CN114164216B - Application of gene in promoting bone formation - Google Patents

Abstract

The invention discloses an application of a gene in promoting bone formation, and belongs to the technical field of genetic engineering. The Etv gene can promote proliferation and differentiation of osteoblasts through inhibiting Etv gene of a mouse osteoblast line MC3T3-E1, so that bone formation is promoted, and reliable theoretical basis and experimental data are provided for clinically treating diseases such as osteoporosis.

Description

Application of gene in promoting bone formation

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to application of a gene in promoting bone formation.

Background

Osteoporosis is a systemic bone metabolic disease, which is manifested by reduced bone density and reduced bone quality, and can aggravate fracture risk, and current medicines for preventing and treating osteoporosis often have poor effects and large side effects. Mature human bone causes bone formation through osteoblasts, and osteoblasts undergo a series of differentiation stages such as proliferation, matrix formation, matrix mineralization, etc., while up-or down-regulation of related marker genes significantly affects the proliferation rate of osteoblasts, reduces bone formation, and eventually leads to the occurrence of osteoporosis.

Therefore, if relevant factors affecting the proliferation of osteoblasts can be researched, a novel medicament with strong specificity and weak side effects can be obtained in the preparation of the osteoporosis medicament, and the novel medicament is greatly helpful for patients with osteoporosis.

Therefore, how to provide an application of a gene in promoting bone formation is a problem to be solved in the art.

Disclosure of Invention

The invention discloses application of a gene in promoting bone formation.

The invention aims to study the influence of ETV4 gene on osteoblast proliferation under the condition of down-regulation of expression, further study the effect of ETV4 gene in osteoporosis, and provide reliable theoretical basis and experimental data for clinically treating osteoporosis diseases.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the application of a gene in bone formation is that the CDS sequence of the gene is shown as SEQ ID NO. 1;

preferably, the gene is a murine Etv gene;

preferably, the use in bone formation includes use in promoting bone formation and use in inhibiting bone formation;

preferably, the use in promoting bone formation and the use in inhibiting bone formation comprise use in promoting bone formation for the preparation of a formulation for promoting bone formation and use in inhibiting bone formation for the preparation of a formulation for promoting bone formation;

preferably, the application of inhibiting bone formation is realized by an siRNA technique, and the siRNA sequence is:

Etv4-siRNA-2：

5’ GGACCUCAGUCACUUCCAAdTdT 3’，SEQ ID NO:3；

3’dTdTCCUGGAGUCAGUGAAGGUU 5’，SEQ ID NO:4；

or (b)

Etv4-siRNA-3：

5’ GCUCGCUGCGAUACUAUUAdTdT 3’，SEQ ID NO:5；

3’dTdTCGAGCGACGCUAUGAUAAU 5’，SEQ ID NO:6；

One or more of the following;

preferably, the bone cells are MC3T3-E1 cells;

preferably, the gene enhances osteoblast proliferation capacity;

SEQ ID NO:1：

atggag cggaggatga aaggcggata cttggaccag cgagtgccct acaccttctg cagcaaatct cccggaaatg ggagcttggg cgaagcgctg atggtcccgc agggaaagct catggacccg ggctccctgc cgccttccga ctcagaagat ctcttccagg acctcagtca cttccaagag acgtggctcg cagaagctca ggtaccggac agtgatgagc agtttgttcc tgatttccat tcagaaaact tagctttcca tagccccacc accaggatca agaaggaacc ccagagtccc cgcacagacc ccgccctgtc ctgcagcagg aagccaccac tcccctacca ccatggagag cagtgccttt actccagtgc ctatgactcc cccagacaaa tcgccatcaa gtcccccgcc cccggtgccc ctggacagtc gcccctgcag cccttttcca gggcagaaca gcagcagagc ctcctgagag cctccagctc ttcccagtcc caccctggcc acgggtacct tggtgagcac agctccgtct tccagcagcc cgtggacatg tgccactcct tcacatctcc tcagggaggg ggccgggaac ctctcccagc cccctatcaa caccaactgt cggagccctg cccaccctac ccccagcaga acttcaagca ggagtaccat gaccccctgt acgaacaggc tggccagccc gcttcaagcc agggtggggt cagtgggcac aggtacccag gggcgggggt ggtgatcaaa caggagcgca cagacttcgc ctacgactca gatgtccctg gatgtgcatc aatgtacctc cacccagagg gcttctctgg accctctcca ggtgatggag tgatgggtta tggctatgaa aaatcccttc gaccattccc agatgatgtc tgcattgtcc ctgaaaaatt tgaaggagac atcaagcagg aagggattgg agctttccgg gaggggccac cctaccagcg ccggggtgcc ttacaactgt ggcagtttct ggtggccctg ctggatgacc caacaaatgc tcatttcatt gcttggacag gccggggaat ggagtttaaa ctaattgaac ctgaagaggt tgccaggctc tggggtatcc agaagaaccg gccagccatg aattatgaca agctgagccg ctcgctgcga tactattatg agaaaggcat catgcagaag gtggctggcg aacgctacgt gtacaagttt gtgtgcgagc cggaggccct gttctctctg gccttcccag ataatcaacg tccagctctg aaggctgagt ttgaccggcc agtcagtgag gaggacacag tccctttgtc ccacttggat gagagtcctg cctacctccc agaactcact ggccccgctc cgcccttcgg ccacagaggt ggatattctt actag。

