Patent Application: US-201614990202-A

Abstract:
there is provided nucleic acids for use in treating or preventing bone loss in a patient . also there is provided a method for reducing the levels of endogenous mir - 138 in a cell .

Description:
the term “ nucleotide ” refers to any of the four deoxyribonucleotides , da , dg , dt , and dc ( constituents of dna ), and the four ribonucleotides , a , g , u , and c ( constituents of rna ). ina , lna , and any other nucleic acid capable of specific base pairing are also suitable in accordance with the present invention . each natural nucleotide comprises or essentially consists of a sugar moiety ( ribose or deoxyribose ), a phosphate moiety , and a natural / standard base moiety . natural nucleotides bind to complementary nucleotides according to well - known rules of base pairing ( watson and crick ), where adenine ( a ) pairs with thymine ( t ) or uracil ( u ); and where guanine ( g ) pairs with cytosine ( c ), wherein corresponding base - pairs are part of complementary , anti - parallel nucleotide strands . the base pairing results in a specific hybridization between predetermined and complementary nucleotides . modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties . sugar modifications include , for example , replacement of one or more hydroxyl groups with halogens , alkyl groups , amines , and azido groups , or sugars can be functionalized as ethers or esters . moreover , the entire sugar moiety can be replaced with sterically and electronically similar structures , such as aza - sugars and carbocyclic sugar analogs . examples of modifications in a base moiety include alkylated purines and pyrimidines , acylated purines or pyrimidines , or other well - known heterocyclic substitutes . nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages . analogs of phosphodiester linkages include phosphorothioate , phosphorodithioate , phosphoroselenoate , phosphorodiselenoate , phosphoroanilothioate , phosphoranilidate , phosphoramidate , and the like . the term “ nucleic acid molecule ” also includes e . g . so - called “ peptide nucleic acids ,” which comprise naturally - occurring or modified nucleic acid bases attached to a polyamide backbone . nucleic acids can be either single stranded or double stranded . in an aspect of the present invention , ‘ nucleic acid ’ is meant to comprise antisense oligonucleotides ( aso ), small inhibitory rnas ( sirna ), short hairpin rna ( shrna ) and microrna ( mirna ). the term antimir ( or antagomir ) refers to an oligonucleotide complementary to a microrna , such as mir - 138 . the term blockmir refers to an oligonucleotide that is complementary to the microrna target sequence on the mrna it regulates . microrna modulation can be achieved either by using antimirs or by using blockmirs . the term bone loss disorder in accordance with the present invention should be interpreted broadly . in certain embodiments , the bone loss is associated with ankylosing spondylitis , renal osteodystrophy ( e . g ., in patients undergoing dialysis ), osteoporosis , glucocorticoid - induced osteoporosis , paget &# 39 ; s disease , abnormally increased bone turnover , periodontitis , bone fractures , rheumatoid arthritis , osteoarthritis , periprosthetic osteolysis , osteogenesis imperfecta , metastatic bone disease , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . in certain embodiments , the bone loss is associated with ankylosing spondylitis , renal osteodystrophy ( e . g ., in patients undergoing dialysis ), osteoporosis , glucocorticoid - induced osteoporosis , paget &# 39 ; s disease , abnormally increased bone turnover , bone fractures , osteoarthritis , periprosthetic osteolysis , osteogenesis imperfecta , metastatic bone disease , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . in certain embodiments , the bone loss is associated with ankylosing spondylitis , renal osteodystrophy ( e . g ., in patients undergoing dialysis ), osteoporosis , glucocorticoid - induced osteoporosis , abnormally increased bone turnover , periodontitis , bone fractures , periprosthetic osteolysis , osteogenesis imperfecta , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . in certain embodiments , the bone loss is associated with osteoporosis , glucocorticoid - induced osteoporosis , paget &# 39 ; s disease , abnormally increased bone turnover , periodontitis , bone fractures , rheumatoid arthritis , osteoarthritis , periprosthetic osteolysis , osteogenesis imperfecta , metastatic bone disease , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . in certain embodiments , the bone loss is associated with osteoporosis , glucocorticoid - induced osteoporosis , paget &# 39 ; s disease , abnormally increased bone turnover , bone fractures , osteoarthritis , periprosthetic osteolysis , osteogenesis imperfecta , metastatic bone disease , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . in certain embodiments , the bone loss is associated with osteoporosis , glucocorticoid - induced osteoporosis , abnormally increased bone turnover , periodontitis , bone fractures , periprosthetic osteolysis , osteogenesis imperfecta , hypercalcemia of malignancy , multiple myeloma , bone loss associated with microgravity , langerhan &# 39 ; s cell histiocytosis ( lhc ), bone loss associated with renal tubular disorders , or bone loss associated with bed - ridden conditions . with the aim to identify specific mirnas with a potential to improve bone formation in vivo the present inventors first performed mirna microarray to compare mirna expression profile of undifferentiated hmscs to hmscs differentiated into osteogenic lineage . microarray analysis and further validation of mirna expression by qrt - pcr revealed significant downregulation of mir - 138 during osteoblast differentiation of hmscs . overexpression of mir - 138 by transfection with pre - mir - 138 decreased osteogenic differentiation capacity of hmscs in vitro , demonstrated by alp activity assay and gene expression analysis . additionally , overexpression of mir - 138 reduced ectopic bone formation in vivo by 85 %. in contrast , silencing mir - 138 by anti - mir - 138 increased osteogenesis of hmscs in vitro and ectopic bone formation in vivo by 60 %. target prediction analysis suggested focal adhesion kinase ( fak / ptk2 ) as a potential target for mir - 138 . since the fak pathway has been reported to play a role in promoting osteoblast differentiation it is likely that mir - 138 regulates bone formation by targeting ptk2 and inhibiting fak pathway and subsequently osteogenesis . in conclusion , the inventors have shown that mir - 138 plays an important role in enhancing bone formation in vivo , possibly through inhibition of fak signaling pathway . inhibition of mir - 138 is a new strategy for enhancing bone formation and osteoblast differentiation of msc in vivo thereby treating osteoporosis . telomerase immortalized human bone marrow - derived mesenchymal stem cells ( hmsc - tert4 ) ( 3 , 36 ) were cultured in minimum essential medium ( mem ) ( gibco invitrogen corporation ), with glutamax i ( gibco invitrogen corporation ) supplemented with 10 % fetal bovine serum ( fbs ) ( gibco invitrogen corporation ) and 1 % penicillin / streptomycin ( p / s ) ( gibco invitrogen corporation ) at 37 ° c . in a humidified atmosphere of 5 % co 2 . cells were induced to osteoblast differentiation when cultures were 70 - 80 % confluent . the osteoblast differentiation medium ( mem supplemented with 10 % fbs , 10 − 8 m dexamethasone , 0 . 2 mm l - ascorbic acid , 10 mm β - glycerophosphate , 10 mm 1 . 25 - vitamin - d 3 , 1 % p / s ) was refreshed every 3 days . cells were simultaneously cultured with mem , 10 % fbs and 1 % p / s for control . the osteoblast phenotype was evaluated by determining alkaline phosphatase ( alp ) activity , alp and alizarin red staining , and expression of osteogenic genes by quantitative rt - pcr analysis . for detection of alp , cells were washed with pbs , fixed with acetone / citrate buffer ( centralapoteket ) ph 4 . 2 for 5 minutes at room temperature and stained with naphtol - as - tr - phosphate solution for 1 hour at room temperature . naphtol - as - tr - phosphate solution consist of naphtol - as - tr - phosphate ( sigma ) diluted 1 : 5 in h 2 o and fast red tr ( sigma ) dissolved in 0 . 1 m tris buffer ( bie and berntsen ), ph 9 . 0 , in the ratio 1 . 2 : 1 . the solutions were mixed 1 : 1 . cells were rinsed with destilled h 2 o ( dh 2 o ) and counterstained with mayers - hematoxylin ( bie and berntsen ) for 5 minutes at room temperature . alizarin red staining was performed to detect matrix mineralization . cells were fixed with 70 % ice - cold ethanol for 1 h at − 20 ° c . and stained with 40 mm alizarin red s ( ar - s ; sigma - aldrich corp . ), ph 4 . 2 for 10 min at room temperature . alkaline phosphatase activity was quantified as previously described ( 37 ). briefly , cells were cultured on 96 - well plates at a density of 10 000 cells / cm 2 and induced to osteogenic differentiation as described above . cells were rinsed with tbs and fixed in 3 . 7 % formaldehyde - 90 % ethanol for 30s at room temperature . cells were incubated for 20 min at 37 ° c . with reaction substrate solution ( 1 mg / ml p - nitrophenylphosphate in 50 mm nahco 3 , ph 9 . 