The Study Of MiR-141 Therapeutic Role In Osteoporosis Model Of Aged Female Rhesus Monkeys

Osteoporosis is a common disease worldwide. Despite many studies have been conducted, the drugs for it are far from ideal. It is very valuable for us to investigate a new drug for osteoporosis. In this study, we explored the therapeutic role of miR-141 in aged female rhesus. Moreover, we identified the mechanism of miR-141 regulating bone mass. This observation provides the first preclinical insight into the contribution of a miRNA to the osteoporosis therapy.In this study, we first collected bone specimens from 23 aged women(69~89years), who were diagnosed with osteoporosis. And, we detected the expression of miR-141 and bone density. Moreover, we choose bone specimens from 32 aged female rhesus monkeys, whose age were over 12 years. In line with human, we also detected the expression of miR-141 and bone density. Next, we chose 3 female rhesus monkeys for ovariectomy(OVX) experiment and another 3 female rhesus monkeys as sham operation(SHAM), which were about 10 years old. Moreover we chose 12 aged female rhesus monkeys, who were over 18 years, as the model of osteoporosis. Then,we detected monkeys’ weight, density of lumbar vertebra and hip and serum NTx(urinary amino-terminal telopeptide of type I collagen), CTx(carboxyl-terminal telopeptide of type I collagen) and TRAP(tartrate-resistant acid phosphatase). We divided 12 rhesus monkeys into 2 groups randomly, marked miR-NC(control group)and miR-141(experimental group). We administrated miR-141 or miR-NC(20mg/kg)to aged female rhesus monkeys by intravenous injection once a week totally for 12 weeks. We recorded the weight and behavior every other week. Moreover, we performed CT to following the change of bone density. We also extracted 3 m L blood to detect serum bone resorption bone marker: NTx, CTx and TRAP5 b. Rhesusmonkeys were sacrificed by euthanasia after 12 weeks, collecting the tissues, such as hearts, livers, spleens, kidenies, lunbar vertebra, hip and legs. For heart, liver, spleen and kidney, we performed HE stain to observe the pathological change. For tibia, we performed micro CT to detect the change of bone density. For lumbar vertebra and hip,we performed HE stain and TRAP stain to detect the change of bone trabecula and osteoclasts. We extracted 3m L bone marrow from a 3 months-year old female rhesus monkey, separating bone marrow macrophage. Based on this, we induced rhesus osteoclast differentiation with M-CSF(50ng/m L) and RANKL(100ng/m L) in vitro.We transfected miR-NC or miR-141 and inhibitors(Anti-miR-NC and Anti-miR-141)into osteoclast during osteoclast differentiation. We detected the specific genes expression level of osteoclast differentiation by Q-PCR, TNFRSF11 A, TRAP and CTSK. To confirm the above, we also performed TRAP stain. Moreover, we treated rhesus monkey osteoclast with either miR-141 or antmiR-141(a miR-141 inhibitor), to detect the expression of TNFRSF11 A, TRAP and CTSK m RNA. Finally, we predicted the targeted gene of Calcr by the most commonly used software algorithms(Target Scan). Therefore, we constructed luciferase reporter plasmids containing miR-141 target gene 3’UTR of Calcr. We cotransfected vectors of miR-NC, miR-141 and miR-141 mutation(miR-141-MUT1 and miR-141-MUT2) into rhesus monkey osteoclasts and detected the activity of luciferase. Moreover, we detected CALCR expression level by Q-PCR, and detected CALCR protein by Western blot. We also detected target protein level in vitro and in vivo.In this study, we assayed the expression of miR-141 in bone specimens from aged patients and rhesus monkeys and identified that miR-141 was positively correlated with BMD. We performed systemic administration of miR-141 in aged female rhesus monkeys. The results indicate that miR-141 could increase bone mass by actively inhibiting bone resorption. Our findings further demonstrate that ectopic expression of miR-141 in rhesus monkey osteoclast could inhibit osteoclastogenesis, which coincides with the results in vivo. Finally, we identify CALCR as a direct functional target of miR-141. This observation provides the first preclinical insight into the contribution of a miRNA to the pathophysiological regulation of bone resorptionduring the progression from osteopenia to osteoporosis in nonhuman primates. This study identify that administrate miR-141 to aged female rhesus monkeys could effectively alleviate loss of bone mass in aged female rhesus, which provide a new direction for osteoporosis treatment.