the mRNA sequence corresponding to CDS is shown as SEQ ID NO. 2, and is specifically as follows:

1 ggaggccaag gcaaaggaaa tgcaccaatc agctgctccc ccgggctcac aactgtctgc

61 tgcgcccgga aaacaagccg gggctctggg gacccggggc tcaggccgcc tcgctccggc

121 ctagccccgc caccttagtt gtgtcatccc ccgggcatgc tgagcatccc cccgcggctc

181 cggcacagac gcccggacct caggtctctg cctccgcgcg ggggcccggc cctgtggccg

241 gagggagcgg ccggatggag cggaggatga aaggcggata cttggaccag cgagtgccct

301 acaccttctg cagcaaatct cccggaaatg ggagcttggg cgaagcgctg atggtcccgc

361 agggaaagct catggacccg ggctccctgc cgccttccga ctcagaagat ctcttccagg

421 acctcagtca cttccaagag acgtggctcg cagaagctca ggtaccggac agtgatgagc

481 agtttgttcc tgatttccat tcagaaaact tagctttcca tagccccacc accaggatca

541 agaaggaacc ccagagtccc cgcacagacc ccgccctgtc ctgcagcagg aagccaccac

601 tcccctacca ccatggagag cagtgccttt actccagtgc ctatgactcc cccagacaaa

661 tcgccatcaa gtcccccgcc cccggtgccc ctggacagtc gcccctgcag cccttttcca

721 gggcagaaca gcagcagagc ctcctgagag cctccagctc ttcccagtcc caccctggcc

781 acgggtacct tggtgagcac agctccgtct tccagcagcc cgtggacatg tgccactcct

841 tcacatctcc tcagggaggg ggccgggaac ctctcccagc cccctatcaa caccaactgt

901 cggagccctg cccaccctac ccccagcaga acttcaagca ggagtaccat gaccccctgt

961 acgaacaggc tggccagccc gcttcaagcc agggtggggt cagtgggcac aggtacccag

1021 gggcgggggt ggtgatcaaa caggagcgca cagacttcgc ctacgactca gatgtccctg

1081 gatgtgcatc aatgtacctc cacccagagg gcttctctgg accctctcca ggtgatggag

1141 tgatgggtta tggctatgaa aaatcccttc gaccattccc agatgatgtc tgcattgtcc

1201 ctgaaaaatt tgaaggagac atcaagcagg aagggattgg agctttccgg gaggggccac

1261 cctaccagcg ccggggtgcc ttacaactgt ggcagtttct ggtggccctg ctggatgacc

1321 caacaaatgc tcatttcatt gcttggacag gccggggaat ggagtttaaa ctaattgaac

1381 ctgaagaggt tgccaggctc tggggtatcc agaagaaccg gccagccatg aattatgaca

1441 agctgagccg ctcgctgcga tactattatg agaaaggcat catgcagaag gtggctggcg

1501 aacgctacgt gtacaagttt gtgtgcgagc cggaggccct gttctctctg gccttcccag

1561 ataatcaacg tccagctctg aaggctgagt ttgaccggcc agtcagtgag gaggacacag

1621 tccctttgtc ccacttggat gagagtcctg cctacctccc agaactcact ggccccgctc

1681 cgcccttcgg ccacagaggt ggatattctt actaggcacc agtggcttcc ccttgacatg

1741 gtggggttgc tcagtgtata tatcaactga tttggtattg gtgaaggccc tctttctgat

1801 gcctgtagaa gtctctgggg tcagagctcc actatcccat ctgatactcc tggccagact

1861 cagctgctaa ccagagtctg cgggaaagac agtggaggca ggccaaatct aaaggcagta

1921 gccgaagttc gctgtggctc acctgtacct tcagttcagc ttggcctctg cctaggtctt

1981 gctcagaggc caagttcctc acccccacca cagagatcca gtgttctatt ctggggacat

2041 acagggactt cccttgttta ttatggcaac agggccaagg ggattctcag aacaccctgt

2101 gtctcccctc tcccaacccc ccatgggaga caaagttctg cctgggttct gccctgaaca

2161 ggggggtcct gtgttcttgg tgctgtgctc tgggaggcag gagcatgtgg gcggcagctg

2221 gggggggggg ttgggtggaa gagtagggat ggctctctgc cctaggccta cccaggccta

2281 attccacctt tgcctcttat gccagacctt aataaagcct ctgcttctcc ggtaaaaaaa

2341 aaaaaaaaaa aa。

in conclusion, the invention discloses application of a gene in promoting bone formation, and the Etv gene can promote proliferation and differentiation of bone cells and promote bone formation by inhibiting the Etv gene of MC3T3-E1, so that reliable theoretical basis and experimental data are provided for clinically treating diseases such as osteoporosis.