6 , 1 mm mgcl 2 ), and absorbance was measured at 405 nm using elisa - reader . total rna was extracted using trizol - phenol - choroform method ( invitrogen ) according to the manufacturer &# 39 ; s protocol . rna concentration was measured on a nd - 1000 spectrophotometer ( nanodrop ® technologies ) and quality of total rna was determined on an agilent 2100 bioanalyzer ( agilent technologies ). microrna microarray was performed at molecular medicine partnership unit ( mmpu ), university of heidelberg , germany , based on exiqon &# 39 ; s microarray platform ( version 8 . 0 ) with lna capture probes in quadruplicates . microarray probes were lna - modified oligonucleotide ( mircury exiqon ) capture probes with sequence complementary to mirnas . slides were scanned using genepix 4000b laser scanner ( axon instruments ). artifact - associated spots were eliminated by software ( tigr spotfinder 3 . 1 . 1 ). image intensities were measured as a function of the median of foreground minus background . negative values and values below 50 were normalized to one . further data analysis was performed using microsoft excel with significant analysis of microarrays ( sam ) excel software using multiclass response dataset analysis . the data was normalized using the limma package for statistical programming language r ( version 2 . 5 . 1 ). medians of four background - corrected replicas for each mirna capture probe were uploaded into the microarray analysis software for more advanced analysis for qrt - pcr analysis of mrna expression total rna was extracted using trizol reagent ( invitrogen ) according to manufacturer &# 39 ; s protocol . cdna was prepared using revertaid h minus first strand cdna synthesis kit ( fermentas ). sybr green qrt - pcr was performed using the primers alp forward ( 5 ′- acgtggctaagaatgtcatc - 3 ′), alp reverse ( 5 ′- ctggtaggcgatgtcctta - 3 ′), col1a1 forward ( 5 ′- tgacgagaccaagaactg - 3 ′), col1a1 reverse ( 5 ′- ccatccaaaccactgaaacc - 3 ′), runx2 forward ( 5 ′- tcttcacaaatcctcccc - 3 ‘), runx2 reverse ( 5 ′- tggattaaaaggacttgg - 3 ′), ptk2 forward ( 5 ′- gcgctggctggaaaaagagcaa - 3 ′), ptk2 reverse ( 5 ′- tcggtgggtgctggctggtagg - 3 ′), β - actin forward ( 5 ′- agccatgtacgttgcta - 3 ′) and β - actin reverse ( 5 ′- agtccgcctagaagca - 3 ′). expression levels were analyzed by qrt - pcr ( sybr green supermix and icycler iq detection system , bio - rad ) using conventional protocols . qrt - pcr consisted of 40 cycles ( 95 ° c . for 30 seconds , 60 ° c . for 30 seconds and 72 ° c . for 1 min ) after an initial denaturation step ( 95 ° c . for 3 min ). qrt - pcr products were quantified by comparative ct ( δδct ) method . for mirna qrt - pcr , total rna was extracted using trizol reagent ( invitrogen ). primers specific for human mir - 26a , mir - 26b , mir - 30c , mir - 101 , mir - 138 , mir - 143 , mir - 222 and internal control snornu44 were purchased from applied biosystems . amplification and detection were performed using 7500ht fast real - time pcr system ( applied biosystems ), using 40 cycles of denaturation at 95 ° c . ( 10 s ) and annealing / extension at 60 ° c . ( 60 s ). this was preceded by reverse transcription at 42 ° c . for 30 min and denaturation at 85 ° c . for 5 min . lna oligonucleotides were synthesized as unconjugated lna / dna mixmers with a complete phosphorothioate backbone ( idt , usa ). the antimir - control ( mir - c ) was purchased from idt , usa . lna modified antimirs sequences : antimir - 138 3 ′- attcacaacaccagc - 5 ′ and antimir - c 3 ′- tgtaacacgtctata - 5 ′, where uppercase letters refers to lna and lowercase letters refers to dna . synthetic pre - mir - 138 sequence 3 ′- agcugguguugugaaucaggccg - 5 ′ was rna oligonucleotides . transfections of 25 nm antimir oligonucleotide ( idt , usa ) or pre - mir ( ambion ) with lipofectamine 2000 ( invitrogen ) were performed according to the manufacturer &# 39 ; s instructions . cells were seeded in 6 - well plates and transfections were performed when cells reached 60 - 70 % confluence . one hour prior transfection , the medium was changed to 1 ml opti - mem i reduced serum ( invitrogen ). cells were incubated with transfection medium , which was changed four hours after transfection to normal mem medium with 10 % fbs and 1 % p / s . the transfection efficiency was evaluated by transfection of a 5 ′ fam - labeled lna oligonucleotide . hmscs were lysed by ripa buffer ( sigma - aldrich corp .) and total protein concentration was determined with pierce coomassie plus bradford assay kit ( thermo fisher scientific inc .). proteins were separated by 10 % sds - page and electrotransferred into nitrocellulose filters . after