CN104189921A

CN104189921A - Application of Linc-RAM for treating muscle diseases

Info

Publication number: CN104189921A
Application number: CN201410446245.7A
Authority: CN
Inventors: 朱大海; 于潇华; 张勇; 李婷婷
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS
Priority date: 2014-09-03
Filing date: 2014-09-03
Publication date: 2014-12-10

Abstract

The invention relates to application of Linc-RAM for treating muscle diseases. Specifically, the invention relates to application of Linc-RAM in preparation of medicines for treating muscle diseases, particularly muscle injury and rhabdomyosarcoma. Furthermore, the invention relates to application of the Linc-RAM in preparation of medicines for promoting differentiation of muscle stem cells, as well as reagents and medicines for promoting transition of fibroblast to myoblast.

Description

The purposes of Linc-RAM in treatment muscle disease

Technical field

Linc-RAM is inventor's first identified and its function and molecular mechanism is studied, and the present invention relates to Linc-RAM in the effect for the treatment of muscle disease, particularly muscle injury and rhabdomyosarcoma.

Background technology

Skeletal muscle is the histoorgan of human body maximum, and 40% of about percentage of liveweight plays very important effect in the vital movement of organism.The function that it is not only brought into play motion and supports, it is also an important metabolism organ simultaneously.The sugar that health absorbs and lipid material be metabolism produce power in skeletal muscle mostly, and this plays very important effect to maintaining the poised state of whole health.Being one and in order and the process of strict regulation and control being not only subject to the regulation and control of various muscle specific transcription factor of skeletal muscle is subject to the finely regulating that ncRNAs and chromatin such as modified and reinvented at the epigenetic simultaneously.

In numerous muscle diseases, as periodic paralysis, muscular dystrophy, myositis etc. all exist muscle injury in various degree, current medicine is less, side effect is larger, and effect is poor.As duchenne muscular dystrophy (DMD), be that a kind of x-linked recessive inheritance is sick, have another name called for false loose type muscular dystrophy, be the most serious muscular dystrophy of symptom.Because causing muscle cell, gene mutation defect can not normally produce a kind of protein that is called dystrophin (Dystrophin), can make calcium ion infiltrate cell, cause waterfall reaction, cause patient's whole-body muscle unable, again because lacking dystrophin in muscle cell, cause cell tissue meat fiber to become unable and fragile, its myocyte is constantly in injury repairing process, after long-term stretching, extension, this disappearance muscle cell tissue will produce the factors such as mechanicalness injury and destroy, and finally cause muscle cell dead.In average every 3500 the newborn boy babies in the whole world, just have a people to suffer from this disease, patient will constantly degenerate and occur muscle weakness or atrophy because of skeletal muscle preschool, causes inconvenience walking.Probably in the time of 7 years old to 12 years old, can thoroughly lose locomotor activity, conventionally will the death because of cardiac muscle, lung myasthenia by more than 20 year old.For this disease, medical circle there is no effective therapy at present.

In recent years, adult stem cell is implanted in the Mice Body of DMD muscle disease, obtain remarkable therapeutic effect, referring to Benchaouir R, Meregalli M, Farini A, D'Antona G, Belicchi M, Goyenvalle A, Battistelli M, Bresolin N, Bottinelli R, Garcia L, Torrente Y.Cell Stem Cell.Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cellsinto dystrophic mice.2007, 1 (6): 646-57.But Myoblast Transplantation for Repair exists rejection and the poor limitation such as cells survival rate, Partridge TA.Cells that participate in regeneration of skeletal muscle.Gene Ther.2002,9 (11): 752-3.Utilize gene therapy that Dystroglycan (Dystrophin) is imported in patient body, make it to express Dystroglycan, concrete enforcement is very difficult, mainly because of carrying DNA fragmentation greatly and they will being placed in to large genomic correct position, and foreign DNA needs to be translated as destination protein after first transcript mRNA again, and this process can be subject to adjusting and the impact of various mechanism in body.Thereby search out the nucleic acid drug that is directly used in gene therapy and more easily treat or alleviate the effect of disease.