Drawings

FIG. 1 shows qRT-PCR detection of the knockdown effect of siRNA on Etv gene in osteoblast line;

FIG. 2 shows the proliferation activity of osteoblasts after knockdown of the Etv gene in CCK-8 cell proliferation assay;

FIG. 3 is a graph showing the relative cell numbers of osteoblasts after the EdU labelling assay Etv knockdown;

FIG. 4 shows the amino acid sequence conservation analysis of human ETV4 protein and murine Etv protein.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

Bioinformatics analysis

ETV4 was found to be very conserved between human and mouse by bioinformatic analysis, with up to 92% protein identity as shown in figure 4.

Culture of osteoblasts (MC 3T 3-E1)

MEM-alpha culture medium containing 10% FBS (fetal bovine serum) and 1% P/S (double antibody) is prepared, the water bath is preheated for 20min, and the culture medium is uniformly planted in a 10cm culture dish according to a control group and an experimental group, shaken uniformly and placed in a culture box with the temperature of 37 ℃ and the concentration of 5% for culture. The liquid is changed for the first time for 24 hours, PBS is used for cleaning, fresh culture liquid is added for continuous culture, the liquid is changed every 2-3 days later, and the cell state is observed regularly.

Cell transfection

(1) Discarding old culture medium in a culture dish, adding trypsin after PBS (phosphate buffer solution) to clean cells, stopping digestion after the cells become round and float, transferring cell digestive juice into a 15ml centrifuge tube, centrifuging at 1000rpm at room temperature for 5min, obviously observing cell precipitation at the bottom of the centrifuge tube, sucking the supernatant by using a vacuum pump, adding 6ml of culture medium to resuspend the cells, sucking 20 mu l of cell suspension, dripping the cell suspension into a cell counting plate, detecting the concentration of the cell suspension by an automatic cell counter (selecting three different fields for counting and finally taking an average), diluting the cell suspension according to the expected cell planting quantity, inoculating the cell suspension into the culture dish, and predicting that the cell density reaches 40% -50% when the cell culture plate is transfected the next day, and placing the cell culture plate into a cell culture box.

(2) According to transfection reagent Lipofectamine ^TM 3000 Instruction manual for Reagent, different doses of Lipofectamine are used in different specifications of culture dishes ^TM 3000 Reagent, siRNA and Opti-MEM ^TM The Medium, the amount of reagent required, is planned. First, two 1.5ml centrifuge tubes were added with equal amounts of Opti-MEM ^TM Adding appropriate amount of Lipofectamine into one of the centrifuge tubes ^TM 3000 Reagent, blow and stand for 5min at room temperature; adding siRNA (specific siRNA oligo information is shown in Table 1) into another centrifuge tube, homogenizing by pipetting, and adding Opti-MEM containing transfection reagent ^TM Adding Medium solution into Opti-MEM containing siRNA or plasmid ^TM In the Medium solution, the mixture is blown evenly and kept stand for 5min at room temperature.