blocking with 5 % not - fat milk for 1h at room temperature , membranes were incubated with primary antibodies against anti - rabbit fak and pfak ( santa cruz biotechnology ) and anti - mouse erk perk and anti - rabbit α - tubulin ( cell signaling ) overnight at 4 ° c . membranes were incubated with horseradish - peroxidase ( hrp ) conjucated anti - mouse or anti - rabbit secondary antibody for 45 min at rt and protein bands were visualized with amersham ecl chemiluminescence detection system ( ge healthcare bio - sciences corp .). a 655 bp fragment of the fak ( ptk2 ) 3 ′ utr , containing the predicted binding site for hsa - mir - 138 , was amplified from human genomic dna using primers with a short extension , containing cleavage sites for xhoi ( 5 ′- end ) and noti ( 3 ′- end ). ptk2 forward ( 5 ′- atactcgagaaactggcccagcagtatg - 3 ′), ptk2 reverse ( 5 ′- atagcggccgcttgcaactgaagggtgttc - 3 ′). amplicons were cleaved with xhoi and noti and cloned in between the xhoi and noti cleavage sites of the psicheck ™- 2 vector ( promega ) downstream of the renilla luciferase reporter gene . huh7 cells were grown to 85 - 90 % confluence in white 96 well plates in dulbecco &# 39 ; s modified eagle medium ( dmem ) ( invitrogen ) supplemented with 10 % fbs , 1 % non - essential amino acids , l - glutamine and penicillin / straptamicin , at 37 ° c . under 5 % co 2 . cells were transfected with 20 ng of either the empty psicheck ™- 2 vector , or the psicheck ™- 2 - ptk2 3 ′ utr reporter , for four hours in reduced - serum and antibiotics - free opti - mem ( invitrogen ) with lipofectamine 2000 ( invitrogen ). cells were co - transfected with the pre - mir - 138 or a negative control ( mir - c ) ( applied biosystems ) at concentrations of 0 , 10 or 20 nm . after transfection , opti - mem was replaced by normal culture medium and cells were incubated for 24 hours . firefly and renilla luciferase were measured in cell lysates according to manufacturer &# 39 ; s protocol using a dual - luciferase reporter assay system ( promega ) on a fusion ™ plate reader ( perkin elmer ). firefly luciferase activity was used for normalization and as an internal control for transfection efficiency . hmscs were transfected as described above , loaded on hydroxyapatite / tricalcium phosphate ( ha / tcp ) ceramic powder ( zimmer scandinavia ) and implanted subcutaneously into 8 - week old nod / mrkbom tac - prkdc scid mice ( taconic ) as previously described ( 22 , 23 ). briefly , cells ( 5 × 10 5 ) were resuspended in 500 μl medium , transferred to 40 mg of wetted ha / tcp and incubated at 37 ° c . overnight . the following day , cells loaded on ha / tcp vehicles were implanted subcutaneously on the dorsal surface of the nod / scid mice . each mouse received four identical implants , two on each side . mice were anaesthetized by intra - peritoneal injection of ketaminal ® ( ketamine 100 mg / kg ) ( intervet ) and rampun ® ( xylazine , 10 mg / kg ) ( bayer healthcare ). after the surgery , mice received a subcutaneous injection of temgesic ® ( buprenorphin , 0 . 3 mg / ml ) ( schering - plough ) for pain relief . implants were removed after 1 or 8 weeks . one - week implants were subjected to rna extraction while 8 - week implants were fixed in 4 % paraformaldehyde ( bie & amp ; berntsen ), decalcified in formic acid ( local pharmacy , odense university hospital ) and embedded in paraffin using conventional histopathologic methods . samples were cut into 4 μm sections and stained with hematoxylin and eosin y ( bie & amp ; berntsens reagent laboratories ). total bone volume per total volume was quantified as previously described ( 23 ). four implants per treatment were engrafted into mice and three sections of each implant were quantified to minimize variation within the implants . a search for predicted target mrnas was performed using the databases targetscan and pictar . targetscan requires perfect complementarity with a mirna seed sequence , whereas pictar allows for targets with imperfect seed matches given that they pass a defined binding - energy threshold . additionally , pictar implements a maximum likelihood approach to incorporate the combinatorial nature of mirna targeting ( 38 ). data are presented as mean ± sd . comparisons were made by using a two - tailed t - test or 1 - way anova for experiments with more than two subgroups . probability values were considered statistically significant at p & lt ; 0 . 05 . in fig1 telomerized mscs ( hmsc - tert4 ) were induced to osteoblast differentiation . a ) osteoblast differentiation confirmed by qrt - pcr analysis of osteoblast marker genes ( runx2 , alp and oc normalized to β - actin ). b ) alp activity was measured during the course of differentiation . white bars represent non - induced and black bars represent induced samples . *** p & lt ; 0 . 