Effect Of Runx2 On The Osteosarcoma Cell And It's Related Mechanism: In Vitro And In Vivo

Objective:To investigate the effect of transfecting or interferencing Runx2 gene on the in vitro bio-characteristics of mice high pulmonary metastatic osteosarcoma cell line K7M2,mice osteosarcoma cell line K7 and human osteosarcoma cell line HOS ,we observed the effect on the RANKL and related cytokines after transfecting Runx2 gene into K7M2 cell;we made BALB/C mice osteosarcoma model to observe the biological characteristics changes and the osteolysis state induced by of K7M2 cells which Runx2 was interfered.Methods:Dividing K7M2,K7 and HOS cells into four groups: K7M2 group,K7M2-neo-plasmid group,K7M2-Runx2 group and K7M2-RNAi group,we transfected neo-plasmide,plasmid conjucted Runx2 and plasmid carrying the gene intervened Runx2 into cells applying the liposome mediated plasmid transfection assay.detected whether or not Runx2 transfected and intervened succeedly with Westernblot method,the survival capacity,migration,invasion and adhesiveness were observed as well,concomitantly,the apoptosis rate and cell cycle distribution of cells in each group were detected with flow cytometry.we chose K7M2 cell transfeced and interferenced succeedly, Detected the level of IL-1α, IL-6, IL-11 and PGE2 in supernatant with ELISA assay in K7M2 group,K7M2-neo group , Runx2 transfected succeedly K7M2 group,the expression of RANKL gene of MC3T3 cells with RT-PCR method which cultured with K7M2,K7M2-neo ,K7M2-Runx2 and K7M2-Runx2 added IL-6 antibody cells supernatant.IL-6R was detected with Westernblot method as well.injected K7M2,K7M2-neo and K7M2-RNAi into BALB/C SPF mice,observed the rate of tumorigenesis ,pulmonary metastasis,surviving and survival period ,concomitant ly,the GS/D,120℃dry weight,lime weight,Ca²⁺ and Pi contents were observed.Results:it can transfected or intervened Runx2 gene with above method. Detected the protein level of K7,K7M2 and HOS cell at 6h,12h,24h,48h,60h and 72h six time point with westernblot method,we found the Runx2 protein expressed stable from 24h after transfection and interference in K7 cell,however,48h in K7M2 and HOS cell line.the level of Runx2 decreased to 40% of pretreatment in K7 cell after 24h interference; the level of Runx2 decreased to 55% of pretreatment in K7M2 cell after 48h interference;the level of Runx2 decreased to 60% of pretreatment in HOS cell after 48h interference; the level of Runx2 increased to 2.5 times of pretreatment in K7 cell after 24h transfection, the level of Runx2 increased to 2 times of pretreatment in K7M2 and HOS cell after 48h transfection.the survival capacity increased but migration,invasion and proliferative index decreased ,comparing the K7M2 group and K7M2-neo-plasmid group,the difference shows statistical deviation.in contrast to K7M2-RNAi group,however,K7M2 cells in K7M2-Runx2 group shows opposite characteristics .Transfecting Runx2 gene can increase the expression of Runx2 protein ,level of IL-6 increased in supernatant after transfecting Runx2 ,the supernatant from K7M2-Runx2 can elevated the RANKL expression ,IL-6 antibody can antagonize the RANKL increasing of MC3T3.K7M2 cells can form tumor in mice,Runx2 interfered can decrease the tumorigenesis rate,increased the survival rate ,pulmonary metastasis rate and suivival time ,most important, interference of Runx2 can attenuate the extent of osteolysis induced by K7M2 cells.Conclusion:Runx2 can change the bio-characteristics ,it can increase survival capacity,migration,invasion mediated by changing the cell cycle distribution so as to make cells slide into G2/S stage more quickly. Runx2 gene can increase the expression of RANKL,it can be mediated by IL-6. the tumorigenesis rate induced by K7M2 which Runx2 interfered was decreased,the survival rate ,survival time were elevated but pulmonary metastasis rate and osteolysis extent were mitigated .