Effects Of CXCR4 On Pulmonary Metastasis Of Osteosarcomatous Cell Subline

Osteosarcoma is the most common osteogenic malignant tumor,lung is the most major metastatic site(about 90%of all distant metastases). Approximate 85%patients have different extent of pulmonary metastases at the time of preliminary diagnosis.Pulmonary metastasis has been the mainest cause of death in osteosarcomatous patients.Therefore,the studies of osteosarcomatous pulmonary metastasis are very important to the improvement of prognosis and the elevation of survival rate.At present,there are many researches with regard to osteosarcoma,but the studies of osteosarcomatous pulmonary metastasis are few.Chemokine receptor 4(CXCR4)is an extraordinary conservative seven-transmembrane,G protein-coupled receptor of stromal cell-derived factor-1(SDF-1).Many studies disclosed that SDF-1/CXCR4 axis had played a key role in driving,guiding and locating the organic specific metastatic behavior of several tumors,such as breast carcinoma,pulmonary carcinoma,ovarian cancer,prostatic carcinoma,pancreatic carcinoma and malignant tumor of hematological system.But until very recently,the researches of SDF-1/CXCR4 axis concerning osteosarcoma have been very rare. Part one:Expression of CXCR4 in osteosarcoma and its clinical significanceObjective To investigate the expression and significance of CXCR4 in human osteosarcomatous tissue.Methods Immunohistochemistry was performed to detect the expressions of CXCR4 in 5 pulmonary lump(exceedingly suspect pulmonary metastasis)and 11 non-pulmonary lump osteosarcomatous specimens diagnosed by x-ray chest film.Results All of 16 specimens showed positive staining of CXCR4 protein.However,the levels of expression of the specimens with pulmonary lumps(exceedingly suspect pulmonary metastasis) (80.52±9.93)were higher than the specimens without pulmonary lumps(65.56±12.75),the difference was significant,P<0.05.Conclusions The level of CXCR4 expression in osteosarcomatous tissue with pulmonary lump(exceedingly suspect pulmonary metastasis) is high,it suggests that CXCR4 may participate in osteosarcomatous pulmonary metastasis,it also provides a clue for the next study. Part two:The correlated study of expression of CXCR4 and exosomatic invasive capability in osteosarcomatous cell sublineObjective To study the influence of CXCR4 expression on the exosomatic invasive capability of osteosarcomatous cell subline.Methods We separated and cultivated osteosarcomatous cell sublines by the unicell cloning technique from human osteosarcomatous cell line MG-63,selected two cell sublines with high and low CXCR4 protein expressions by immunohistochemistry,detected the expressions of CXCR4 mRNA by RT-PCR,detected the exosomatic invasive capabilities by transwell invasive assay in vitro,observed the effects of CXCR4 monoclonal antibody on exosomatic invasive capabilities,analyzed the relationships between the expressions of CXCR4 and exosomatic invasive capabilities.Results(1)Two osteosarcomatous cell sublines with high(B8)and low(G5)expressions of CXCR4 protein were obtained from MG-63;(2) The expression of CXCR4 protein of B8 cell subline(94.74±17.35)was higher than the G5 cell subline(41.86±6.54),the difference was significant,P<0.05;(3)The expression of CXCR4 mRNA of B8 cell subline(1.37±0.36)was higher than the G5 cell subline(0.44±0.13),the difference was significant,P < 0.05;(4)The exosomatic invasive capability of B8 cell subline(53.60±15.37)was higher than the G5 cell subline(19.60±8.20),the difference was significant,P<0.05;(5)The exosomatic invasive capability of cell subline after using CXCR4 monoclonal antibody(21.80±8.53)was lower than the capability before using antibody(53.60±15.37),the difference was significant,P<0.05;(6) The exosomatic invasive capability has positive correlation with the expression of CXCR4 protein,P<0.05;(7)The exosomatic invasive capability has positive correlation with the expression of CXCR4 mRNA,P<0.05.Conclusions Osteosarcomatous cell with high expression of CXCR4 protein and mRNA can easily generate invasion and metastasis in vitro, the invasion and metastasis can be inhibited by CXCR4 specific antibody,it suggests that CXCR4 plays a significant role in regulating the invasive and metastatic behavior in vitro of osteosarcomatous cell.Part three:Effects of small interfering RNA on CXCR4 expression and exosomatic invasive capability of osteosarcomatous cell sublineObjective To explore the effects of small interfering RNA(siRNA) on the expression of CXCR4 mRNA and exosomatic invasive capability of osteosarcomatous cell subline.Methods We designed and chemically synthesized sequencespecific siRNA targeting CXCR4 gene and transfected siRNA into osteosarcomatous cell subline with high CXCR4 expression by using Lipofectamine^TM2000,detected the change of CXCR4 mRNA expression by RT-PCR,observed the change of exosomatic invasive capability by transwell invasive assay in vitro.Results(1)CXCR4 siRNA was successfully synthesized and transfected into osteosarcomatous cell subline with high CXCR4 expression,the transfection efficiency was 43.82±3.02%;(2)The expression of CXCR4 mRNA after transfection was lower than the negative control group(0.75±0.08)and blank group(0.72±0.08),the differences were significant,P < 0.05;(3)The exosomatic invasive capability after transfection(31.20±7.19)was lower than the capability before transfection(53.60±15.37),the difference was significant,P<0.05.Conclusions The CXCR4 siRNA could successfully inhibit the expression of CXCR4 mRNA and exosomatic invasive capability of osteosarcomatous cell,it suggests that CXCR4 should play an important role in the invasion and metastasis in vitro of osteosarcomatous cell and CXCR4 siRNA may be potentially useful in the next research of preventing pulmonary metastasis of osteosarcoma.Part four:Effects of CXCR4 on pulmonary metastasis of osteosarcomatous cell subline in nude miceObjective To investigate the effects of CXCR4 on pulmonary metastasis of osteosarcoma cell subline in nude mice.Methods We injected osteosarcomatous cell sublines with different CXCR4 protein and mRNA expressions into cervical and dorsal subcutaneous tissues of nude mices,killed the nude mices and examined the pulmonary metastases after four weeks,serial sections of the fixed pulmonary tissues were done to grade.Results(1)The grade of nude mice's pulmonary metastases of osteosarcomatous cell subline with high CXCR4 protein and mRNA expression(84.40±14.79)was higer than that of low CXCR4 expression(32.00±8.00);(2)The pulmonary metastatic capability of osteosarcomatous cell in nude mice has positive correlation with the expression of CXCR4 protein,P<0.05;(3)The pulmonary metastatic capability of osteosarcomatous cell in nude mice has positive correlation with the expression of CXCR4 mRNA,P<0.05.Conclusions The osteosarcomatous cell with high expression of CXCR4 protein or mRNA can easily generated pulmonary metastasis in nude mice,it suggests that CXCR4 should play a significant role in the pulmonary metastasis of osteosarcomatous cell in nude mice.