CN101638653A - RNA interfering target of osteoclast proton pump sub-gene a3 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecule biology and biomedical science, and relates to targets in an a3 sequence for RNA interference, siRNA obtained in various ways for the targets and application thereof in preparing new medicines for treating osteoporosis. A slow virus is used for infecting osteoclast and expressing shRNA, and a method of osteoclast function detection after invitro gene is knocked down is also used; thus, three targets for RNA interference in an a3 gene are screened, and the No.2 target is preferred. ShRNA for the No.2 target can obviously inhibit the expression of a3 protein, stably inhibit the capabilities of extracellular acidification and bone resorption of the osteoclast, and does not affect the formation of filiform actin rings of the mature osteoclast. Based the target sequence, biological medicines for treating osteoporosis can be prepared.

Description

RNA disturbance target point and the application thereof of osteoclast proton pump subunit a3

Technical field

The invention belongs to molecular biology and biological medicine technology field, relate on the osteoclast proton pump subunit a3 sequence and be used for RNA interferential disturbance target point, and at the siRNA molecule that obtains in every way of these target spots with and application in the medicine of preparation treatment osteoporosis.

Background technology

Osteoporosis is sickness rate height, mortality ratio height, the big modal bone metabolism disease of health care cost consumption.According to estimates, osteoporotic fracture can take place in the women more than half and about 1/3rd the male sex in its lifetime.Fracture brings great misery for people's life, and the lighter descends people's quality of life, and weight person will cause paralysis (as Fracture of femur) and cause a lot of complication thus even cause death.The generation of these diseases is relevant with the overacfivity of osteoclast (Osteoclast).Osteoclast and scleroblast (Osteoblast) are two class important cells in the bone, are responsible for bone resorption and bone forming respectively, and balance has between the two guaranteed the constant of adult animals and human bone amount; Yet when the activity of osteoclast was active greater than scleroblast, osteoclast, caused bone density to descend and osteoporosis finally occurs greater than osteoblastic bone forming effect the absorption of bone, must suppress the activity of osteoclast for these treatment of diseases.

Osteoclast V-ATP enzyme is responsible for the extracellular acidization of osteoclast, makes the bone demineralization and provides acidic micro-environment for proteasome degradation bone organic component.A3 is the special subunit that osteoclast V-ATP enzyme has, and this subunit in 1996 found first by the contriver and successful clone, and in contriver in 1999 with the a3 gene knockout, in mouse, observed the effect of this gene in the osteoclast bone resorption.The result shows, all a3 sudden change homozygotes (/-) the mouse growth retardation, the long bone growth failure has appearred, and the osteosclerosis of disappearance is built and reproduced to bone, and tooth disintegrates, the about 4 week death in age in birth back; A3-/-mouse shortage medullary space, the osteogenesis face enlarges, and extend in the calcified cartilage zone; A3-/-osteoclast of type mouse attached on the bone and number normal, but can not form the bone resorption lacuna.The a3-of vitro culture/-mouse osteoclast shortage extracellular acidifying function, can not make the bone demineralization.These results show that the a3 gene is the element of the special proton pump of osteoclast.Therefore, a3-/-serious osteosclerosis that mouse occurs is because osteoclast has lost extracellular acidifying function.A3 only expresses in osteoclast, a3-/-proton pump of mouse kidney is unaffected, illustrates that a3 is the gene of the single-minded expression of osteoclast.Therefore, a3 can be used as the activity of the special inhibition osteoclast of most promising drug target.The present invention's design and external slow virus are expressed the bone resorption activity that a3-shRNA is used to suppress osteoclast, to reach the purpose for the treatment of bone photo related disorders such as osteoporosis.

Summary of the invention

One object of the present invention is to provide the mRNA disturbance target point of osteoclast proton pump subunit a3, designs artificial sequence at this target spot, and this sequence is expressed the acquisition siRNA molecule in expression vector, and makes up the recombinant slow virus expression vector.Therefore purpose of the present invention also is to provide the siRNA molecule at a3, comprise the expression vector of this siRNA molecule and by the slow virus expression vector of its conversion, in addition, express to produce by expression vector, be also contained among the present invention as the bob clamping structure RNA molecule of siRNA molecule precursor.