001 between non - induced and induced sample . c ) alp and alizarin red staining were performed at day 15 . n = 3 for all experiments . in fig2 the effect of mir - 138 on osteoblast differentiation was studied . hmscs transfected with 25 nm mir - c , pre - mir - 138 or antimir - 138 were induced into osteoblast differentiation for 15 days . a ) osteoblast differentiation was evaluated with qrt - pcr analysis of osteoblast marker genes ( runx2 , alp and oc , normalized to β - actin ) at day 15 . b ) alkaline phosphatase activity was measured at day 10 of osteoblast differentiation . c ) alkaline phosphatase and alizarin red staining were performed at day 15 . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 , n = 3 for all experiments . in the experiment of fig3 hmsc were transfected with 25 nm mir - c , pre - mir - 138 or antimir - 138 and implanted into nod / scid mice . a ) qrt - pcr analysis of osteoblast genes ( runx2 , col1a1 , alp and oc normalized to ( 3 - actin ) was performed after 1 week of implantation . b ) h & amp ; e staining was performed after 8 weeks of implantation . bone formation was quantified as total bone volume per total volume from h & amp ; e staining and expressed as fold change of mir - c . * p & lt ; 0 . 05 , ** p & lt ; 0 . 01 , four implants per treatment were engrafted into mice and three sections of each implant were quantified to minimize variations within the implants . in the experiment of fig4 a ) ptk2 gene expression profile during osteoblast differentiation of hmsc was quantified with qrt - pcr . b ) computational analysis was performed for the complementarily of mir - 138 seed sequence to the 3 ′ utr of ptk2 and conservation of the putative binding site in vertebrates . c ) huh7 cells were transfected with 20 ng of either the empty psicheck ™- 2 vector , or the psicheck ™- 2 - ptk2 vector . cells were co - transfected with 0 , 10 or 20 nm of the pre - mir - 138 or a negative control . firefly and renilla luciferase were measured in cell lysates and values are normalized to the psicheck vector and presented as fold change of mir - c . ** p & lt ; 0 . 01 , *** p & lt ; 0 . 001 in the experiment of fig5 hmsc were transfected with 25 nm mir - c , pre - mir - 138 or antimir - 138 and induced to osteoblast differentiation and western blot analysis for fak protein , phosphorylation of fak and erk1 / 2 were performed at day 2 . graphs represent quantifications of western blot results . in fig6 mir - 138 is expressed in undifferentiated msc and suppresses fak translation , thereby decrease phosphorylation of fak and its downstream target erk1 / 2 . subsequently , inhibition of the cascade results in decreased expression of osx and suppression of osteoblast differentiation of msc . in fig7 there is shown an expression analysis of microrna - 138 ( mir - 138 ) and protein tyrosine kinase 2 ( ptk2 ). ( a ) expression of mir - 138 was measured in primary human stromal ( mesenchymal ) stem cells ( hmscs ) and calvarial preosteoblastic mc3t3 - e1 cells during osteoblast differentiation . ( b ) mir - 138 expression was evaluated in 5 - d - old mouse tissues . ( c ) expression of focal adhesion kinase ( fak ; ptk2 ) in primary hmscs . expression of ptk2 in primary hmscs during osteoblast differentiation evaluated at day 1 , 5 , 10 , and 15 by quantitative rt - pcr ( qrt - pcr ). in fig8 there is shown effects of mir transfection into cells . ( a ) expression of mir - 138 in transfected hmscs . hmscs were transfected with mir - c , premir - 138 , and antimir - 138 and induced to osteoblast differentiation . the mir - 138 expression levels in premir - 138 —( left ) and antimir - 138 — transfected ( right ) hmscs were quantified by qrt - pcr and normalized to sn / snornu44 , and values are presented as log2 fold change over mir - c . ** p & lt ; 0 . 01 ; *** p & lt ; 0 . 