The Establishment Of MiR-142-5P Overexpression And Inhibition Mice Models And Analysis Of Bone Fracture Phenotypes

Part one The establishment of miR-142-5p overexpression and inhibition transgenic mice modelsObjectiveTo establish the miR-142-5p overexpression and inhibition transgenic mice models.Methods(1) We synthesized pre-miR-142and miR-142-5p sponge cDNA and subcloned them into the Sall-EcoRI site in the plasmid containing the osteocalcin2promoter. The constructed plasmids were named as Bglap-pre-miR-142and Bglap-miR-142-S and then sequenced.(2) The MC3T3-E1cells were transfected with Bglap-pre-miR-142or Bglap-miR-142-S plasmids using Lipofectamine2000, and then cultured in osteogenic medium for72hours. We detected the expression of miR-142-5p by qRT-PCR.(3) The plasmids were then injected into fertilized eggs of C57BL/6mice using the pronuclear injection method. Positive founder mice were identified through PCR at3weeks old.(4) MiR-142-5p levels in bone and other tissues were determined by qRT-PCR of pre-miR-142and miR-142-5p sponge transgenic mice.Results1. The expression plasmids of pre-miR-142-5p and miR-142-5p sponge containing the osteocalcin promoter were constructed successfully. They can express in MC3T3-E1during osteoblast differentiation. MiR-142-5p was overexpressed after Bglap-pre-miR-142transfected, while inhibited after Bglap-miR-142-S transfected in MC3T3-E1.2. We established the miR-142-5p overexpression and inhibition transgenic mice models successfully. In Bglap-pre-miR-142transgenic mice, miR-142-5p was specifically over-expressed in bone, while in Bglap-miR-142-S transgenic mice, miR-142-5p was specifically inhibited in bone.ConclusionWe established the miR-142-5p overexpression and inhibition transgenic mice models successfully, which may be used for bone fracture research. Part Two The phenotype analysis of bone fracture in miR-142-5p overexpression or inhibition transgenic mice modelsObjectiveTo determine the role of miR-142-5p during fracture healing in vivo. Methods(1) Unilateral open femoral shaft transverse osteotomy with locking screw fixation procedures were performed on wild type (WT), over-expression of miR-142-5p (Mir-142-ob-TG) and osteoblast-specific deficiency using miR-142-5p sponge (Mir-142-ob-S) mice models.(2) X-ray and HE stainings of callus sections were used to evaluate the characteristics of bone fracture healing of these mice at7days,14days and21days after bone fracture.(3) qRT-PCR was used to detect the expression patterns of miR-142-5p and osteocalcin mRNA at0days,7days,14days and21days after bone fracture.(4) To verify if WWP1was still the target of miR-142-5p in vivo, qRT-PCR and Western blot were used to detect the expression of WWP1mRNA and protein in the transgenic mice after bone fracture. Results1. We established the bone fracture models successfully in miR-142-5p overexpression and inhibition transgenic mice.2. X rays showed that over-expression of miR-142-5p specifically in osteoblasts promoted fracture healing while deficiency of miR-142-5p inhibited fracture healing.3. Histological staining showed mineralization, bone formation and remodeling of bone callus were promoted in Mir-142-ob-TG mice, while inhibited in Mir-142-ob-S mice.4. Mir-142-5p and osteocalcin expression of bone callus reached the peak at21days after bone fracture in WT mice, while in Mir-142-ob-TG mice, it was at14days. However, in Mir-142-ob-S mice, both mir-142-5p and osteocalcin were decreased at different time points after bone fracture.5. The mRNA level of WWP1increased and reached the peak at21days after fracture in bone callus. However, the WWP1protein increased more striking in Mir-142-ob-S mice. ConclusionOver-expression of miR-142-5p specifically in osteoblasts promoted fracture healing while deficiency of miR-142-5p inhibited fracture healing. MiR-142-5p regulates WWP1expression at posttranscriptional levels in vivo. Part Three The therapeutical effect of miR-142-5p in aged mice with delayed fracture healingObjectiveTo establish the bone fracture model in aged mice and explore the therapeutical effect of agomir-miR-142-5p.Methods(1) Unilateral open femoral shaft transverse osteotomy with locking screw fixation procedures were performed on aged mice.(2) qRT-PCR was used to detect the expression patterns of miR-142-5p and osteocalcin mRNA at0days,7days,14days,21days and28days after bone fracture.(3) Agomir-142-5p was injected to the fracture site of18-month aged mice twice a week between1and28day post fracture. X-ray and HE stainings of callus sections were used to evaluate the characteristics of bone fracture healing of these mice at28days after bone fracture.Results1. We established the bone fracture models successfully and found mir-142-5p and osteocalcin expression of bone callus both decreased at all time points in aged mice. Furthermore, the expression of mir-142-5p reached the peak at28days after bone fracture in aged mice.2. X ray showed that the callus from agomir-142-5p-treated aged mice were calcified and mostly bridged at28day post fracture. The boundary between the newly formed hard calls and cortical bone also became obscure. However, the callus from agomir-NC treated aged mice and vesicle-treated aged mice were partly bridged and the gap remained existed.3. Histological analysis at28day post fracture showed that fractures in aged mice treated with agomir-142-5p had reduced cartilage and increased bone compared with agomir-NC-treated and vesicle-treated aged mice. ConclusionAgomir-142-5p treatment resulted in improved bone formation and fracture repair in aged mice.