Another object of the present invention is to provide the application of RNA disturbance target point in preparation treatment medicine for treating osteoporosis of a3.

According to an aspect of the present invention, according to osteoclast proton pump subunit a3 mRNA sequence, the segmental dna sequence dna of expressed double-chain small disturbance RNA (SiRNA) of external synthetic inhibition a3 genetic expression, and be cloned on the expression plasmid of bob clamping structure RNA (ShRNA), express and screen osteoclast is absorbed the stronger a3-SiRNA of restraining effect, determine the RNA interfered target sequence, SEQ ID NO.1～3 are the RNA interfered target sequence at a3, and preferred RNA disturbance target point has the nucleotide sequence shown in SEQ ID NO.2.SEQ ID NO.4～5 are the dna sequence dna that is used to express SiRNA at the design of interfered target sequence 1, SEQ ID NO.6～7th is at the dna sequence dna that is used to express SiRNA of interfered target sequence 2 designs, SEQ ID NO.8～9th, at the dna sequence dna that is used to express SiRNA of interfered target sequence 3 designs, every pair of sequence is complementary sequence.Concrete sequence sees Table 1 and table 2.

The SiRNA molecule is a double stranded rna molecule, and a strand contains the RNA target sequence and at 3 ' terminal UU dinucleotide tail (shown in SEQ ID NO.10), another strand is a complementary strand, and dinucleotide tail UU is positioned at 3 ' end equally.SiRNA enters behind the cell directly jamming target expression of gene.

Table 1 a3 RNAi target sequence

The synthetic dna sequence dna that is used to express siRNA of table 2

RNA target sequence SEQ ID NO.2 of the present invention is for having 19nt length, and after forming the SiRNA molecule, 3 ' end adds that UU dinucleotide tail formation has the double-stranded RNA of 21bp respectively and brings into play function.It will be understood by those skilled in the art that, the SiRNA sequence that increases or reduce several bases can have same function in gene silencing, for example the SiRNA sequence of 21～23bp has been proved to be and has had said function in the silence of other genes (Nature 2001, (411): 24,494-98).Be understandable that equally, also can design longer sequence, play a role by when bringing into play function, being cut into short RNA sequence.

After the gene order of external synthetic inhibition a3 genetic expression is cloned into expression vector, express ShRNA, ShRNA contains antisense sequences, the bob clamping structure sequence (sequence is shown in SEQ ID NO.11) of interfered target sequence, interfered target sequence.The bob clamping structure sequence preference that forms the ShRNA sequence adopts the loop ring sequence UUCAAGAGA of nine bases, also can use TTCG tetraloop (LeukemiaResearch 30 (2006) 1013-1017).Bob clamping structure RNA, express generation by expression vector, need the interior Dicer enzyme of cell to be cut into the siRNA molecule during performance function and disturb expression of gene, Fig. 1 shows by composition sequence and is cloned into expression vector, produce ShRNA, be reprocessed into the process of the SiRNA with function.

According to a further aspect in the invention, provide the application of RNA disturbance target point in the preparation medicine for treating osteoporosis of a3 gene, the siRNA molecule of slow virus mediation can suppress the bone resorption activity of osteoclast, is used for the treatment of bone photo related disorders such as osteoporosis.

The technique means that realizes the object of the invention is:

The first, at the external synthetic dna sequence dna that suppresses the double-stranded SiRNA of a3 genetic expression, and be cloned on the shRNA expression plasmid;

The second, screen the stronger a3-SiRNA of osteoclast bone resorption restraining effect with the detection method of extracellular acidifying function and osteocomma absorptive function.

The 3rd, illustrate the mechanism that a3 strikes low back osteoclastic bone receptivity defective by the osteoclast functional experiment.

(1) a3-SiRNA's is synthetic

1.SiRNA design

Adopt the specific target sequence of Dharmacon siDESIGN center (http://www.dharmacon.com) design, choose 3 of specific target sequences, and target sequence is similar to people's respective target sequence 90% at a3mRNA.