In addition, rhabdomyosarcoma (rhabdomyosarcoma) is a kind of malignant tumor occurring from embryo's mesenchymal tissue, its cell is similar to sarcoplast (myoblast), most human rhabdomyosarcoma cells is expressed Myostatin Gene (myogenic differentiation 1, MyoD), yet human rhabdomyosarcoma cells does not but possess from propagation to the ability that end breaks up eventually, thereby infinite multiplication, form malignant tumor.Rhabdomyosarcoma accounts for 15% of Children with Solid Tumors, 50% of soft tissue sarcoma.The difference of the multiformity of clinical manifestation, the multiplicity of pathological change and site of pathological change, becomes in tumors in children rhabdomyosarcoma the most complicated a kind of.Rhabdomyosarcoma is divided into following 4 kinds of hypotypes: embryo type, acinus type, Fructus Vitis viniferae bunch type and pleomorphic type.Early stage radical surgery method comprises amputation, pelvis and orbital tumor enucleation.In the past few decades, the measure that adopts chemotherapy, radiotherapy to combine with operation for the different happening parts of tumor and the scope of expansion thereof, patient's survival rate has had raising significantly, because the treatment of rhabdomyosarcoma has characteristic, it is the object that the topical therapeutic of surgery and radiation does not often reach long term survival, only have application general chemotherapy could obviously improve survival rate, the chemotherapy on human body of this general produces harm greatly.

Therefore, find and suppress human rhabdomyosarcoma cells propagation, and reduce the study hotspot that the medicine of patient body injury is become to current treatment of cancer, at present, have been reported and show that anti-miR-122 has got permission to enter clinical treatment hepatocarcinoma in the U.S., points out increasing nucleic acid drug to enter the probability of clinical practice, and nucleic acid drug is easy to use because of himself, easy a large amount of production, the features such as high specificity, for the selection of clinical application provides potential.

Long-chain non-coding RNA (long non-coding RNA, lncRNA) be extensively present in various organisms, referring to Stefani G, Slack FJ.Small non-coding RNAs in animal development.Nat Rev Mol Cell Biol.2008,9 (3): 219-30.Most by rna plymerase ii, transcribed and through alternative splicing.LncRNA is the RNA molecule that a class transcript length surpasses 200nt, they are encoding proteins not, but with the form of RNA in multiple aspect, as epigenetic regulation, referring to Mazo A, Hodgson JW, Petruk S, Sedkov Y, Brock HW.Transcriptional interference:an unexpected layer of complexity in gene regulation.J Cell Sci.2007,120 (Pt 16): 2755-61; Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, et al.Antisense transcription in the mammalian transcriptome.Science, 2005,309 (5740): 1564-6.The expression of the controlling gene such as transcriptional control and post-transcriptional control.

Nearest research shows, some lncRNAs participate in transcription factor important in regulation and control skeletal development processes and the expression of signaling molecule, thus propagation and the differentiation of regulation and control Skeletal Muscle Cell.As, Myostain processes after mice, the expression of Malat1 in gastrocnemius significantly lowered, further the up-regulated at C2C12 cell and primary muscle cells differentiation phase Malat1 mRNA is found in research, knock out after Malat1, cell proliferation is suppressed, referring to Watts R, Johnsen VL, Shearer J, Hittel DS Myostatin-induced inhibition of the long noncoding RNA Malat1 is associated with decreased myogenesis.Am J Physiol Cell Physiol.2013, 304 (10): C995-1001.It is the SRAP hypotype of non-coding (SRA ncRNA) and coding that steroid receptor RNA activates son (SRA) transcribed, SRAncRNA can enhances skeletal myocyte break up, after SRA ncRNA is combined with MyoD simultaneously, the non-Skeletal Muscle Cell transdifferentiation that can strengthen MyoD mediation is Skeletal Muscle Cell, but this effect can be suppressed by SRAP, further research is found, the inhibitory action of SRAP need to be by being achieved with the interaction of SRA ncRNA, referring to Caretti G, Schiltz RL, Dilworth FJ, Di Padova M, Zhao P, Ogryzko V, Fuller-Pace FV, Hoffman EP, Tapscott SJ, Sartorelli V.The RNA helicases p68/p72and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.Dev Cell.2006, 11 (4): 547-60, Hub é F, Velasco G, Rollin J, Furling D, Francastel C.Steroid receptor RNA activator protein binds to and counteracts SRA RNA-mediated activation of MyoD and muscle differentiation.Nucleic Acids Res.2011,39 (2): 513-25.

