CD147 Expression In Human Osteosarcoma And Inhibitatin Of The Antisense RNA To Osteosarcoma Invasion And Metastasis

Osteosarcoma is the most frequent highly malignant tumor in adolescent. The traditional ways of osteosarcoma treatment are amputation and exsect of tumor segment, patients who accept these treatments usually died of pulmonary metastasis within early stage so the survival ratio is very low. With the development of surgical and imaging technology, the improvement of neoadjuvent chemotherapy, radiotherapy and biotherapy, and the understanding of tumor biology character, the long-term survival rate is markedly increased. But now no effective ways can be used to treat tumor patients in later period, 90% of whom still died of complication associated with tumor invasion and metastasis. Invasion and metastasis is continually killer of tumor patients.Past studies have showed that tumor cells must have ability of degradating extracellular matrix and basal membrane, going in and out through vasal wall and moving to achive invasion and metastasis. Later studies demonstrated that MMPs, a proteinase existed widely in round about tumor, can almost degradate all component of extracellular matrix and basal membrane and play a important role in tumor invasion and metstasis. MMPs also involved in tumor magligant conversion and tumor-induced angiogenesis, consequently become focus of tumor study. CD147, a glyoprotein existed in surface of tumor cell, can stimulate interstitial cells neighbor rumor-mostly fibroblasts(fb)- to produce MMPs by interaction with them and thereby contribute to invasion and metastasis of tumor.Besides, CD 147 aslo involved in the movement of cells and forming of pseudopod by changing carboxyl terminal within cells. It involved in each step of adhesion, degradation and movement of tumor invasion, accordingly become new target of treating tumor invasion and metastasis.Objective: To investigate the expression of CD 147 in osteosarcoma, and the specific inhibition of CD 147 expression , MMPs producing , as well as tumor cells invasion and metastasis by antisense RNA.Methods: 1. The clinic pathologic information of seventy-six cases of osteosarcoma was studied retrospectivele. Immuno-histochemical staining of above pathologic slice was performed using anti-CD 147 monoclone antibody and analyzed statistically. RT-PCR was performed to clone CD 147 coding cDNA from osteosarcoma cell line MG63. Western Blot was performed to investisate CD 147 protein expression in osteosaroma cells. 2. The cloned gene was reversely insert to EcoR I and Kpn I site of plasmid PcANA3.1(+) to construct CD 147 antisense RNA expressing vector and transfect the recombinated vector to MG63 cells. The transfected cells were selected by G418 to construct stable cell lines. Immunohistochemical staining was performed to investigate inhibition action of antisense RNA. To identify positive clone by cytoflowing method. To draw cells growing linear. MG63 cells and fb cells was co-cutured and the co-cutured cells was immunohistochemical stained using CD 147, MMP-2 and MMP-9 monoclone antibodies. Gelatinase Zymography was used to detect proMMP-2 and activated MMP-2 expression in co-cultured extraction. Western Blot technology was used to detect MMP-2 and it's activator-MTl-MMP and MT2-MMP-expression in co-cultured extraction. 3. Milipore culture cabinet and Matrigel gel was used toimitate extra-orgnismal invasion experiment in order to detect the activity of MG63 cells and transfectants breaking through membrane. The result was analyzed statistically. To construct nude mice subcutaneous and in situ metastasis tumor model, tumorigenicity and lung metastasis as well as growing speed of transfectants was analyzed by these models. Lung metastasis rate was measured by injecting MG63 cells and transfectants into nude mice tail vein.Results: 1. The expression rate of CD 147 in osteosarcoma was 90.8% (69/76). The expression of CD147 was not correlated with sexuality, age, tumor size and pathology typing (p>0.05), but it was significantly correlated with soft tissue invasion, Enneking surgical staging, Price grading and WHO typing (p<0.01). The survival time of patients highly expressing CD 147 is shorten than that of patients lowly expressing CD 147. About 810bp CD 147 coding cDNA was acquired from MG63 cells by RT-PCR. After purified, the cDNA was reversely insert into EcoR I and Kpn I site of plasmid PcANA3.1(+) and was proved correct by sequencing. Western Blot technology was used to detect extraction of cells membrane of MG63, LX-1 and fibroblasts. The results showed that there is a clear band at 50kDa position in MG63 and LX-1 group, but it was not detected in fibroblasts group. The results verified that MG63 cells highly express CD 147 the same as LX-1 cells. 2. After enzyming by EcoR I and Kpn I, each of constructed antisense RNA expressing vector can acquired 2 fragment of 820bp and 5.4kb. The results verified that the recombined vector was constructed successfully. It was further verified by sequencing and was named PcDNA3.1-(+)/asCD147. The recombined plasmids were transfected into MG63 cells and stable transfectants was constructed after selected by G418. The transfectants immunohistochemical staining showed negative results of CD 147 expression, in contrast with it, none-transfectedMG63 cells and ones transfected by empty vector showed CD 147 stain strong positive results. The transfectants finite diluted, 6 clones was acquired. Cytoflowing results showed that 4 of 6 clones present positive CD 147 expression and one of the positive transfectants appear the best inhibition and named MG63/PcDNA3.1(+)/asCD147. The cells growing curve showed that MG63/PcDNA3.1(+)/asCD147 grow more slowly than MG63, but MG63 transfected by empty vector grow quickly the same as MG63. Immunohistochemical staining of co-cultured cells showed that MG63 transfected by empty vector and MG63 present positive CD 147 expression, co-cultured fb present negative, MG63/PcDNA3.1(+)/asCD147 present negative. In MG63 transfected by empty vector and MG63 + fb co-cultured groups, fibroblasts present positive MMP-2 and MMP-9 expression, osteosacomas cells co-cultured present negative expression. In MG63/PcDNA3.1(+)/asCD147+fb group, both of the cells present negative MMP-2 and MMP-9 expression. The results of Gelatinase Zymography and Western Blot of co-cultured extraction showed that proMMP-2(68kD), activated MMP-2(62kD), MMP-2 activator-MTl-MMP(60kD) and proMT2-MMP (68kD) as well as MT2-MMP(62kD) present high expression, but when anti-CD 147 antibody was mixed into co-cultured system, the production of above MMPs was inhibited. Mixing of IgG into co-cultured system did not inhibit the production of MMPs. The production of MMPs was not observed in cultured alone. In MG63/PcDNA3.1(+)/asCD147+fb co-cultured group, the production of MMPs was apparently inhibited. 3. Extra-orgnismal invasion experiment demonstrated that the cell numbers of MG63/PcDNA-3.1(+)/asCD147 breaking through membrane was less than that of the none-transfected MG63 cells(p<0.01). The nude mice subcutaneous tumor model showed that the growing rate of transfectants...