001 ( n = 3 ). ( b ) osteoblast differentiation of mir - 138 — transfected primary hmscs . primary hmscs were transfected with mir - c , premir - 138 , and antimir - 138 and induced to osteoblast differentiation . differentiation was evaluated at day 15 by alkaline phosphatase ( alp ) staining . ( c ) morphology of mir - 138 — tranfected hmscs . morphology was evaluated by photography of primary hmscs transfected with mir - c , premir - 138 , and antimir - 138 under the microscope at day 1 . ( d ) proliferation of mir - 138 — tranfected primary hmscs and mc3t3 - e1 cells . proliferation was evaluated by cell count in primary hmscs and mc3t3 calvarial cells transfected with mir - c , premir - 138 , and antimir - 138 . osteoblast differentiation of hmsc was induced by using standard osteoblast - induction medium containing dexamethasone , ascorbic acid , β - glycerolphosphate and vitamin - d 3 and evidenced by increased expression of genes associated with osteoblast differentiation ; runx2 , alkaline phosphatase ( alp ) and osteocalcin ( oc ) at day 1 , 10 and 15 after induction ( fig1 a ). the osteoblast phenotype was also confirmed by demonstration of increased alkaline phosphatase activity ( fig1 b and c ) and alizarin red staining for matrix mineralization ( fig1 c ). increased expression of osteoblast - associated genes and the observed osteoblast phenotype were in accordance with previous reports describing hmsc differentiation into osteoblasts ( 1 - 3 ). to identify differentially expressed mirnas during osteoblast differentiation we carried out mirna array profiling of hmsc 10 days after induction to osteoblasts using lna microarrays ( 24 ). expression levels of 33 mirnas were significantly altered between differentiated and non - differentiated cells . of these , 7 mirnas were selected for validation by qrt - pcr , based on their relative difference score and up - or down - regulation ( si table 1 ). microarray analysis and qrt - pcr data showed that the expression of mir - 26a , 26b , 30c , 101 and 143 were up - regulated and mir - 138 and 222 were down - regulated during osteoblast differentiation of hmscs . to evaluate the biological effect of the differentially expressed mirnas on osteogenesis , antimirs and mirna over - expression for selected mirnas were applied in a pilot functional screening for the impact on osteoblast differentiation in vitro and ectopic bone formation in vivo ( data not shown ) and pointed out to mir - 138 as a novel negative regulator of osteoblastic differentiation . we over - expressed or inhibited mir - 138 levels utilizing synthetic pre - mir and lna - modified anti - oligonucleotides designed as complementary sequences to mature mirnas , so - called antimirs . antimir oligonucleotides can be used as specific inhibitors of mirna function , thus providing a valuable tool to access the biological function of specific mirnas in vitro and in vivo ( 25 , 26 ). transfection efficiency of 80 % was observed using a 5 ′ fam - labeled control lna oligonucleotide ( data not shown ). the degree of mirna inhibition and over - expression was monitored by qrt - pcr after transfection of antimir - 138 or pre - mir - 138 to hmscs at 25 nm , respectively . the mature mir - 138 levels were elevated ˜ 900 - fold relative to control - treated cells 24 hours post transfection , with the levels still being 16 - and 8 - fold higher than in the control 10 and 15 days after pre - mir - 138 transfection , respectively ( fig7 ). by comparison , treatment of hmscs with antimir - 138 led to inhibition of mir - 138 by 1 . 1 -, 1 . 5 - and 2 - fold 1 , 10 and 15 days after transfection ( fig7 ). to study the impact of mir - 138 on hmsc osteoblast differentiation , hmscs were induced to differentiate to osteoblasts after transfection with either pre - mir - 138 or antimir - 138 . inhibition of mir - 138 significantly enhanced osteogenic differentiation , as indicated by higher expression of the osteoblast - specific genes runx2 , alp and oc and ( fig2 a ), and increased alp activity and enhanced in vitro matrix mineralization visualized by alizarin red staining ( fig2 b , c ) in antimir - 138 transfected hmscs as compared to control transfected cells . in contrast , alp activity , matrix mineralization and the osteoblast marker gene expression were reduced in pre - mir - 138 transfected hmscs ( fig2 a , b , c ). taken together , our results indicate that mir - 138 is a negative regulator of osteoblast differentiation of hmscs . to study whether silencing of mir - 138 enhances ectopic bone formation also in vivo , untransfected control hmscs and hmscs transfected with mir - 138 , antimir - 138 or mir - control were loaded on hydroxyapatite implants in nod / scid mice for 1 or 8 weeks . no major changes were observed by histology of the implants with untransfected hmscs compared to mir - c transfected hmscs . gene expression of osteoblast marker genes was analyzed after one week of implantation . qrt - pcr analysis revealed up - regulation of runx2 , alp , col1a1 and oc in the antimir - 138 treated implants as compared to implants transfected with mir - c ( fig3 a ), corroborating the results obtained from in vitro cell culture assays . additionally , we determined the ability of mir - 138 inhibition or over - expression to regulate ectopic bone formation in vivo by quantifying the area of bone per total area after 8 weeks . bone formation was increased 2 . 