SEMA3B protein. Specifically, the MVD value in the hepatocellular carcinoma cells was112±7.5, and this value was significantly down-regulated in the normal tissues (85.1±4.4, P<0.05). The negative correlation between the level of SEMA3B protein and the expression of MVD may reflect that the importance of SEMA3B in the regulation of tissue vascularization.3.SEMA3B is important to characterize the clinical and pathological features of hepatocellular carcinoma cells (HCCs) To unveil the possible role of SEMA3B in cancer diagnosis, we statistically analyzed the correlation between the expression of SEMA3B and the clinical and pathological features of the56patients with hepatocellular carcinoma. In this study, we analyzed the differences between SEMA3B-positive and SEMA3B-negative patients in the following clinical and pathological features such as gender, age, hepatitis B, AFP, liver cirrhosis, the differentiation of the tumor cells, size, with or without a complete capsule, the number of tumor nodules, CLIP score, and TNM stage. These analyses showed that the number of the tumor nodules in the SEMA3B-positive group was significantly higher than those in its negative group (P=0.045). The size of tumors in the SEMA3B-positive group were statistically larger than those in the SEMA3B-negative group (P=0.034). Furthermore, the number of the completely encapsulated tumors in SEMA3B-positive patients were much more than that in SEMA3B-negative patients (P=0.009), while the CLIP score in SEMA3B-positive group was significantly lower than that in the SEMA3B-negative group (P=0.013).4. To study the relationship between expression of SEMA3B and prognosis, we continuously monitored the56patients with hepatocellular carcinoma (24SEMA3B-Positive,32SEMA3B-negative). Clinical follow-up studies showed that the cancer recurrence rate in the SEMA3B-positive group was significantly lower than that in the SEMA3B-negative group (P=0.009).Furthermore,the survival rate in SEMA3B-positive group was significantly higher than that in the SEMA3B-negative group (P-0.020).Conclutions:The expression of SEMA3B was decreased in HCC tissues, and its expression was closely associated with tumor progression, angiogenesis and prognosis, indicating that it might be a predictor of prognosis and a possible novel target of antiangiogenic therapy for patients with HCC.