The RNAi Targeting Atp6i A Specific Inhibitor Of Osteoclast Resorption In Rats

The osteoclast(OC) is the functional cell in the bone absorption.Dys -function of OC often causes unbalances of bone reconstruction.Normal bone remodeling requires precise control over the rates of bone formation by osteoblast(OB) and degradation by OC.It is a common pathological change in bone diseases resulting from the OC dysfunction and over-activity of bone resorption,such as osteoporosis,Paget disease,giant cell tumor of bone, rheumatoid arthritis and tumor periodontal disease.The gene Atp6i of vacuolar type H⁺ adenosine triphosphatase(v-ATPase),which is the specific and highest expression in the ruffled border membrane of activated OC,is essential for OC function,such as bone resorption and metabolism balance with normal function of osteoblast.Estrogen,calcitonin and diphosphonate have been used clinically to suppress differentiation of OC and its abnormal bone resorption. These medicines,however,often bother clinicians due to side-effects and non-specificity.In order to seek nontoxicity,non side-effect and higher specific target molecular methods and strategy,we focus on post-transcration level inhibition of Atp6i gene in OC-like cell(OCL).Lentivirus mediated RNA interference(RNAi) technique has been used to knock-down the rat Atp6i gene in OCL specificly and to inhibit bone absorption.Biomedication could be made inhibiting Atp6i expression via RNAi,which is considered to be a medication method to control overactive osteoclast function in clinical therapeusis.1 Design and construction of a lentivirusal vector driving shRNA for Atp6iFirst of all,the lentivirus(pGCL-GFP) shuttle plasmid containing target Atp6i gene was constructed by gene engineering,and highly efficient short hairpin RNA(Atp6i-shRNA) shuttle vector was selected by Western Blotting from 293T cell which over-expressed Atp6i gene fusion protein(contained by pEGFP-C1-Atp6i).Five short interfering RNA(siRNA) targets had been desighed. Finally,Atp6i-shRNA(NO.5) shuttle vector was the best one used to make high titer Atp6i-shRNA lentivirus in the presence of packaging plasmid.2 Establish a system for rat OCL culture in vitroThree to four week-old male Sprague -Dawley(SD) rats were sacrificed and femurs and tibias were dissected free from adherent tissue.The marrow cells were washed withα-MEM and diluted at a density of 1.5×10⁶ cells/ml on plates with either cover slips or bone slices in complete medium(α-MEM with 20% FBS,10nM of 1,25-(OH)₂D₃,100U/ml penicillin and 100ug/ml streptomycin).For stimulation of osteoclast formation,marrow cells were continuously incubated with 1,25-(OH)₂D₃.The medium were replaced half with fresh complete medium every two days.After 5 days in culture,mononuclear precursor and multinuc -leated OCL phenotype were verified by tartrate-resistant acid phosphatase (TRAP) staining and bone-resorptive capacity.The number of the muture OCL achieved the peak on the 8^th day of culture,and OCL was closed to death on the 12^th day.The resorption lacunae on bone slices were formed from the 7^th day of culture,and their number reached maximum at 10-11 days.Therefore, it was the best time to do the RNAi experiment of Atp6i of OCL on the 6^th day of culture.3 RNAi experiments in vitroUsing the SD male rats of 3-4 weeks and making 1,25-(OH)₂D₃ as ininducer, it was to set up OCL in vitro,which was cultured with cattle femurs bone slices. The OCL was divided into 3 groups:transfection group(positive Atp6i-shRNA lentivirus),negative control group(negative Atp6i-shRNA lentivirus),and blank group(no siRNA).The Atp6i-shRNA lentivirus vector was transfected into the groups on the 6^th day marrow cells were cultured.Ninety-six hours after transfection,total RNA of the OCL of each group was harvest,used for real time polymerase chain reaction(PCR) analysis to detect the different expression levels of products of Atp6i gene.Quantities of the areas of absorption lacuna were also analyzed. Ninety-six hours after Atp6i-shRNA lentivirus transfected,compared with negative groups and blank group,we can find the extinction level of products of real time PCR in transfection group was significantly reduced by 57.73% (P＜0.01),and the areas of absorption lacuna in transfection group was reduced by 60.6%(P＜0.01).In summary,this study successfully constructed lentivirus Atp6i-shRNA vector and established system for rat OCL culture in vitro.We found that it efficiently suppressed the expression level to Atp6i gene of OCL,and bone absorptive function of OCL of rat was also identified.Atp6i gene is indispensable to maintain the bone absorption function of OC.