2, the synthetic and purifying of strand SiRNA

By Invitrogen company synthesizing and purifying

3, the preparation of double-stranded SiRNA and expression ShRNA clone's structure

Article two, complementary strand SiRNA handles through the heating after annealing and forms double-stranded SiRNA, and double-stranded siRNA is cloned on the plasmid of expressing ShRNA.

(2) screen the stronger a3-SiRNA of osteoclast bone resorption restraining effect with the detection method of extracellular acidifying function and osteocomma absorptive function

The extracellular acidifying function that the in-vitro screening employing is widely used and the detection method of osteocomma absorptive function: normal osteoclast has extracellular acidifying and bone resorption function, osteoclast proton pump with the extracellular acidifying after acridine orange dyeing be orange, the osteocomma after being absorbed by osteoclast can be observed a large amount of navy blue bone resorption lacunas after toluidine blue dyeing.The slow virus that in the former foster osteoclast of being commissioned to train, adds various expression a3-shRNA respectively, the extracellular souring ability of acridine orange dyeing observation of cell after 3-5 days, and lacuna number and form on the osteocomma are observed in toluidine blue dyeing; According to bone resorption lacuna number and form, select bone resorption suppress effect preferably a3-SiRNA be used for other functional experiment of osteoclast.

The present invention at drug target a3 at first find and clone by the applicant, it is the necessary gene of osteoclastic bone absorptive function.Though a3 is acknowledged as the osteoporotic very promising drug target of treatment, but also there be not the osteoclast inhibitor of report so far at a3, so the selected SiRNA target sequence of the present invention is the new drug target at a3, and similar to people's respective target position sequence 90%.

Existing nearly 20 years of the appearance of the RNAi technology that the present invention utilizes, but the experimental result of utilizing the RNAi technology to obtain often is published on the very high periodical of influence power; This has also illustrated the importance of RNAi in fundamental research and application.Along with people to the understanding of RNAi principle and the expansion of its range of application, this technology has broken through the scope of fundamental research, strides forward towards application.The initial research in disease treatment field shows, by this technology of expression of virus-mediated ShRNA expression inhibiting Disease-causing gene because the advantage of himself will become the new growth point of medicinal design.And slow virus expression ShRNA itself has a lot of advantages than adenovirus and retrovirus, and slow virus can infect division or Unseparated Cell and terminally differentiated cells, and retrovirus can only infect somatoblast, and adenovirus can infect division or Unseparated Cell.Therefore, with respect to traditional new drug development, the RNAi technology of slow virus mediation has many good qualities: with strong points, at the gene of curing the disease; Specificity is good; Need not to know proteic three-dimensional structure; Need not to screen a large amount of candidate compounds; It is a system of defense treatment disease of utilizing Mammals self, does not find side effect so far; Long action time, it plays a role by being incorporated into to express in the cellular genome, and body has the mechanism of RNAi amplification.

The expression with slow virus mediate rna i technology inhibition disease gene of initiative of the present invention, we can say to have very strong perspectively, the technology of expressing the slow virus infection medullary cell of special a3 ShRNA can be expected to apply to develop slow virus type biotechnological formulation with the treatment osteoporosis.Simultaneously, the present invention will provide brand-new thinking for pharmacy industry.

Description of drawings

Fig. 1 is the structure of pSUPER-SiRNA plasmid; Show the structure iron after forming at the SiRNA molecule of target sequence 2 and ShRNA molecule;

Fig. 2 is the structure of PLB-SiRNA carrier, with the H1-SiRNA sequence clone on the pSUPER-siRNA plasmid to the pLB plasmid and replace the U6 promotor;

Fig. 3 is the expression vector PLB-SiRNA carrier structure figure that contains SiRNA;

Fig. 4 is retroviral packing;

Fig. 5 strikes low efficient for Wetern blotting detects Lenti-a3;

Fig. 6 strikes differentiation and the sophisticated influence of low back to osteoclast for detecting a3;

Fig. 7 strikes the influence of low back to acidifying of osteoclast extracellular and bone resorption for detecting a3;

Fig. 8 strikes the influence that low back forms the actin filament ring for detecting a3.

Embodiment

Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.

Use various substratum and reagent to be commercially available purchase if no special instructions among the embodiment.

