The Experimental Study On Inhibition Of Invasive Ability Of Human Lung Cancer Cell A549 By Heparanase Antisense Oligodeoxynucleotide

Background and ObjectiveLung cancer is one of the most common malignant tumor which seriously threaten human life and health in the world. Lung cancer has become the leading cause of death. In the big cities of Europe, America and China, the incidence has taken the first place in every kind of male tumor statistically. The 5-year survival rate is lower than 15%, because the majority of patients with lung cancer were already in the progressive phase as they are diagnosed, and have missed the optimal surgery opportunity. The accurate molecular mechanism of lung cancer has not fully elucidated. Therefore it is of great importance to explore the formation, development and metastasis molecular mechanisms of lung cancer and create a convenient method for diagnosis and prevention at the gene level. Recent researchers have demonstrated that multiple gene changes result in tumor genesis and progression. In the complicated courses, many genes are involved, including activation of proto-oncogenes and inactivation of antioncogenes. In clinical tumor therapy, the major factor which influences patients' prognosis is the conditions of tumor invasiveness and metastasis, and tumor biological behavior is also a major element in determination of tumor therapy plan. Many tumor experts have always been working hard at the early diagnosis of tumor invasiveness and metastasis, In order to improve tumor prognosis and prolong patients' life as much as possible. Therefore, tumor markers which can provide information on the conditions of tumor invasiveness and metastasis have always been emphasized by many tumor experts and become a hot researching subject, among which are HPSE. Heparanase (HPSE) is a type of Heparin lyase which was cloned by Vlodavsky in 1991 and able to degrade the heparanase of the extracellular matrix and basement membrane. The recent research shows that HPSE is expressed highly in the malignant tumor which plays an important role in facilitating the invasion and metastasis of tumor and has a close relationship with the tumorigenesis as well. Lung cancer is the most usually malignant tumor. Whether the expressions of HPSE and VEGF-C gene relate to the invasive growth and lymph node metastasis of the lung cancer are not reported.The research tested expression of HPSE gene of 52 cases in lung cancer tissues, adjacent tissues of cancer and normal lung tissues by immunohistochemical staining method so as to investigate the relationships between the expressions of HPSE gene and the invasiveness, lymph node metastasis and prognosis of the lung cancer; To evaluate the inhibitory effect of HPSE ASODN on the invasiveness of human lung cancer A549 cell lines;To study the inhibitory effects of heparanase antisense oligonucleotide (HPSE ASODN) on the growth and angiogenesis of tumor xenografts derived from A549 human lung cancer cell lines in nude mice.MethodsThe expression of HPSE protein in 52 cases of lung cancer was tested by immunohistochemical method and analysed with clinico-pathological characteristics and prognosis of lung cancer. HPSE ASODN which was complementary with initiation codon region of HPSE mRNA was designed and synthesized. After embedded by cation lipofectin, it was transfected into A549 cells of human lung cancer. The transfection of ASODN into A549 was divided into three groups as follows:ASODN group (transfected with the lipofectin expressing ASODN), lipofectin group (transfected with lipofectin only) and control group. The expression of HPSE protein and HPSE mRNA in A549 cell lines were detected by Western blot and RT-PCR. Meanwhile Transwell invasive assay was used to measure the inhibitory effect of HPSE ASODN on the invasiveness of human A549 cell lines. The transfected cells in three groups were injected subcutaneously into nude mice to establish the model of human lung cancer xenografts respectively. The growth curve of the tumor xenografts was observed and the inhibitory rate of tumor calculated. Furthermore, the expression of heparanase protein and micro-vascular density (MVD) were detected by immunohistochemistry method in the tumor. Grading were carried out according to the dying intensity and the positive cell number.Results were expressed as mean value standard deviation (x±s). T test and one-way ANOVA test were used to evaluate differences among the groups. SPSS 13.0 software was used to make statistics analysis. A value less than 0.05(P<0.05) were taken as having statistics significance.ResultsPart I The expression of heparanase in human lung cancer tissue and its significanceThe results of immunohistochemical method show that the rate of expression of HPSE protein in lung tumor tissues was 63.5%. It was shown that HPSE protein expression did significantly not correlate with the type(66.7%,56.3%) (P>0.05), and grade of the tumor (63.6%,57.9%,68.2%) (P>0.05), but it correlated with the clinical TNM stage (80.0%,61.5%,45.5%) (P<0.05) and survival time of the patients (82.6%,48.3%) (P<0.05).Part II Inhibition of invasiveness of human lung cancer cell lines by heparanase antisense oligodeoxynucleotideThe HPSE protein and HPSE mRNA expression and invasiveness of human A549 cells treated with ASODN of different concentrations were significantly decreased as the ASODN concentration increasing. There was a significantly difference between control group and each group of ASODN respectively (P<0.01), between lipofectin group and each group of ASODN respectively (P<0.01), and among the groups of ASODN (P< 0.01). Besides, the difference of inhibitory effect was significantly among the cells treated with different ASODN concentrations (P<0.01). The inhibition rates of A549 cell invasiveness were 49.2%,73%,87.5% treated by ASODN at final concentration of 100nmol/L,200nmol/L and 400nmol/L respectively.Part III Inhibitory effects of heparanase antisense oligodeoxynucleotide on human lung cancer cells transplanted subcutaneously in nude mice.The formation of tumor xenografts in the ASODN group was apparently late, and the volume of the tumor was smaller than that in the control group and lipofectin group(P< 0.01), the inhibitory rate was 64.3% at the time of the sixth week. The MVD counts were 13.5±1.8,24.3±2.5,24.7±2.6 in the three groups respectively, and compared with the control groups, the MVD in the ASODN group was apparently less(P<0.01). Conclusions1. The results suggested that there be overexpression of HPSE protein in lung cancer tissues, and which perhaps participate in regulation of progression in lung cancer. HPSE play a important role in the invasiveness and metastasis of human lung cancer. The expression of HPSE protein be used as an useful marker of the biological behavior of lung cancer and as an independent prognosis factor for the patients with lung cancer.2. By down-regulating the expression of HPSE mRNA and HPSE protein, HPSE ASODN has significant inhibitory effect on the invasiveness of human A549 cell line in a dose-dependent manner.3. The heparanase antisense oligonucleotide can inhibit the tumor growth and angiogenesis of the exnografts of human lung cancer cell lines A549 in nude mice, and it is very promising to be used as a potential way of gene therapy for human lung cancer.