Effect Of RNAi Silencing OPG Gene On The Expression Of RANKL/OPG In Bone Marrow Mesenchymal Stem Cells

Objective:Preliminary explore the feasibility of increasing the osteoclast activity by using the RNAi technique to silence the OPG gene on bone marrow mesenchymal stem cells,to provide new ideas for the study of bone degradation of bone substitute materials.Methods:Lentiviral vector construction;BMSCs isolation?cell cultureand identification;BMSCs were set up in the control group comprised untransfected cells,shScr group?BMSCs transfected with empty lentiviral vector,which expressed scrambled RNA,was used in the mock group?and shOPG group?BMSCs transfected with recombinant plasmid containing OPG interference sequence-shRNA?,MTT assay was used to detect the proliferation of BMSCs after transfection;The osteogenic differentiation of BMSCs after OPG silencing was detected by ALP staining;On days 3,7,14 a nd 21 after transfection,compared the relative mRNA levels of OPG and RANKL by RT-PCR,detected the protein expression of OPG and RANKL by western blot.Rat nonadherent bone marrow cells?as OC progenitor cells?cocultured with BMSCs generated osteoclast-like cells,which were measured by tartrate-resistant acid phosphatase?TRAP?staining.Results:1.MTT results:There was no significant difference in cell viability among the three groups?P>0.05?.2.ALP staining results:The three groups of cells all had brown-black alkaline phosphatase granules.3.Results of RT-PCR:?1?Expression of OPG mRNA:The OPG mRNA inhibitory effect of the shOPG group peaked at 3 days after infection?inhibition rate was 73%?and then gradually decreased until the 21st day the inhibition rate dropped to 26%.OPG mRNA expression in the shOPG group was 23%,37%,54%and 74%of that in the control group on the 3^rd,7^th,14^th and 21^st day after transfection,respectively?There were significant differences between the two groups at 3,7,14 and 21 d ays after transfection,P<0.05?.There was no significant difference in the relative expression of OPG mRNA between the shScr group and the control group on the 3rd,7th,14th and 21st days after transfection,?P>0.05?.?2?Expression of RANKL mRNA:The expression of RANKL mRNA in the shOPG and shScr groups showed a transient peak on the 3rd day after transfection,and then gradually tended to be the same as the blank group.RANKL mRNA expression in the sh OPG group was 390%,135%,97%and 84%of that in the control group on the 3^rd,7^th,14^th and 21^st day after transfection,respectively?There was a significant difference between the two groups on the3rd and 7th day after transfection,P<0.05;and no significant difference between the two groups on the 14th and 21st days after transfection,P>0.05?.RANKL mRNA expression in the shScr group was compared with the control group on the3^rd,7^th,14^th and 21^st day after transfection,respectively(There was a significant difference between the two groups on the 3^rd day,P<0.05;and no significant difference between the two groups on the 7^th,14^th and 21^st day,P>0.05).4.Results of western blot:?1?Expression of OPG protein:The inhibitory effect of OPG in the shOPG group peaked at 3 days after infection?inhibition rate was 69%?,and then gradually decreased until the 21st day the inhibition rate dropped to 1 3%.OPG protein expression in the shOPG group was 31%,45%,68%,and 87%of that in the control group on the 3^rd,7^th,14^th and 21^st day after transfection,respectively(There was a significant difference between the two groups on the 3^rd,7^th and 14^th day after transfection,P<0.05;and no significant difference on the 21^st day after transfection,P>0.05).There was no significant difference in the relative expression of OPG protein between the shScr group and the control group on the 3^rd,7^th,14^th and 21^st days after transfection,?P>0.05?.?2?Expression of RANKL protein:There were no significant difference among the three groups on the 3^rd,7^th,14^th and 21^st days after transfection?P>0.05?.5.Changes in RANKL/OPG ratio:?1?Ratio of RANKL/OPG at the mRNA level:the RANKL/OPG ratio was significantly increased in the sh OPG group on the 3^rd,7^th and 14^th day?Compared with the control group,there were significant differences,P<0.05?,and increased by approximately 17?3.8 and1.8-fold,respectively;There was no significant difference between the 21st day and the control group?P>0.05?.The shScr group presented with a sensitive2.1-fold increase in the RANKL/OPG ratio on the 3^rd day?Compared with the control group,there was significant differences,P<0.05?,followed by a decreased ratio that was no significantly different from the control group?P>0.05?.?2?At the protein level,the RANKL/OPG ratio was significantly increased in th e shOPG group on the 3^rd,7^th and 14^th day?Compared with the control group,there was significant differences,P<0.05?,and increased by approximately 3.7?2.5 and 1.44-fold respectively;there was significant differences on the 21^st day?P<0.05?.The shScr group was no significantly different from the control group on the 3rd,7th,14th and 21st days after transfection?P>0.05?.6.Osteoclastogenesis Assay:The number of TRAP-positive multinucleated cells was significantly increased during the coculture of nonadherent bone marrow stromal cells with the sh OPG BMSCs then the shScr group and control group?P<0.05?;The BMSCs in the sh Scr coculture group did not significantly differ from those in the control group?P>0.05?.Conclusions:RNAi technology silenced the OPG gene on bone marrow mesenchymal stem cells,which can be used to enhance the activity of osteoclasts by increasing the ratio of RANKL/OPG.