TABLE 1 siRNA oligo sequence

RNA extraction

(1) Taking cells: cells transfected for 48h were removed, old medium in the dish was pumped in vacuo, and the cell reject was washed with PBS and repeated twice.

(2) Lysing the cells: adding a proper amount of Buffer RLT Plus solution to cover the bottom surface of the culture dish, and vortex and shake for 15s.

(3) Removal of genomic DNA: gDNA Eliminator spin column is placed in a fresh 2ml collection tube, the cell lysate from step (2) is transferred to gDNA Eliminator spin column, centrifuged at 8000rpm for 1min, and gDNA Eliminator spin column is discarded, leaving the filtrate in the collection tube.

(4) To the filtrate, 350. Mu.l of 70% alcohol was added, the pipette was used to blow the mixture, 700. Mu.l of the mixture was added to RNeasy spin column, centrifuged at 8000rpm for 15s, and the solution was discarded, leaving RNeasy spin column. If the volume of the mixed solution is more than 700. Mu.l, the mixed solution can be transferred to the same RNeasy spin column in multiple times, and the above operation is repeated.

(5) To RNeasy spin column, 700. Mu.l of Buffer RW1 solution was added, centrifuged at 8000rpm for 15s, and the filtrate was discarded, leaving RNeasy spin column.

(6) To RNeasy spin column, 500. Mu.l Buffer RPE solution was added, centrifuged at 8000rpm for 15s, the filtrate was discarded, and RNeasy spin column was retained.

(7) To RNeasy spin column, 500. Mu.l Buffer RPE solution was added, centrifuged at 8000rpm for 2min, the filtrate was discarded, and RNeasy spin column was retained.

(8) The 2ml collection tube was replaced with RNeasy spin column and centrifuged at 12,000 rpm for 1min.

(9) RNeasy spin column was placed in an unused 1.5ml centrifuge tube, 50. Mu.l deionized water was added to RNeasy spin column to elute RNA, and the RNA in the RNeasy spin column filter membrane was first incubated at room temperature for 1min to allow sufficient dissolution in deionized water, centrifuged at full speed (14,000 rpm) for 1min, RNeasy spin column was discarded, and the filtrate was retained.

(10) RNA concentration determination: RNA concentration determination was performed using NanoDrop 2000. First, the NanoDrop 2000 software was turned on, and the concentration detection mode (nucleic acid- > RNA) was selected. Lifting a sample arm, cleaning a NanoDrop 2000 detection base by using deionized water, adding 1 μl of deionized water, putting down the sample arm for blank quantification, adding 1 μl of a sample to be detected for measuring absorbance, wiping the detection base clean by using clean dust-free paper after each sample detection is finished, and performing the same operation each time. And (5) observing the spectrum of the sample displayed by the image panel, and recording the corresponding sample concentration and absorbance.

Reverse transcription

Reverse transcription of RNA into cDNA the reverse transcription reaction system is shown in Table 2 (the whole preparation process of the reaction system is operated on ice) by means of High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor kit, and after the preparation of the reverse transcription system is completed, the procedure is set on the PCR apparatus (shown in Table 3) to complete the reverse transcription.

TABLE 2 reverse transcription reaction system

TABLE 3 reverse transcription procedure

Real-time fluorescent quantitative PCR

Real-time fluorescent quantitative PCR is a PCR amplification process that is detected in real time by fluorescent signals. In the PCR exponential amplification phase, the Ct value of the fluorescent signal is linearly related to the initial amount of the target gene, so that the target gene is quantified by using the Ct value. Primers for the target gene were designed (as shown in Table 4), a reaction system for real-time fluorescent quantitative PCR was prepared as shown in Table 5 (the reaction system was prepared by the whole process of ice-making operation), and a corresponding reaction program was run on a BIORAD CFX96 PCR instrument (as shown in Table 6).

TABLE 4 qRT-PCR primer sequences

TABLE 5 qRT-PCR reaction System

TABLE 6 qRT-PCR procedure

qRT-PCR results show that the mRNA level of Etv-siRNA-2 and Etv-siRNA-3 in osteoblasts is obviously reduced in three siRNAs targeting Etv, and the success of siRNA knockdown is determined, so Etv4-siRNA-2 and Etv4-siRNA-3 are selected for the subsequent verification experiment.

