CN108559773A - Application of miR-218 in preparation of medicine for treating osteoporosis - Google Patents

Abstract

The invention discloses miR-218 down-regulation in the process of differentiating RAW 264.7 cells into osteoclasts. miR-218 overexpression inhibits osteoclast differentiation, and miR-218 expression inhibition promotes the process. Bioinformatic analysis and luciferase reporter gene showed that TNFR1 is the target gene for miR-218. Therefore, miR-218 inhibits the regulation negative of an NF-kB signal channel through targeting TNFR1 to regulate osteoclastic differentiation, and the targeted miR-218 is suggested to be a potential treatment method for postmenopausal osteoporosis and can be applied to preparation of a medicament for treating the postmenopausal osteoporosis.

Description

MiR-218 is preparing the application in treating osteoporosis agents

Technical field

The invention belongs to field of biomedicine technology, and in particular to miR-218 is in preparing treatment osteoporosis agents Application.

Background technology

Postmenopausal Osteoporosis is a kind of common disease, related with estrogen deficiency, leads to bone loss and bone tissue Change, cause poor bone quality and risk of bone fracture to increase, seriously affect the Health and Living quality of the elderly, it has also become is current important Health problem.The characteristics of Postmenopausal Osteoporosis is bone density reduction, and bone structure deteriorates, and bone is fragile, leads to brittleness bone The risk of folding increases.Postmenopausal Osteoporosis is mainly due to osteoclastic bone resorption and osteoblast bon e formation dysequilibrium It is caused.

microRNA（miRNA）It is a kind of non-coding single strand RNA molecule by endogenous gene codes, there are about 22 nucleosides Acid plays important adjustment effect process in the development of various biologies.MiRNAs mainly by the post-transcriptional control of gene come The expression for regulating and controlling target gene, to participate in tumour generation, biological development, organ generation, virus defense and metabolism.There is research in the recent period Show that miRNAs can be participated in the ripe differentiation of osteoclast.PDCD4 is combined by targeting（Apoptosis egg White 4）, miR-21 can regulate and control the osteoclast differentiation pathway of the Monocytes/Macrophages precursor of derived from bone marrow, and osteoclastic In the detection of cell-progenitor cells strain RAW364.7, researcher has found that miR-223 is expressed and can be passed through inhibition wherein NF1-A（Nuclear factor 1 A）And M-CSFR（Macrophage colony-stimulating factor receptor）Level is sent out to regulate and control the differentiation of osteoclast It educates.Opposite, miR-155 is in high level expression in macrophage, and induction into osteoclast atomization expression by It gradually reduces, prompts it that may may play the part of the effect of inhibiting factor in osteoclast atomization.Therefore miRNAs is in sclerotin Important regulating and controlling effect is played in loose relevant osteoclast differentiation, and its mechanism of action also needs us and further studies.

Currently, being concentrated mainly on the effect during the occurrence and development to tumour to the research of miR-218, research is found： MiR-218 is a kind of tumor suppressor, has and prevents tissue glioma cell migration, migrate, be proliferated and cancer cell is dry thin The function of born of the same parents.

However, the regulatory mechanism of osteoclast is still not clear in miR-218, therefore miR-218 is inquired into osteoclast point The effect of change and molecular mechanism therein obviously have highly important theory significance and application value, contribute to as post menopausal bone The treatment of matter osteoporosis provides new approaches, and is likely to become the effective means of Postmenopausal Osteoporosis treatment.

Invention content

Goal of the invention：Present invention aims in view of the deficiencies of the prior art, the tune of the miR-218 in osteoclast is studied Control mechanism provides applications of the miR-218 in the drug for preparing treatment osteoporosis.

It is a further object of the present invention to provide miR-218 to prepare treatment osteoporosis by regulating and controlling target gene TNFR1 Drug in application.

Technical solution：The purpose of the invention is achieved by the following technical solution：

Applications of the miR-218 in the drug for preparing treatment osteoporosis.

The osteoporosis is postmenopausal osteoporosis.

The miR-218 precursor sequences are：5′-TTGCGGATGGTTCCGTCAAGCA-3′.

The present invention also provides a kind of reagents or drug for treating osteoporosis, and the reagent or drug include：

（a）MiR-218 analogies and/or miR-218 agonists；

（b）Receptible virus, carrier and/or auxiliary material in pharmacy.