Embodiment one: former generation osteoclast cultivation

With reference to Yang, S.and Li, Y.P. (2007) J.Bone Miner.Res., 22,45-54 is former be commissioned to train nourishing the bone marrow mononuclearcell and induce it to be divided into mature osteoclast.6-8 C57BL6 mouse in age in week draws neck to put to death, and behind the routine disinfection, gets its hind leg femur and shin bone and puts into containing of ice precooling of antibiotic RIMP-1640 training liquid.The connective tissue of blood vessels of bone surface is separated totally with tweezers with scissors, the antibiotic RIMP-1640 training liquid that contains of ice precooling washes.Scissors cuts off femur and shin bone two ends, with asepsis injector (1ml) medullary cell is blown in the α that the contains 10%FBS-MEM training liquid of ice precooling.Syringe needle is inhaled repeatedly and is washed into single cell suspension.4 ℃ centrifugal, and (1100rpm * 6min) is with containing M-CSF (10ng/ml) and RANKL (10ng/ml) (R﹠amp; D sysyems) α-MEM+10%FBS training liquid re-suspended cell.Cell is inoculated in 24 orifice plates with 1-2 * 105/ hole, is inoculated in 6 orifice plates with 1 * 106/ hole.Cell culture medium is α-MEM+10%FBS+10ng/ml RANKL+10ng/ml M-CSF.Cell is cultivated the sophisticated osteoclast that obtained in 4-6 days under 37 ℃, 5%CO2 condition.

Embodiment two: antibody is prepared

Synthetic polypeptide a3 ⁸¹⁶CFYSGTGYKLSPFTFTVDSD ⁸³⁴Be coupled to KLH, immune male new zealand white rabbit obtains the polyclonal antibody of anti-a3, and the monoclonal antibody of anti-GAPDH is available from cellsignaling company.

Embodiment three: screen effective SiRNA sequence

Adopt the little interference siRNA of Dharmacon siDESIGN center (http://www.dharmacon.com) design, select three special target sequences at Atp6v0a3 mRNA

a3siRNA1：5’-AGATGAAGGCAGTGTACCT-3’(SEQ?ID?NO.1)；

a3siRNA2：5’-CTCGGCGTTTCATCTGTGG-3’(SEQ?ID?NO.2)；

a3siRNA3：5’-ACGGACTGCTCATGTTTCT-3’(SEQ?ID?NO.3)。

Negative control is special target sequence si-LacZ:5 '-CTCGGCGTTTCATCTGTGG-3 ' at the LacZ gene mRNA.The target sequence of the bob clamping structure of chemosynthesis is connected to pSUPER carrier (OligoEngine) and goes up between the BglII/HindIII site after the annealing renaturation.It is by at human embryonic kidney cell line 293T (HEK293T, ATCC No.CRL-11268 that the efficient that low a3 expresses of striking bob clamping structure RNA (shRNA) detects ^TM) in co-expression plasmid pFlag-CMV-4-a3 (Flag-a3) and plasmid pSUPER-siRNA finish.That is: the clone there are shRNA expression plasmid and the Flag-a3 plasmid co-transfection HEK293T cell of siRNA with lipofectamine reagent (Invitrogen company), after the transfection 48 hours, collecting cell (lysate: 50mM Tris-HCl, pH6.8,100mMdithiothreitol, 2%SDS, 0.001%bromphenol blue, 10%glycerol), carry out protein blot experiment (western blotting).

Embodiment four: the packing retrovirus

First day (9-10am): 293T cell inoculation, every 10cm culture dish inoculation 2-2.5 * 10 ⁶The 293T cell; Second day (9-10am): transfection, prepare calcium-phosphoric acid precipitates (1ml/10cm plate)

Transfer?vector(PLB，Addgene)-20μg

Packaging?plasmid(dR8.2，Addgene)-15μg

Envelope?plasmid(vsvg，Addgene)-6μg

Add 2.5M CaCL2 50ul, add ddH2O to 500 μ l, mixing;

Add 500 μ l, 2 * HBS, the piping and druming mixing;

Incubated at room 20 minutes will precipitate and dropwise be added in the cultured cells mixing;

