Cloning And Functional Study Of A Novel MicroRNA Regulating Osteoclast Differentiation

PART ONE Cloning and functional study of a novel microRNA specifically expressed in osteoclastObjectives:To find novel microRNAs (miRNAs) specifically or preferentially expressed in mouse osteoclast. Study the expression pattern of the new microRNA in various mouse tissues and cells and the role of the new microRNA during osteoclastogenesis.Methods:Primary osteoblasts and bone marrow cells from C57BL/6mice were co-cultured to generate primary osteoclasts. Isolate and clone total small RNAs in primary osteoclasts. Each RNA sequence was subjected to BLAST analysis against the mouse genome, and a novel microRNA was identified. The new microRNA was submitted to miRBase for registry and was termed miR-9718. Northern Blot was applied to measure the expression levels of miR-9718in various mouse tissues and cells. RAW264.7cells were induced to differentiate into osteoclasts with RANKL and M-CSF. Northern Blot and qRT-PCR was applied to determine the expression pattern of miR-9718during osteoclastogenesis. Expression vector of miR-9718(pre-miR-9718) was constructed by inserting a sequence of miR-9718precursor into pSilencer4.1-CMV vector.2’-O-methyl antisense inhibitory oligoribonucleotide (anti-miR-9718) was used to inhibit the effect of miR-9718in RAW264.7cells. RAW264.7cells were transfected with pre-miR-9718or anti-miR-9718to upregulate or downregulate miR-9718expression levels and were induced to differentiate into osteoclasts by RANKL and M-CSF. Total RNA was extracted for measurement of osteoclastic specific markers TRAP and NFATc1mRNA levels with qRT-PCR. TRAP staining was performed to observe osteoclast differentiation. Results:(1) We cloned a novel microRNA in mouse primary osteoclast, and the new microRNA was submitted to miRBase for registry and was termed miR-9718.(2) We found that miR-9718was selectively highly expressed in osteoclasts, while expression of it was not detected in other tissues or osteoblasts.(3) During RANKL and M-CSF induced osteoclastogenesis of RAW264.7cells, miR-9718expression increased as time grew.(4) Expression vector of miR-9718(pre-miR-9718) was constructed with pSilencer4.1-CMV vector and was transfected into RAW264.7cells. Northern Blot proved the elevated expression of miR-9718in RAW264.7cells.(5) RAW264.7cells transfected with pre-miR-9718or anti-miR-9718were induced by RANKL and M-CSF. Compared to control, transfection of pre-miR-9718increased the number of TRAP+multinuclear giant cells apparently and elevated expression levels of NFATc1and TRAP mRNA.(6) Transfection of anti-miR-9718decreased formation of TRAP+multinuclear giant cells and expression levels of NFATc1and TRAP mRNA.Discussion:We identified a novel microRNA in mouse primary osteoclast. Expression of the new miRNA increased gradually during osteoclast differentiation of RAW264.7cells and played a positive regulatory role in osteoclast differentiation. PART TWO Study on regulatory mechanism of miR-9718during osteoclast differentiationObjectives:Predict the target gene of miR-9718in osteoclast differentiation and study the regulatory mechanism of miR-9718during osteoclast differentiation of RAW264.7cells.Methods:Rna22software was used to predict the possible target gene of miR-9718in osteoclast differentiation and PIAS3was predicted to be a probable target of miR-9718. Then a segment of PIAS3CDS was cloned into pGL3vector to construct wild type PIAS3luciferase reporter gene (WT-pGL3-PIAS3). The QuickChange site-directed mutagenesis kit was used to introduce point mutations into the predicted miR-9718binding site of WT-pGL3-PIAS3, resulting in MUT-pGL3-PIAS3. Either the WT-pGL3-PIAS3or MUT-pGL3-PIAS3was co-transfected with pre-miR-9718or miR-C into RAW264.7cells, and then the luciferase activity was detected. Pre-miR-9718was transfected into RAW264.7cells, and qRT-PCR and Western Blot was applied to detect the mRNA and protein levels of PIAS3. Wild type and mutant PIAS3CDS expression vector was constructed with pcDNA3.1(+) expression vector. Then either WT-PIAS3-CDS or MUT-PIAS3-CDS was co-transfected with pre-miR-9718or miR-C into RAW264.7cells and then RAW264.7cells were induced by RANKL and M-CSF. Western Blot and qRT-PCR was used to detect protein expression levels of PIAS3and mRNA levels of NFATcl and TRAP. Pre-miR-9718or anti-miR-9718was transfected into RAW264.7cells and then RANKL and M-CSF was used to induce osteoclast differentiation. Western Blot and qRT-PCR was used to detect protein expression levels of PIAS3, MITF, NFATc1, c-FOS and NF-κB and mRNA levels of PIAS3respectively.Results:(1) PIAS3was predicted to be the target gene of miR-9718in osteoclast differentiation by Rna22software.(2)Wild type and mutant PIAS3firefly luciferase reporter gene were constructed. Compared to miR-C transfection control group, co-transfection of pre-miR-9718with WT-pGL3-PIAS3suppressed the luciferase activity, while co-transfection of pre-miR-9718with MUT-pGL3-PIAS3had no effect on the luciferase activity.(3) Compared to miR-C transfection control, transfection of pre-miR-9718in RAW264.7cells decreased the protein levels of PIAS3, while no change in PIAS3mRNA levels was observed.(4) Wild type or mutant PIAS3CDS expression construct was co-transfected with pre-miR-9718or miR-C into RAW264.7cells, and was induced by M-CSF and RANKL. Induction of TRAP and NFATc1mRNA by pre-miR-9718was rescued by the mutant PIAS3CDS construct. Western blot showed that the mutant PIAS3CDS construct was able to rescue the pre-miR-9718induced downregulation of PIAS3protein.(5) Compared with control, expression levels of NFATc1, MITF, c-Fos and NF-κB protein was significantly up-regulated in RAW264.7cells transfected with pre-miR-9718, while the protein levels of PIAS3was decreased without change in PIAS3mRNA levels. By contrast, expression levels of NFATc1, MITF, c-Fos and NF-κB protein was down-regulated in RAW264.7cells transfected with anti-miR-9718, while the protein levels of PIAS3was increased.Discussion:MiR-9718promoted osteoclast differentiation by repressing PIAS3gene expression at the post-transcriptional level.