Advantageous effect：The present invention has studied the level of the miR-218 during 264.7 cell differentiations of RAW are osteoclast Variation；And miR-218 analogies or inhibitor are transfected in 264.7 cells of RAW, have studied the miR-218 in osteoclastic differentiation Effect；The target gene of miR-218 is identified and verified using bioinformatic analysis and luciferase reporter gene.The present invention is sent out 264.7 cell differentiations of present RAW are miR-218 downwards during osteoclast.MiR-218, which is overexpressed, inhibits osteoclast Differentiation, and miR-218 expression is inhibited then to promote this process.Bioinformatic analysis and luciferase reporter gene show TNFR1 is the target gene of miR-218.Therefore, miR-218 inhibits the adjusting negativity tune of NF- κ B signal accesses by targeting TNFR1 Osteoclastic differentiation is saved, targeting miR-218 is prompted to be likely to become the potential treatment method of Postmenopausal Osteoporosis.

Description of the drawings

Fig. 1（A）Being dyed for TRAP confirms osteoclast differentiation（0,1,3,5 day）；Fig. 1（B）For NFATC1 and TRAF-6 eggs The variation of white expression（0,1,3,5 day）；Fig. 1（C）It is NFATC1 and TRAF-6 mRNA in osteoclastic differentiation 0,1,3,5 day Expression.Fig. 1（D）For miR-218 expression in osteoclastic differentiation expression in 0,1,3,5 day.N=3（P ＜ compared with the control group 0.05）.

Fig. 2（A）Left figure is light microscopic observation transfection efficiency, and right figure is that qRT-PCR methods detect 264.7 cell transfecting miR- of RAW The variation of miR-218 expressions when 218 analogies, inhibitor or miR-NC；Fig. 2（B）For 264.7 cell transfectings of RAW Row TRAP dyeing in 3 days after being induced with RANKL after miR-218 analogies, inhibitor or miR-NC；Fig. 2（C）It is detected for PCR NFATc1 and TRAF6 expressions are in miR-218 analogies, the change of inhibitor or miR-NC groups.Fig. 2（D）It is detected for WB Change of the NFATc1 and TRAF6 protein expression levels in miR-218 analogies, inhibitor or miR-NC groups.N=3（With NC phases Than p ＜ 0.05）.

Fig. 3（A）For TargetScan, miRanda, and PicTar predict potential target gene；Fig. 3（B）Schematic diagram is shown TNFR13 '-UTR have the binding site of miR-218；Fig. 3（C）For 3 ' UTR luciferase reports of cotransfection HEK293T cells TNFR1 It accuses（Binding site in wild type or saltant type）；Expression significantly inhibits 3 ' UTR uciferase activities of WT TNFR1.N=3（With Blank group is compared, p ＜ 0.05）.

Fig. 4（A）Variation of TNFR1, p65 and ph-p65 express spectra in osteoclastic differentiation 0,1,3 and 5 day is detected for WB.Fig. 4 （B）For WB TNFR1, the variation of p65, ph-p65 in miR-218 analogies, inhibitor or miR-NC groups.N=3（With NC phases Than p ＜ 0.05）.

Fig. 5 is that miR-218 breaks up regulatory mechanism to osteoclast.

Specific implementation mode

Technical solution of the present invention is described in detail below by attached drawing, but protection scope of the present invention is not limited to The embodiment.

Experimental method：

Material：

264.7 cells of RAW（Mouse osteoclast precursor cells）For the research of osteoclastic differentiation, from rich permanent cell centre（On Sea, China）Purchase.With Nuclear factor kappa B ligand receptor activator（RANKL）（50 ng/ml）Stimulate the osteoclastic differentiation of cell（When induction Between 0,1,3,5 day）.Under standard cell culture conditions（5% CO₂With 95% humidity）Cultivate cell.By TRAP dyeing and Detect osteoclast differentiation marker gene（TRAF6 and NFATc1）Expression verify the successful osteoclast of induction.Pass through light Learn the multinucleated osteoclast that TRAP stained positives are counted under microscope（>3 cores）.

