Experimental Study Of Blocking The Way Of RANKL Sign By RNA Interference To Inhibit Transformation Of Pre-osteoclast

【Objective】Ligand of receptor activator of NFκB(RANKL) plays a important role in proliferation, differentiation, activation, survival of osteoclast. RNA interference(RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism. Now RNAi has been adopted as a functional genomics tool and a gene therapy approach. Suppression of the expression of RANKL by RNAi is likely to have great impact as a therapeutic tool in osteolysis such as osteoporosis. The objective of this experiment include:(1) to synthesize and screen effective siRNA targeting RANKL.(2) To explore time-course changes of siRNA down regulation of RANKL mRNA and protein, and to observe the effect of RANKL gene silencing on the function of osteoblast and transformation of pre-osteoclast.(3) To investigate the rhythm of rankl and opg expression in bone and BMD of ovariectomized(OVX) S-D rat for the further experiment.【Method】(1) Osteoblasts were transfected with 4 chemically synthesized siRNA. RANKL mRNA levels were analyzed by real time reverse transcription-polymerase chain reaction(RT-PCR). The optimal siRNA were selected for next experiment.(2) The optimal siRNA were transfected into osteoblasts, and the levels of mRNA and protein of RANKL and OPG were detected at different times. Proliferation, ALP activity and I type collagen expression of osteoblasts were observed at the same time. Osteoblasts transfected with siRNA and pre-osteoclasts were co-cultured, and the influence of osteoclastogenesis was investigated.(3) OVX model of S-D rats was established. Bone mineral density(BMD) and the levels of mRNA and protein of RANKL and OPG in condyles of femur were detected at different times. The relationship between the expression of RANKL and OPG and osteoporosis was investigated.【Result】(1) 4 chemically synthesized siRNA can suppress the expression of RANKL mRNA in osteoblasts. The inhibitory rates of siRNA4 was 89%, and compared with the blank control group(no transfected group), significant statistical difference existed(P<0.05).(2) Compared with the blank control group, the expression rate of RANKL mRNA was 35.3%,11.1%,25.9%,49.0%,66.9%(P<0.05) respectively at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, and the expression rate of RANKL protein was 59.1%,39.5%,26.6%,40.0%,57.3%(P<0.05) respectively at the sane time. The optimal silencing effect at mRNA and protein level was at 2nd and 3rd day post-transfection, respectively.(3) The expression rate of OPG mRNA and protein is lower than the blank control group at 1st, 2nd, 3rd, 5th, 7th day after the optimal siRNA transfection, however no statistical difference existed(p>0.05). Proliferation, ALP activity and I type collagen expression of osteoblasts were similar to the expression profile of OPG(P>0.05).(4) After transfected osteoblasts and bone marrow cells were co-cultured 7 days, the number of osteoclasts was less than the blank control group(P<0.05).(5) The femoral condyles BMD in OVX was significantly lower than that in sham control since 6 weeks after OVX(P<0.05).(6) The expression level of RANKL mRNA peaked at 4 weeks after OVX, and that of RANKL protein peaked at 6 weeks after OVX, then both maintain at a high level. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05). The expression level of OPG mRNA peaked at 4 weeks after OVX, and that of OPG protein peaked at 2 weeks after OVX, then both decreased rapidly. Compared with sham control, statistical difference existed in the expression level at each time point after OVX(P<0.05).【Conclusion】(1) 4 chemically synthesized siRNA targeting RANKL can suppress the expression of RANKL mRNA in osteoblasts, and 5'→3' UCC CAU CGG GUU CCC AUA is the most effective sequence.(2) Effective siRNA targeting RANKL can suppress the expression of RANKL mRNA and protein in osteoblasts, and the suppression is up to 7 days.(3) Effective siRNA targeting RANKL doesn't influence the expression of OPG mRNA and protein, and has no significant effect on proliferation, ALP activity and I type collagen expression of osteoblasts.(4) Transfected osteoblasts are co-cultured with bone marrow cells, which can inhibit transformation of pre-osteoclast.(5) The femoral condyles BMD in OVX is significantly lower than that in sham control since 6 weeks after OVX, and the femoral condyles BMD is sensitive to OVX.(6) The expression level of RANKL maintains at a high level and that of OPG increases transiently and decreases rapidly, which is the direct reason of postmenopausal osteoporosis.