|Year : 2013 | Volume
| Issue : 2 | Page : 250-252
The expression of Heparanase mRNA and its clinical significance in Hepatocellular carcinoma
Qiang Wang, Yu Shen, Peng Wang, Xiaowei Li
Department of General Surgery, Jiangsu Shengze Hospital, Wujiang, Jiangsu, China
|Date of Web Publication||13-Jun-2013|
Department of General Surgery, Jiangsu Shengze Hospital, 9 Shichangzhonglu Road, Wujiang, Jiangsu
Source of Support: None, Conflict of Interest: None
Objective: To investigate the clinico-pathologic importance of Heparanase mRNA expression in hepatocellular carcinoma and its relationship with prognosis.
Materials and Methods: Fresh tumors and noncancerous liver tissues were obtained from 30 hepatocellular carcinoma patients after hepatectomy. Expression levels of Heparanase and glyceraldehyde-3-phosphate dehydrogenase mRNA were quantitatively analyzed by real-time reverse transcriptase-polymerase chain reaction.
Results: The relative Heparanase mRNA expression level (Heparanase: glyceraldehyde-3-phosphate dehydrogenase ratio) in hepatocellular carcinoma was lower than that in noncancerous tissue (P < 0.05).
Conclusion: Reduced Heparanase mRNA expression might result in abnormal cell growth and correlate with hepatocellular carcinoma progression.
Keywords: Heparanase, Hepatocellular carcinoma, Real-time polymerase chain reaction
|How to cite this article:|
Wang Q, Shen Y, Wang P, Li X. The expression of Heparanase mRNA and its clinical significance in Hepatocellular carcinoma. J Can Res Ther 2013;9:250-2
| > Introduction|| |
The incidence rate of hepatocellular carcinoma ranks fifth in malignancy cancer worldwide, and the mortality rate of systemic cancer ranks the third.  Hepatocellular carcinoma has become the most fatal tumor in China. Most of the patients are in advanced stage by the time the diagnosis is confirmed, and the prognosis is poor.  The 5-year survival rate for hepatocellular carcinoma patients was within 5%. The main reason is that the cancer cells invade portal vein thus lead to dissemination which cause high metastasis rate and high postoperative recurrence.  In the last decade most hepatocellular carcinoma metastasis studies focused on protease whose substrate is structural protein. The knowledge of Heparanase whose substrate is glycosaminoglycan or heparansulfateproteoglycan is preliminary.  For this reason, 30 cases of hepatocellular carcinoma were detected by Heparanase mRNA to know its express in hepatocellular carcinoma as well as if the clinical and pathology stages was significantly correlated with metastasis and recurrent after operation.
| > Materials and Methods|| |
Choice of Cases
30 cases of primary hepatocellular carcinoma proved by pathology all came from January to December 2010 in Jiangsu Shengze Hospital. There were male 24 and female 6. Their age was from 28 to 78, and the mean operation age was 46 years old. Among these, one of the men was found intrahepatic diffuse spread and a few organizations was taken and send pathology examination. The others were performed liver cancer resection, one of which was recurrent liver cancer, and all of these did not do interventional therapy.
After removing of liver cancer in surgery, a small piece of hepatocellular carcinoma tissue (about 0.5 cm × 0.5 cm × 0.5 cm) was cut immediately in a sterile conditions, and necrotic tissue and bleeding area was avoided. Non-tumor tissue around the liver cancer tumors were taken from liver 2 cm from the carcinoma edge, which didn't infiltrated by tumor. The specimens was immediately frozen in liquid nitrogen and stored in -75℃.
Total RNA Extraction
The RNA was extracted by TRIzol (Shanghai biological products company) from 50 mg tissue and the operation was according to the manufacturers recommend steps. The operation of retrovirus reagents (Revert Aid TM First Strand cDNA Synthesis Kit, Jingmei Co) were according to the manufacturers recommend steps.
Real-Time PCR for Heparanase mRNA Expressions
With reference to amplification production of housekeeping gene GAPDH, sample templates were standardized on quantity. Heparanase and GAPDH amplification were in two tubes. The primer sequence was designed to the literature of Koliopanos A et al., and combined by Shanghai Biological Engineering Company.
upstream primer: 5'-TCACCA TTg Acg CCA ACC T-3',
Downstream primers: 5'- CTTTgC AgA ACC Cag gAg gA T-3',
TaqMan probe: 5'-FAM2CCA Cgg ACC CgC ggT TCC T-3'-TAMRA
upstream primer: 5'-GAA GGT GAA GGTCGG A GT C-3'
Downstream primers: 5'-GAA GA T GGTGA T GGG A TT TC-3',
TaqMan probe: 5'-FAM2CAA GCT TCC CGT TCT CA G CC-3'-TAMRA.
PCR condition: 94°C denaturation for 45 s,60 °C anneal for 15 s,72℃ 45 s,50 circulations; 72°C fully stretching for 5 minutes.
