Biology Behavior Effecrts Of BMP9on143B Cell Line

Osteosarcoma is one of the most common malignant bonetumor.Although treatment modalitiesa have been improved,it is still atumor with a high mortality rate ascribed to tunor cell invasion andmetastasis. Bone morphogenetic proteins,(BMPs) are members of theTransforming growth factorβ(TGFβ) superfamily of signaling moleculesand have previously been shown to be associated with the biologicalbehavior of osteosarcoma.And has been reported, in osteosarcoma clinicalspecimens, BMP2-8were abnormal expression with differentlevel.However,the effects of BMP9on osteosarcoma development andprogression are unknown.In this study,cell lines with relatively low BMP9were infected after characterizing the endogenous expression of BMP9inosteosarcoma cell lines,BMP9expression levels observed afterupregulation of osteosarcoma cells on cellproliferation,apoptosos,migration, clony formation and nude mousexenografts experiment and other aspects. explore theory basis for theclinical diagnosis and treatment of osteosarcoma. Methods1. Amplification of the reco mbinant adenovirus AdS100A4anddetermine the optimal virus titer.2. Through RT-PCR and Western Blot to identify the expression ofendogenous BMP9in143B and MG63cells lines，then to detect the changeof it after infecting osteosarcoma cell lines143B which relatively lowBMP9expression。3. After infection of143B with appropriate amount of virusrespectively, to detect cell proliferation by MTT, to assay cell migration bywound healing assay and Transwell migration, to detect cell invasion bytranswell invasion experiment and to detect cell cycles and apoptosis byHoechst staining and flow cytometry,to detect cell clony forming by clonyforming assay,4. To inject with well-infected virus respectively according to certainnumber of each cells group to BALB/c nude mice, put the mice to deathwith cervical dislocated after20d calculating tumors weight, volume, toobserve the effects of BMP9on nude mice into tumor ability.Results1. There were expression of green fluorescent protein in HEK293cellinfected by AdBMP9, the infection rate at72hour was more than90percent; the AdBMP9titer was2×1011IU/ml. 2. AdBMP9mRNA and protein were all found in143B and MG63with RT-PCR and Western Blot; but relatively low BMP9expression werefound in143B cell lines.3. BMP9expression with relatively low endogenous are significantlyincreased after infection with AdBMP9, indicating that AdBMP9can beeffective intervention tools.4. MTT assay showed that: The proliferation of cell line143Bdecreased by17%(P <0.05) respectively than control group after infectionof AdBMP948hour,and38%(P <0.05) of72hour, suggesting that BMP9can inhibit the proliferation of143B cell lines.5. Wound healing showed that:The wound healing rate of143Btreated by AdBMP9in24hour decreased by45%(P <0.05), respectively,than the control group, These results suggested that BMP9can inhibit thecell migration143B cell lines.6. Transwell invasion experiment showed that: The number ofpenetrating143B cells treated by AdBMP9in24hour decreased by45%(P <0.05),than the control group, These results suggest that BMP9cansignificantly inhibit the cell invasion ability of143B cell lines.7. Hoechst stain and flow cytometry showed that: The24hourapoptosis ratio of143B treated by AdBMP9increased46%(P>0.05)respectively, than the GFP group;The48hour apoptosis ratio of of 143B treated by AdBMP9increases63%(P>0.05) than the control group.These results prompt that BMP9can promote apoptosis of143B cell lines.8. Flow cytometry test showed that:The143B cell were more easierblocked in G2/M phase by AdBMP9treated cells. The result suggested thatBMP9can significantly block the cell cycle of143B cell lines.9. Clonoy formation assay showed that:The ability of cell line143Bclony decreased by48%(P <0.05) respectively than control group afterinfection of AdBMP918days. The result suggested that BMP9cansignificantly inhibit the cell clonoy formation of143B cell lines.10. BMP9on nude mice into tumor ability test showed that: Theweight and volume of143B treated by AdBMP9injection into nude micedecreased53%、49%(P>0.05)respectively, than the GFP group. Theseresults prompt that BMP9can inhibit the ability on nude mice of143B celllines.Conclusion1. AdBMP9can be successfully amplified in HEK293cell.2.143B and MG63both have BMP9expression,143B has lowerexpressionlevel.3. AdBMP9was successfully expression and upreglate BMP9mRNA andprotein level in143B cell lines.4. BMP9could significantly inhibit the proliferation, migration,invasionand clony forming ability of143B cell and promote its apoptosis. Meantime,BMP9can inhibit the ability of tumor formation in nude mice invivo. This suggested that BMP9may be involved in osteosarcomaprogress.