The transfection of miRNA analogies or inhibitor

Analogies, inhibitor and the negative control of the miR-218 of fluorescent marker（miR-NC）By RiboBio（Guangzhou, China）It closes At.264.7 cell inoculations of RAW are in six orifice plates（20 × 104 cells/wells）Transfect the miR- of 100 nm respectively by Lipo3000 218 analogies, the miR-218 inhibitor and miR-NC of 150 nm.After 36 hours are incubated, in fluorescence microscopy, transfection is imitated under the microscope Rate.Then it will cultivate in cell fresh culture, be incubated 3 days by RANKL.

The extraction of miRNA

With Trizol total serum IgE is extracted from 0,1,3,5 day cell of the differentiation of collection.In brief, cell is collected manages in EP, It is cracked with 1 milliliter/pipe Trizol, chloroform is then added（0.2 milliliter/milliliter Trizol）.12000 turns 4 °C 15 points of centrifugation Zhong Hou is abandoned in supernatant to new EP pipes and is mixed isopropanol（0.5 milliliter/milliliter Trizol）.Then, by mixture in 12000 Rpm centrifuges 4 °C 10 minutes；Upper strata aqueous phase is dropped to obtain RNA precipitate.RNA precipitate is washed twice in 75% ethyl alcohol, in 12000 rpm After 4 °C of 15 minutes removal upper strata aqueous phases of centrifugation, RNA precipitate is dried in air, at room temperature, water-soluble with 20-50 μ l DEPC Solve RNA precipitate.Total serum IgE is analyzed for RT-qPCR.

RT-qPCR

Trizol methods extract total serum IgE.RT is carried out using PrimeScript RT kits（TaKaRa）.Real-time RT-qPCR is special Property primer from raw work biotechnology purchase.

Following primer is detected for RT-qPCR：

U6：forward: 5′-AGAGAAGATTAGCATGGCCCCTG-3′,

reverse: 5′-ATCCAGTGCAGGGTCCGAGG-3′；

miR-218：forward: 5′-TTGCGGATGGTTCCGTCAAGCA-3′,

reverse: 5′-ATCCAGTGCAGGGTCCGAGG-3′;

TNFRSF1A：forward: 5′-GGGGATACATCCATCAGGGGT-3′,

reverse: 5′-GCTCGGACAGTCACTCACC-3′;

NFATc1：forward: 5′-GACCCGGAGTTCGACTTCG-3′,

reverse: 5′-TGACACTAGGGGACACATAACTG-3′;

TRAF6：forward: 5′-AAAGCGAGAGATTCTTTCCCTG-3′,

reverse: 5′-ACTGGGGACAATTCACTAGAGC-3′;

GAPDH：forward: 5′-TGGCCTTCCGTGTTCCTAC-3′,

reverse: 5′-GAGTTGCTGTTGAAGTCGCA-3′)。

Amplification and detection are carried out using SYBR Premix Ex Taq II kits（TaKaRa）And Applied Biosystems StepOnePlus Real-Time PCR systems.U6 and Gapdh are used as internal reference.

MicroRNA target prediction

Using TargetScan, miRanda, and PicTar predict the potential target genes of miR-218.We have found that candidate target In gene, the 3 ' UTR of TNFR1 have the binding site of miR-218, TNFR1 and osteoclast signal path closely related.

Luciferase reporter gene is analyzed

By detecting in the HEK293T cell cotransfection UTR luciferases of miR-218 and 3 ' TNFR1（The knot of wild type or saltant type Close site）.Renilla luciferase transfections are used as control from view of profit.Luciferase detection system is utilized according to the instruction of manufacturer System detection uciferase activity（Promega companies, the U.S.）.

Western

Liquid is cleaved in cell to crack and using BCA protein quantification kit measurement protein concentrations（The green skies, China）.Always Albumen（80μG）It mixes and boils 5 minutes with 1 × buffer solution, be placed on ice, with 10% sodium dodecyl sulfate polyacrylamide gel Electrophoresis, and it is transferred to pvdf membrane.5% 4 °C of skim milk 1 hour is closed, then in 4 °C of overnight incubation primary antibodies（NFATc1, TRAF6, TNFR1, P65, ph-p65, GAPDH）After removing unbonded antibody and being washed three times with TBST buffer solutions, film is used 37 °C of secondary antibody is incubated 1 hour, then uses three changes of TBST buffer solutions.Exposure analysis.