The results was analyzed by Wilcoxon Matched-Pairs Signed-Ranks Test and rank sum test of two independent samples, which was done by SPSS11. 0 statistical software.
| > Results|| |
The expression of Heparanase mRNA in different tissues
According to the findings of RT-PCR, Heparanase mRNA expressed in non-tumor tissues surrounding the liver cancer tumors was superior to that in hepatocellular carcinoma tissue (28/30). Heparanase mRNA expression in non-tumor tissue was inferior to that in hepatocellular carcinoma tissue (4/30). The gene expression differences was significant in the two types of carcinoma tissues (P < 0.05). Heparanase mRNA expression in non-tumor tissue was superior to that in hepatocellular carcinoma tissue.
The relationship between the Heparanase mRNA expression, the clinico-pathological features, metastasis, and recurrence of hepatocellular carcinoma
The expression of Heparanase mRNA does not depend on the size of cancer focal (Edmondson classification and TNM classification) (P > 0.05). Its expression was bound up with AFP level, HBsAg condition, and prognosis (P < 0.05), see [Table 1].
| > Discussion|| |
0Heparanase is a general designation of endogenous glucuronidase, which is cracking heparan sulphate of ECM and heparan sulphate proteoglycan in an epithelial basement membrane. The complete structure of human Heparanase cDNA is a mature protein with high activity. It is formed by 386 amino acid residues on C-terminal after cutting 543 amino acid residues, molecular weight 50 kDa. Moreover, the hydrolysis activation of precursor enzyme includes participating in degradation of the ECM protein and cell infiltration enzymes. Different tissues and cells, such as human placenta, platelets, and metastatic tumor cells still express the same Heparanase.
The metastasis of tumor occurs when the tumor cell breaks through the restriction of extracellular matrix to diffuse into other organs in order to continue with its proliferation. Cancer is termed as a fatal disease because of its uncontrolled proliferation and metastasis. In the process of metastasis, cell adhesion, cell movement, degradation of ECM, angiogenesis determine tumor metastasis. According to the research of Hulett MD et al., the activity of Heparanase is related to metastatic ability of tumor cells in animals. In the report of Ikuta M et al., the incidence rate of tumor metastasis in patients with oral cancer is related to the high level expression of Heparanase mRNA. In addition, Koliopanos A et al. reported there was a significant negative correlation between the survival rate of pancreatic cancer patients after surgery and the expression of Heparanase mRNA. These results suggest that there is a positive correlation between the expression of Heparanase mRNA and the invasive ability of tumor cell. However, the research on the Hepatocellular carcinoma indicates an exactly opposite result. This study shows the expression of the gene is much lower in tumor tissue of Hepatocellular carcinoma than that in the normal hepatic tissue. Moreover, greater the degree of aggregation in tumor, lower is the expression of the tumor Heparanase mRNA. Ogawa K et al. reported Hepatocellular carcinoma cell line of rats, and found decreased expression of Heparanase mRNA in one of the cell line. The high metastases rate of lungs was found after the cell was injected into the nude mice through the skin. The research results prove that the biological significance of the expression of Heparanase mRNA in tumor cell might have complex content.
In recent years, the biological significance of Heparan sulfate proteoglycans (HSPGs) was reported. HSPGs is the main constituent of extracellular matrix and basement membrane. It is ubiquitin macromolecule combined with the cell surface, which is connected to bFGF and its acceptor. The compound composed of these three molecules plays an important part in the course of signal transduction. Kuniyasu H et al. found heparan sulfate could increase the invasiveness of rectal cancer cell line. Liu D et al. reported that Heparinase III could reduce the tumor volume of rats and the rate of pulmonary metastasis after injecting B16BL6 melanoma cells. These results indicate that HSPGs play an important role in the course of metastasis and advancement of tumor. Moreover, the over expression of heparan occurs by damaging the bFGF/HSPG/RTK compound restraining the tumor progress. However, there was a report showed heparan could promote the tumor vessels formed. Suppose heparan decreased the connect of HSPGs and bFGF or VEGF in the extracellular matrix. As a result, the dissociative bFGF or VEGF could show the strong activity of angiogenesis promoting effect. El-Assal ON et al. reported that there was a significant positive relation between the expression degree of heparan mRNA in hepatocellular carcinoma and microvessel density of tumor.
Angiogenesis requires the interaction between these various factors. In the progress of angiogenesis, heparan may play a role, however, it is difficult to determine whether heparan plays an importance role of tumor angiogenesis in clinical practice. Surprisingly, Ginath S et al. found that heparan protein was highly expressed in ovarian cancer on the basis of immunohistochemistry. So, they considered apoptosis was the main mechanism through which heparan migrated from intracellular to extracellular matrix. Liu D et al. reported that the number of apoptotic cell markedly increased in the rats, which were subcutaneously injected with B16BL6 melanoma cells. This phenomenon shows that the degradation of bFGF/HSPG/RT compound by heparan may induce apoptosis as it reduces the signal transduction of bFGF path. Therefore, the heparan may control cell growth signal transduction. And the expression of heparan mRNA is necessary for the mutation of normal cells. Furthermore, a reduction in the mRNA expression of the heparan may cause abnormal cell growth. In other words, the low expression of heparan mRNA is related with the progression of tumor.
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