Cell proliferation assay

(1) CCK-8 detection of cell proliferation

a) Inoculating cells: cells were seeded by cell counting in 96-well plates (number of seeded cells was determined according to different cell types), 6 wells were repeated for each sample, 100 μl of final volume per well, the "8" shape was shaken to ensure uniform cell seeding, the 96-well plates were placed in a cell incubator, and transfection was performed 24 hours after seeding.

b) CCK-8 incubation: after 3 days of transfection, 96-well plate cells were taken, 10 μl of CCK-8 was added to each well (the operation should be protected from light, CCK-8 should be added below the liquid level of the cell culture medium), "8" was shaken well, incubated in a cell incubator protected from light for 1-4h (darkening of the solution was observed with naked eyes), and absorbance at a wavelength of 450nm was read using an microplate reader.

CCK-8 results show that compared with a control group, the osteoblast proliferation capacity of both the two experimental groups Etv-siRNA-2 and Etv-siRNA-3 is obviously inhibited (P < 0.01), and the difference has statistical significance.

(2) EdU labeling method for detecting cell proliferation

a) Inoculating cells: cells were seeded into 96-well plates by cell counting (number of seeded cells was determined according to different cell types), 6 wells were repeated for each sample, 100 μl of final volume per well, the "8" shape was shaken to ensure uniform cell seeding, the 96-well plates were placed in a cell incubator, transfected 24 hours after seeding, and the EdU labeling experiments were performed 3 days after transfection.

b) EdU-labeled cells: a2 XEdU working solution was prepared in a cell culture medium, 100. Mu.l of 2 XEdU working solution was added to each well of a 96-well plate, the final volume was 200. Mu.l, the final concentration of EdU working solution was 1X, and the cells were incubated in a cell incubator for a suitable period of time (MC 3T3-E1 incubation for 4 hours). The green fluorescent label is used herein.

c) Cell fixation and infiltration promotion:

after the incubation of the EdU working solution was completed, the old medium was aspirated, 50. Mu.l of 4% paraformaldehyde was added to each well, and the solution was discarded after fixation at room temperature for 15 min. Adding 50 mu l of 2mg/ml glycine solution into each hole to neutralize paraformaldehyde, incubating for 20min at room temperature, and discarding the solution; washing twice with 3% BSA in PBS, and discarding; adding 0.5% TritonX-100 in PBS to promote permeation, incubating at room temperature for 20min, and discarding the solution; the 3% BSA in PBS was washed twice and discarded.

d) EdU detection

Adding 100 μl of Click-iT reaction mixture into each well, placing on a shaking table, incubating for 30min at room temperature under dark conditions, and discarding the solution; the 3% BSA in PBS was washed twice and discarded.

e) DNA counterstaining

Adding 100 μl PBS into each well to clean the cells, and discarding the liquid; adding 100 mu l of Hoechst solution with the concentration of 5 mu g/ml, and incubating for 20min at room temperature in a dark place; and adding PBS for washing twice, and discarding the liquid.

f) Imaging and analysis

Using a fluorescence microscope to observe Hoechst marks under ultraviolet light, observing EdU green marks under blue fluorescence, observing the same view under the same focal plane by ultraviolet light and blue fluorescence, processing pictures by using imageJ, and calculating the ratio of EdU/Hoechst in the same view.

The EdU detection result shows that compared with the control group, the number of the nuclei marked by EdU of the two experimental groups Etv-siRNA-2 and Etv-siRNA-3 is obviously reduced, and further proves that the proliferation of osteoblasts is obviously inhibited, and the difference has statistical significance.

The experiments are all carried out for 3 independent experiments, the display results are based on 3 representative independent experiments, the results are expressed by mean and standard deviation, the average comparison between two groups is tested by t test, the comparison between the groups is analyzed by one-way analysis of variance (ANOVA), and the statistical difference is formed by taking P < 0.05.

Etv4 (ETS variable 4) is a transcription factor newly found in ETS family, and is closely related to proliferation and metastasis of cells. According to the invention, the Etv gene is specifically knocked down in the osteoblast by the siRNA technology, the influence of the Etv gene on the proliferation of the osteoblast is observed, and the result shows that the proliferation of the osteoblast is obviously inhibited compared with a control group after the Etv gene is knocked down, and the method has statistical significance.