2 - fold in implants treated with the antimir - 138 compared to mir - c ( fig3 b ), indicating that inhibition of mir - 138 enhances bone formation of hmsc . furthermore , over - expression of mir - 138 decreased bone formation by 6 . 7 fold , compared to mir - c implants ( fig3 b ), supporting the notion that mir - 138 negatively regulates osteoblast differentiation and bone formation in vivo . to understand the molecular mechanisms that underlie mir - 138 - mediated regulation , we searched for potential targets of mir - 138 implicated in osteoblast differentiation using the mirna target prediction algorithms targetscan and pictar ( 27 ). among the predicted targets we identified focal adhension kinase ( fak ), which provides a link between activation of erk1 / 2 and stimulation of the runx2 / cbfa1 transcription factor . to confirm the involvement of fak in osteogenesis of hmsc we studied the expression pattern of fak during differentiation . qrt - pcr analysis revealed that expression of ptk2 , the gene encoding fak , was increased during osteoblast differentiation similar to osteoblast marker genes and coinciding with down - regulation of mir - 138 ( fig4 a ). according to in silico analysis , ptk2 has a 7 nucleotide seed match site for mir - 138 within its 3 ′ utr , and this putative target site is highly conserved among the vertebrates ( fig4 b ). to determine whether mir - 138 inhibits ptk2 gene expression by binding to the predicted target site in the 3 ′ utr , we used a dual luciferase reporter gene system ( 28 ), with renilla luciferase as a reporter gene and firefly luciferase as an internal control . assays were performed in huh7 cells that were chosen based on their low endogenous expression of mirnas ( 29 ). co - transfection of the ptk2 3 ′ utr luciferase reporter with pre - mir - 138 resulted in concentration - dependent down - regulation of luciferase activity , compared to the mock or scrambled oligonucleotide controls ( mir - c ) ( fig4 c ). in comparison , pre - mir - 138 had no effect on the luciferase control reporter without the ptk2 3 ′ utr , implying that ptk2 is a direct target of mir - 138 . gene expression analysis revealed no significant change in ptk2 mrna levels when mir - 138 was either over - expressed or antagonized ( fig8 a ). however , western blot analysis showed reduced fak protein levels in the pre - mir - 138 treated cells at day 2 compared to mir - c transfected samples ( fig5 ). since the fak signaling pathway is suggested to provide a link between activation of erk1 / 2 by ecm proteins in osteogenesis ( 7 ), we next assessed phosphorylation of fak and erk1 / 2 . western blot analysis showed markedly decreased phosphorylation of both fak and erk1 / 2 in mir - 138 over - expressing hmscs , while levels of pfak and perk1 / 2 were increased in the antimir - 138 transfected hmsc ( fig5 ). accordingly , expression of osterix ( osx ), a downstream target gene of the erk1 / 2 pathway ( 7 ), was decreased when mir - 138 was over - expressed and increased in the absence of mir - 138 ( fig8 b ), which is consistent with the notion that mir - 138 suppresses fak downstream signaling by negatively regulating fak at the post - transcriptional level . bone marrow contains a population of stromal ( skeletal , mesenchymal ) stem cells ( hmsc ) that under appropriate in vivo and in vitro conditions can differentiate into osteoblastic cells ( 30 ). differentiation of hmsc into osteoblastic cells is a highly regulated process involving complex pathways and de - regulation may lead to pathological conditions . therefore the molecular mechanisms of this process needs to be explored to ultimately improve therapies for the related diseases . recently , it has been shown that mirnas influence the complexity of the “ sternness state ” in a number of cellular compartments through negative regulation of gene expression at the post - transcriptional level ( 31 ). the present inventors have identified mir - 138 as a negative regulator of hmsc osteoblast differentiation and demonstrated that antimir mediated silencing of mir - 138 significantly enhance ectopic bone formation in vivo . this suggests functional silencing of mir - 138 as a potential novel strategy for anabolic treatment of osteoporosis . the therapeutic feasibility of such an approach has been demonstrated in the work by li and co - workers who demonstrated that a single tail vein injection of antimir - 2861 caused minimum femur bone mineral density in ovarectomized mice ( 15 ). the present inventors conducted