TRAP is dyed

Osteoclast is confirmed by using TRAP staining kits.30 s are fixed with fixer at room temperature, then use deionized water Cleaning is three times.Prepare dyeing liquor, is mixing in one 100 milliliters of beaker of reagent below：45 ml deionized waters（37°C）, 1 milli It rises and adds garnet aqueous slkali（0.5 milliliter）And sodium nitrite solution（0.5 milliliter）, 0.5 milliliter of naphthols AS-BI phosphate solution, The tartaric acid solution that 2 milliliters and 1 milliliter of acetic acid solution.Cell is soaked in dyeing liquor, 37 DEG C be incubated 1 hour, then spend from Sub- water rinses.Microscopically observation TRAP positive cells（Containing 3 cores）.

Statistical analysis

All data are unit mean value means ± SD.Comparison among groups use One-way ANOVA, followed by post-hoc to examine （Least significant difference）.P ＜ 0.05 are conspicuousness.All experiments are in triplicate.

Experimental result

MiR-218 expresses downward in osteoclastic differentiation

We detect the expression of the miR-218 when RANKL is induced 0,1,3,5 day, are used as negative control within 0 day.As expected that Sample, 264.7 cell differentiations of RAW are osteoclast under RANKL inductions, by the dyeing for enhancing TRAP（Figure 1A）And NFATc1 Expression increase with TRAF6 proves, that is, marks mature osteoclast（Figure 1B, 1C）.Then we are detected with real-time quantitative PCR Variations of the miR-218 in osteoclast generation.MiR-218 expression is significantly reduced in osteoclast differentiation, 5 days miR- after induction 218 is horizontal minimum（Fig. 1 D）.These results indicate that miR-218 expresses downward in osteoclastic differentiation.

MiR-218 up-regulated expressions inhibit the differentiation of osteoclast

In order to study effects of the miR-218 in osteoclast differentiation, 264.7 cell transfecting miR-218 analogies of RAW, suppression Preparation or miR-NC.Three groups of transfection efficiency is 60% or more.MiR-218 analogies induce miR-218 expression significantly to increase （About 228 times of miR-NC groups）, and inhibitor significantly reduces miR-218 expression（Nearly 1/5th of miR-NC groups）（Figure 2A）.TRAP dyeing displays, miR-218 overexpressions make declines of the RANKL to osteoclast（Fig. 2 B）.However, inhibiting miR- 218 expression then promotes the differentiation of osteoclast.PCR and Western is inquiring into miR-218 analogies, inhibitor or miR-NC The express spectra variation of group NFATc1 and TRAF6.MiR-218 up-regulated expressions obviously inhibit the differentiation of osteoclast, by osteoclast The decline of specific gene NFATc1 and TRAF6 expression shows（Fig. 2 C, 2D）.However, low miR-218 expression significantly stimulation RAW 264.7 cell osteoclasts break up, this is consistent with the above results.In conclusion miR-218 up-regulated expressions inhibit osteoclast Differentiation.

TNFR1 is the target gene of miR-218

In order to inquire into the molecular mechanism that miR-218 regulates and controls in the differentiation of RAW264.7 cell osteoclasts, we use TargetScan, miRanda, and PicTar find potential target gene（Fig. 3 A）.The relevant candidate target base of osteoclast Because in, it has been found that there are the 3 ' UTR of TNFR1 to have binding site（Fig. 3 B）.Inquiring into miR-218 can be by combining 3 ' UTR to reduce TNFR1 is expressed, with 3 ' UTR luciferase reporter vectors of plasmid co-transfection 293T cells TNFR1 and expression vector.Then, we Whether the variation that mutation miR-218 luciferase check and evaluations are used in combination is that its binding site predicted exists due to cell recognition TNFR1 rather than because of other nonspecific effects TNFR1 seed zones.MiR-218 significantly inhibits WT TNFR1 rather than is mutated TNFR1（Fig. 3 C）.These results indicate that TNFR1 is the target gene of miR-218.