The effect of the Etv gene on osteoblast proliferation capacity was verified by two cell proliferation assay methods, CCK-8 and EdU. The CCK-8 and EdU results show that after Etv gene knockdown, the proliferation of osteoblasts is obviously inhibited, and the results have statistical significance, and the Etv gene plays a promoting role in the proliferation process of osteoblasts.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> university of Beijing aviation aerospace

<120> use of a Gene to promote bone formation

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1461

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atggagcgga ggatgaaagg cggatacttg gaccagcgag tgccctacac cttctgcagc 60

aaatctcccg gaaatgggag cttgggcgaa gcgctgatgg tcccgcaggg aaagctcatg 120

gacccgggct ccctgccgcc ttccgactca gaagatctct tccaggacct cagtcacttc 180

caagagacgt ggctcgcaga agctcaggta ccggacagtg atgagcagtt tgttcctgat 240

ttccattcag aaaacttagc tttccatagc cccaccacca ggatcaagaa ggaaccccag 300

agtccccgca cagaccccgc cctgtcctgc agcaggaagc caccactccc ctaccaccat 360

ggagagcagt gcctttactc cagtgcctat gactccccca gacaaatcgc catcaagtcc 420

cccgcccccg gtgcccctgg acagtcgccc ctgcagccct tttccagggc agaacagcag 480

cagagcctcc tgagagcctc cagctcttcc cagtcccacc ctggccacgg gtaccttggt 540

gagcacagct ccgtcttcca gcagcccgtg gacatgtgcc actccttcac atctcctcag 600

ggagggggcc gggaacctct cccagccccc tatcaacacc aactgtcgga gccctgccca 660

ccctaccccc agcagaactt caagcaggag taccatgacc ccctgtacga acaggctggc 720

cagcccgctt caagccaggg tggggtcagt gggcacaggt acccaggggc gggggtggtg 780

atcaaacagg agcgcacaga cttcgcctac gactcagatg tccctggatg tgcatcaatg 840

tacctccacc cagagggctt ctctggaccc tctccaggtg atggagtgat gggttatggc 900

tatgaaaaat cccttcgacc attcccagat gatgtctgca ttgtccctga aaaatttgaa 960

ggagacatca agcaggaagg gattggagct ttccgggagg ggccacccta ccagcgccgg 1020

ggtgccttac aactgtggca gtttctggtg gccctgctgg atgacccaac aaatgctcat 1080

ttcattgctt ggacaggccg gggaatggag tttaaactaa ttgaacctga agaggttgcc 1140

aggctctggg gtatccagaa gaaccggcca gccatgaatt atgacaagct gagccgctcg 1200

ctgcgatact attatgagaa aggcatcatg cagaaggtgg ctggcgaacg ctacgtgtac 1260

aagtttgtgt gcgagccgga ggccctgttc tctctggcct tcccagataa tcaacgtcca 1320

gctctgaagg ctgagtttga ccggccagtc agtgaggagg acacagtccc tttgtcccac 1380

ttggatgaga gtcctgccta cctcccagaa ctcactggcc ccgctccgcc cttcggccac 1440

agaggtggat attcttacta g 1461

<210> 2

<211> 2352

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ggaggccaag gcaaaggaaa tgcaccaatc agctgctccc ccgggctcac aactgtctgc 60