genome - wide array analysis of the mirna levels during osteogenic differentiation of hmsc and found deregulation of several mirnas during osteoblast differentiation of hmsc in vitro . more detailed analysis suggested mir - 138 as a novel negative regulator of osteoblastic differentiation . in vitro experiment revealed that inhibition of mir - 138 function enhanced osteoblast differentiation , whereas mir - 138 over - expression inhibited the osteogenic potential . these experiments were extended to an in vivo milieu where silencing of mir - 138 by antimir - 138 led to increased ectopic bone formation while over - expression of mir - 138 significantly diminished bone formation . these findings suggest that mir - 138 plays a pivotal role in bone formation in vivo by negative regulation of osteogenic differentiation in hmscs leading to reduced ectopic bone formation . recently , mir - 138 has been implicated in differentiation of human adipose tissue - derived mesenchymal stem cells ( had - mscs ) and mir - 138 was found to be a negative regulator of adipocyte differentiation ( 21 ). these new data , together with our findings suggests a general role of mir - 138 as an inhibitor of hmsc differentiation and maintenance of the “ sternness state ”. to study the molecular mechanism whereby mir - 138 regulates osteogenesis , the present inventors searched for potential target genes that have an established function in promoting osteogenesis . interestingly , the 3 ′ utr of ptk2 possess a seven nucleotides perfect match site to the mir - 138 seed region . the protein encoded by ptk2 , fak , has been shown to function as an activator of extracellular signal - related kinase ( erk1 and erk2 ) via the grb2 - sos - ras pathway during osteogenic differentiation of hmsc ( 32 , 33 ). recently , mir - 138 was found to target rhoc and rock ( 34 ), which are also involved in regulation of osteoblast differentiation and this may be an additional mechanism for mir - 138 as negative modulator of osteoblast differentiation . however , the exact organization of these pathways in developing bone is not well understood , although osteoblasts in osteoporosis and osteoarthritis patients have reduced fak activity ( 35 ). here , the present inventors show that mir - 138 over - expression results in down - regulation of fak at the protein level , whereas functional inhibition of mir - 138 by antimir - 138 leads to de - repression of fak , strongly suggesting that fak is regulated by mir - 138 during osteogenesis . indeed , ptk2 3 ′ utr luciferase reporter assays confirmed that fak is a direct target of mir - 138 . furthermore , over - expression of mir - 138 decreased phosphorylatation of fak and subsequently attenuated activation of fak downstream signaling , as shown by decreased phosphorylation of erk1 / 2 in hmscs . activation of erk1 / 2 pathway has emerged as an important regulator of osteoblast differentiation , where it regulates runx2 phosphorylation and subsequently expression of osterix ( 7 ). significant up - regulation of osterix in the absence of mir - 138 supports the hypothesis that inhibition of osteoblast differentiation by mir - 138 is due to suppression of the downstream pathway of fak ( fig6 ). the impact of mirnas on osteoblastic differentiation of a number of cell types has been investigated by modulation of mirna function by antimirs and over - expression . these approaches have successfully demonstrated that mir - 204 / 211 targets runx2 , stimulates adipocyte differentiation and diminishes osteoblastic differentiation ( 18 ). employing similar approach , enhanced activity of mir - 125b in mouse st2 cells inhibits osteoblastic differentiation ( 13 ) whereas mir - 2861 acts as a positive regulator by targeting hdac5 ( 15 ). luzi et al . 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( 12 ) demonstrated an increase in mir - 196a expression and concomitant decrease of hox8 expression , a negative regulator of smad1 , during osteogenic differentiation of hascs . the present inventors found that mir - 138 repress fak expression , which , in turn , results in suppression of the fak - erk1 / 2 signaling pathway . importantly , our results show that functional inhibition of mir - 138 can accelerate osteogenic differentiation of hmscs leading to increased bone formation in vivo , suggesting that therapeutic approaches targeting mir - 138 could be useful in the enhancing bone formation and treatment of pathological conditions of bone loss . 1 . jaiswal , r . k ., jaiswal , n ., bruder , s . p ., mbalaviele , g ., marshak , d . r ., & amp ; pittenger , m . f . 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