After the transfection 6-8 hour, renew bright training liquid, 6ml/plate;

The 4th day (9-10am): collect virus

Collect culture supernatant

3000rpm/5min/RT is centrifugal

Supernatant 0.45 μ m filters, packing, direct infection cell or frozen in-80 ℃

Embodiment five: the retrovirus titer determination

The 1st day (9-12am): at each hole inoculation 3 * 104 293T cell of 24 orifice plates, 1ml training liquid is cultivated; The 2nd day (4-6pm): the cell (should 6-8 * 104) in a hole of counting, the virus infected cell of 4-6 times of serial dilution adds 4 μ g/ μ l polybrene.The 3rd day (9-12am): add 1ml training liquid; The 5th day observation of cell fluorescent protein expression, and with the cell proportion of facs analysis express fluorescent protein, and calculate titre.

Embodiment six: the retroviral infection osteoclast

BMNC was induced 48 hours in the presence of M-CSF (10ng/ml) and RANKL (10ng/ml), then in the presence of polybrene (4 μ g/ml) with packaged retroviral infection 8 hours.Renewing bright training liquid α-MEM+10%FBS+10ng/ml RANKL+10ng/ml M-CSF continues to cultivate 4-6 days.Collect osteoclast albumen (lysate: 50mM Tris-HCl, pH6.8,100mM dithiothreitol, 2%SDS, 0.001%bromphenol blue 10%glycerol), carries out protein blot experiment (western blotting) and is used to detect a3 and strikes low efficient.

By the cell green-emitting fluorescence of virus infection, under fluorescent microscope, observed fluorescigenic cell in 2 days behind the virus infection, the result shows: the equal green-emitting fluorescence of nearly all cell (Fig. 5) behind the virus infection.Western-blotting Western blot result shows, the a3 expression level of the osteoclast that infects with lenti-LacZ is compared, and Lenti-a3 (target sequence is a3siRNA2) infects that the expression of a3 significantly reduces by 86% (Fig. 5) behind the osteoclast.The protein expression level that can significantly suppress a3 behind the slow virus infection osteoclast of expressing special shRNA at a3 is described.

Embodiment seven: TRAP dyeing

Fixedly the TRAP active coloring test kit with Sigma dyes behind the osteoclast, and the sophisticated osteoclast of precursor osteoclast box (more than three nuclears) presents scarlet, and counts under light microscopic.Every group of ten visuals field are counted, and data are represented (n=10) with mean ± standard deviation.

The painted result of TRAP shows: similar to the cell after lenti-LacZ handles, the cell after Lenti-a3 handles can differentiation and maturation be a multinucleated osteoclast also, and the two shows similar multinucleated osteoclast number and forms per-cent (Fig. 6).Illustrating that a3 strikes does not influence the osteoclast that the BMNC differentiation and maturation is a plurality of nuclears after low.

Embodiment eight: acridine orange dyeing

BMNC is seeded in 24 orifice plates, in the presence of M-CSF (10ng/ml) and RANKL (10ng/ml), induced 48 hours, then in the presence of polybrene (4 μ g/ml) with packaged retroviral infection 8 hours, renew bright training liquid α-MEM+10%FBS+10ng/mlRANKL+10ng/ml M-CSF and continue to cultivate 4 days.Osteoclast in the α-MEM that contains 5 μ g/ml acridine oranges 37 ℃ hatched 15 minutes, wash 10minutes with α-MEM, observation of cell under fluorescent microscope (exciting light 490nm and absorb light 525nm).

The acridine orange coloration result shows: compare with the osteoclast that lenti-LacZ infects, the multinucleated osteoclast cell orange dyeing that Lenti-a3 infects obviously reduces, and the extracellular acidifying obviously is subjected to suppress (Fig. 7).Illustrate that a3 strikes the extracellular acidifying function of hanging down the back osteoclast and obviously descends.