tgcgcccgga aaacaagccg gggctctggg gacccggggc tcaggccgcc tcgctccggc 120

ctagccccgc caccttagtt gtgtcatccc ccgggcatgc tgagcatccc cccgcggctc 180

cggcacagac gcccggacct caggtctctg cctccgcgcg ggggcccggc cctgtggccg 240

gagggagcgg ccggatggag cggaggatga aaggcggata cttggaccag cgagtgccct 300

acaccttctg cagcaaatct cccggaaatg ggagcttggg cgaagcgctg atggtcccgc 360

agggaaagct catggacccg ggctccctgc cgccttccga ctcagaagat ctcttccagg 420

acctcagtca cttccaagag acgtggctcg cagaagctca ggtaccggac agtgatgagc 480

agtttgttcc tgatttccat tcagaaaact tagctttcca tagccccacc accaggatca 540

agaaggaacc ccagagtccc cgcacagacc ccgccctgtc ctgcagcagg aagccaccac 600

tcccctacca ccatggagag cagtgccttt actccagtgc ctatgactcc cccagacaaa 660

tcgccatcaa gtcccccgcc cccggtgccc ctggacagtc gcccctgcag cccttttcca 720

gggcagaaca gcagcagagc ctcctgagag cctccagctc ttcccagtcc caccctggcc 780

acgggtacct tggtgagcac agctccgtct tccagcagcc cgtggacatg tgccactcct 840

tcacatctcc tcagggaggg ggccgggaac ctctcccagc cccctatcaa caccaactgt 900

cggagccctg cccaccctac ccccagcaga acttcaagca ggagtaccat gaccccctgt 960

acgaacaggc tggccagccc gcttcaagcc agggtggggt cagtgggcac aggtacccag 1020

gggcgggggt ggtgatcaaa caggagcgca cagacttcgc ctacgactca gatgtccctg 1080

gatgtgcatc aatgtacctc cacccagagg gcttctctgg accctctcca ggtgatggag 1140

tgatgggtta tggctatgaa aaatcccttc gaccattccc agatgatgtc tgcattgtcc 1200

ctgaaaaatt tgaaggagac atcaagcagg aagggattgg agctttccgg gaggggccac 1260

cctaccagcg ccggggtgcc ttacaactgt ggcagtttct ggtggccctg ctggatgacc 1320

caacaaatgc tcatttcatt gcttggacag gccggggaat ggagtttaaa ctaattgaac 1380

ctgaagaggt tgccaggctc tggggtatcc agaagaaccg gccagccatg aattatgaca 1440

agctgagccg ctcgctgcga tactattatg agaaaggcat catgcagaag gtggctggcg 1500

aacgctacgt gtacaagttt gtgtgcgagc cggaggccct gttctctctg gccttcccag 1560

ataatcaacg tccagctctg aaggctgagt ttgaccggcc agtcagtgag gaggacacag 1620

tccctttgtc ccacttggat gagagtcctg cctacctccc agaactcact ggccccgctc 1680

cgcccttcgg ccacagaggt ggatattctt actaggcacc agtggcttcc ccttgacatg 1740

gtggggttgc tcagtgtata tatcaactga tttggtattg gtgaaggccc tctttctgat 1800

gcctgtagaa gtctctgggg tcagagctcc actatcccat ctgatactcc tggccagact 1860

cagctgctaa ccagagtctg cgggaaagac agtggaggca ggccaaatct aaaggcagta 1920

gccgaagttc gctgtggctc acctgtacct tcagttcagc ttggcctctg cctaggtctt 1980

gctcagaggc caagttcctc acccccacca cagagatcca gtgttctatt ctggggacat 2040

acagggactt cccttgttta ttatggcaac agggccaagg ggattctcag aacaccctgt 2100

gtctcccctc tcccaacccc ccatgggaga caaagttctg cctgggttct gccctgaaca 2160

ggggggtcct gtgttcttgg tgctgtgctc tgggaggcag gagcatgtgg gcggcagctg 2220

gggggggggg ttgggtggaa gagtagggat ggctctctgc cctaggccta cccaggccta 2280

attccacctt tgcctcttat gccagacctt aataaagcct ctgcttctcc ggtaaaaaaa 2340

aaaaaaaaaa aa 2352

<210> 3

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggaccucagu cacuuccaat t 21

<210> 4

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

uuggaaguga cugaggucct t 21

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gcucgcugcg auacuauuat t 21

<210> 6

<211> 22

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

uaauaguauc ugcagcgagc tt 22

Claims (3)

1. Use of an siRNA for the preparation of a formulation for promoting bone formation, wherein the siRNA has the sequence:

Etv4-siRNA-2：

5’GGACCUCAGUCACUUCCAAdTdT 3’，SEQ ID NO:3；

3’dTdTCCUGGAGUCAGUGAAGGUU 5’，SEQ ID NO:4；

or (b)

Etv4-siRNA-3：

5’GCUCGCUGCGAUACUAUUAdTdT 3’，SEQ ID NO:5；

3’dTdTCGAGCGACGCUAUGAUAAU 5’，SEQ ID NO:6；

One or more of the following.

2. The use according to claim 1, wherein the osteoblast is a mouse MC3T3-E1 cell.

3. The use according to claim 1, wherein the osteoblast proliferation potency is enhanced.