MiR-218 inhibits p65 signal activations

The mechanism that miR-218 inhibits osteoclastic differentiation is further inquired into, we analyze TNFR1 signal transduction pathways.According to we institute Know, TNF/TNFR1 is to cause nuclear factor B（NF-κB）The activation of signal path, and demonstrate NF- κ B signals accesses with it is osteoclastic Cells into close is related.The phosphorylation level of TNFR1 expression and p65 was detected at the 1st, 3,5 day.The phosphorylation water of TNFR1 and p65 It puts down and is significantly increased after RANKL inductions（Fig. 4 A）.Then, we inquire into whether miR-218 is by inhibiting NF- κ B signals to inhibit broken Bone breaks up, and we have studied p65 protein levels, p65 is TNFR1 downstream molecules, is proved to closely related with osteoclast formation. MiR-218 is expressed and is inhibited not changing total p65 levels significantly.We also have detected the phosphorylation level of p65；miR- The phosphorylation level of p65 is higher when 218 expression decline；MiR-218 is overexpressed then phosphorylation p65（ph-p65）It is horizontal notable.On It states the result shows that miR-218 is by inhibiting p65 accesses to inhibit the differentiation of osteoclast（Fig. 4 B）.

Present invention research finds that miR-218 downwards can be such that TNFR1 expression rises, and activates TNF signals.TNFR superfamilies at The signal transduction of member's activation TAK1, induced activation IKK complexs, to activate NF- κ B accesses to lead to osteoclastic differentiation.miR-218 Up-regulated expression inhibits the differentiation of osteoclast（Fig. 5 is that miR-218 breaks up regulatory mechanism to osteoclast）.MiR-218 passes through target Inhibit the adjusting negativity of NF- κ B signal accesses to adjust osteoclastic differentiation to TNFR1, prompts targeting miR-218 that can become post menopausal bone The potential treatment method of matter osteoporosis can be applied to the drug for preparing treatment osteoporosis.

As described above, although the present invention has been indicated and described with reference to specific preferred embodiment, must not explain For the limitation to invention itself.It without prejudice to the spirit and scope of the invention as defined in the appended claims, can be right Various changes can be made in the form and details for it.

Sequence table

<110>Jiangsu Prov. People's Hospital（No.1 Attached Hospital, Nanjing Medical Univ）

<120>MiR-218 is preparing the application in treating osteoporosis agents

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213>MiR-218 precursor sequences (DNA)

<400> 1

ttgcggatgg ttccgtcaag ca 22

<210> 2

<211> 20

<212> DNA

<213>The trans- sequences of miR-218 (DNA)

<400> 2

atccagtgca gggtccgagg 20

<210> 3

<211> 23

<212> DNA

<213>U6 precursor sequences (DNA)

<400> 3

agagaagatt agcatggccc ctg 23

<210> 4

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<212> DNA

<213>The trans- sequences of U6 (DNA)

<400> 4

atccagtgca gggtccgagg 20

<210> 5

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<213>TNFRSF1A precursor sequences (DNA)

<400> 5

ggggatacat ccatcagggg t 21

<210> 6

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<212> DNA

<213>The trans- sequences of TNFRSF1A (DNA)

<400> 6

gctcggacag tcactcacc 19

<210> 7

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<212> DNA

<213>NFATc1 precursor sequences (DNA)

<400> 7

gacccggagt tcgacttcg 19

<210> 8

<211> 23

<212> DNA

<213>The trans- sequences of NFATc1 (DNA)

<400> 8

tgacactagg ggacacataa ctg 23

<210> 9

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<212> DNA

<213>TRAF6 precursor sequences (DNA)

<400> 9

aaagcgagcg attctttccc tg 22

<210> 10

<211> 22

<212> DNA

<213>The trans- sequences of TRAF6 (DNA)

<400> 10

actggggaca attcactaga gc 22

<210> 11

<211> 19

<212> DNA

<213>GAPDH precursor sequences (DNA)

<400> 11

tggccttccg tgttcctac 19

<210> 12

<211> 20

<212> DNA

<213>The trans- sequences of GAPDH (DNA)

<400> 12

gagttgctgt tgaagtcgca 20

Claims (4)

1. Applications of the 1.miR-218 in the drug for preparing treatment osteoporosis.
2. 2. application according to claim 1, which is characterized in that the osteoporosis is osteoporosis in postmenopausal women Disease.
3. 3. application according to claim 1, which is characterized in that the miR-218 precursor sequences are：5′- TTGCGGATGGTTCCGTCAAGCA-3′。
4. 4. a kind of drug for treating osteoporosis, the drug include：

   （a）MiR-218 analogies and/or miR-218 agonists；

   （b）Receptible virus, carrier and/or auxiliary material in pharmacy.