Embodiment nine: the osteoclastic bone absorptive function is analyzed

The analysis of osteoclast bone resorption activity is finished by the formation of analyzing absorption lacuna.BMNC is seeded on the ivory osteocomma that is positioned in 96 orifice bores, in the presence of M-CSF (10ng/ml) and RANKL (10ng/ml), induced 48 hours, then in the presence of polybrene (4 μ g/ml) with packaged retroviral infection 8 hours, renew bright training liquid α-MEM+10%FBS+10ng/ml RANKL+10ng/ml M-CSF and continue to cultivate 6 days.Osteocomma ultrasonic cell of going out in the solution of 1M NH4OH after 6 days.Acellular osteocomma was through 1%toluidine blue (1%sodium borate) dyeing 1 minute.Absorption lacuna is mazarine, and observes under light microscopic, and absorption area accounts for the per-cent of total visual field area under the count random visual field, several three visuals field of every batch total, and data are represented (n=3) with mean ± standard deviation.

The bone resorption experimental result shows: form the percentage ratio that bone resorption lacuna (lacuna is a mazarine) area accounts for random observation visual field area with osteoclast that lenti-LacZ infects and compare, the percentage ratio that the absorption lacuna area that the multinucleated osteoclast that Lenti-a3 infects forms on the ivory osteocomma accounts for random observation visual field area obviously reduce ( ^*P＜0.05, n=3), and the absorption lacuna more shallow (Fig. 8) that on the ivory osteocomma, forms of the multinucleated osteoclast that infects of Lenti-a3.Illustrate that a3 strikes low back osteoclastic bone absorptive function and obviously descends.

Embodiment ten: the dyeing of cell actin filament ring

BMNC is seeded in 24 orifice plates, in the presence of M-CSF (10ng/ml) and RANKL (10ng/ml), induced 48 hours, then in the presence of polybrene (4 μ g/ml) with packaged retroviral infection 8 hours, renew bright training liquid α-MEM+10%FBS+10ng/mlRANKL+10ng/ml M-CSF and continue to cultivate 4 days.Cell is fixed 10 minutes in 3.7% Paraformaldehyde 96; The saturating film of 0.2%Triton X-100 was handled 10 minutes; With sealing under 1% lowlenthal serum and the 3%BSA room temperature 1 hour; With hatching 30 minutes under 2U/ml rhodamine phalloidin (Molecular Probes) room temperature; 1 μ g/ml DAPI (Sigma) dyes nuclear and is blue; Cell is observed under fluorescent microscope.

Cell actin filament ring coloration result shows: to form the actin filament ring similar to the mature osteoclast of expressing special LacZ siRNA, the actin filament ring that the mature osteoclast of expressing special a3 siRNA also can formation rule.Show that a3 strikes the low formation that does not influence mature osteoclast bone actin filament ring.

SEQUENCE?LISTING

＜110〉Saier Biological Medical Research Co., Ltd., Zhejiang

＜120〉RNA disturbance target point and the application thereof of osteoclast proton pump subunit a3

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Claims (10)

1, the RNA disturbance target point of a kind of osteoclast proton pump subunit a3 is characterized in that it has the nucleotide sequence shown in SEQID NO.2.

2, a kind of siRNA molecule, it is a duplex molecule, wherein a strand contains the target sequence of RNA shown in the claim 1 and at 3 ' terminal UU dinucleotide tail, another strand is a complementary strand.

3, a kind of expression vector, it contains the siRNA molecule of claim 2.

4, the described expression vector of claim 3 is characterized in that it has structure shown in Figure 3.

5, a kind of recombined lentivirus vector contains the siRNA molecule of claim 2.

6, the described recombined lentivirus vector of claim 5 is characterized in that described lentiviral vectors transforms with the described expression vector of claim 4.

7, the application of the RNA disturbance target point of the described osteoclast proton pump subunit of claim 1 a3 in preparation treatment medicine for treating osteoporosis.

8, the described application of claim 7 is characterized in that with the described lentiviral vectors of claim 5 as the osteoclast absorption inhibitor.

9, a kind of bob clamping structure RNA molecule, it contains the antisense sequences of the described RNA interfered target sequence of claim 1, bob clamping structure sequence and RNA interfered target sequence at least.

10, the described bob clamping structure of claim 9 RNA molecule is characterized in that further having UU dinucleotide tail at 3 ' end.

Images