At present, in the Mechanism Study for lncRNAs regulation and control skeletal development, there is researcher to find that lncRNAs can regulate the expression of miRNAs or interact with miRNAs, thereby further realize the regulation and control to skeletal development.As, YY1 is is just regulating and controlling the expression of the lincRNA--Yam-1 that skeletal muscle is relevant, and Yam-1 is by regulating the expression inhibiting skeletal muscle of miR-715 to generate, referring to Lu L, Sun K, Chen X, Zhao Y, Wang L, Zhou L, Sun H, Wang H.Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis.EMBO J.2013,32 (19): 2575-88.The Skeletal Muscle Cell that H19 knocks out and mice skeletal differentiation of stem cells are suppressed, further research is found, two miRNA of h19 gene first exons coding miR-675-3p and miR-675-5p, both are along with differentiation is induced to express gradually, the two can make up because H19 knocks out caused Skeletal Muscle Cell differentiation and injury repairing and suppress, referring to Dey BK, Pfeifer K, Dutta A.The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration.Genes Dev.2014 Mar 1, 28 (5): 491-501.Long-chain non-coding RNA--the linc-MD1 that Cesana etc. find that by analyzing the genome structure of miR-206 and miR-133b Skeletal Muscle Cell is specific expressed and raise along with Skeletal Muscle Cell expression of differentiation, the genome sequence of linc-MD1 gene and miR-206 and miR-133b is overlapping, Mechanism Study is found, linc-MD1 passes through in conjunction with miR-133 and miR-135, competitive miR-133 and the miR-135 of regulating is combined with its target gene MAML1 and MEF2C, thereby regulation and control Skeletal Muscle Cell differentiation, referring to Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A, Bozzoni I.A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA.Cell.2011Oct 14, 147 (2): 358-69.Prompting, miRNAs, may be to inquiring into the more aobvious shortcut of mechanism of action of lncRNAs the bridge between lncRNAs and target gene.

Equally, the unconventionality expression of some lncRNAs molecules can be detected in muscle disease, prompting lncRNAs participates in the physiological and pathological process of muscle disease.DMD gene locus identifies a series of lncRNAs, point out these lncRNAs may participate in muscular dystrophy, referring to Bovolenta M, Erriquez D, Valli E, Brioschi S, Scotton C, Neri M, Falzarano MS, Gherardi S, Fabris M, Rimessi P, Gualandi F, Perini G, Ferlini A.The DMD locus harbours multiple long non-coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms.PLoS One.2012, 7 (9): e45328.Linc-MD1 is down-regulated expression in Du Shi myocyte, referring to Cesana M, Cacchiarelli D, Legnini I, Santini T, Sthandier O, Chinappi M, Tramontano A, Bozzoni I.A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA.Cell.2011 Oct 14; 147 (2): 358-69.FSHD (FSHD) is autosomal dominant inheritance, AD myonosus, there is the disappearance of the D4Z4 repetitive sequence of 4q35 in most of patient, healthy experimenter D4Z4 raises PcG albumen and suppresses 4q35 gene, FSHD patient is due to the disappearance of D4Z4, the silence effect of PcG complex reduces, cause producing the non-coding RNA that chromatin is relevant--DBE-T, DBE-T raises Ash1L albumen to FSHD site, make H3K36 demethylation, chromatin remodeling, cause the genetic transcription in 4q35 region to activate, referring to Cabianca DS, Casa V, Bodega B, Xynos A, Ginelli E, Tanaka Y, Gabellini D.A long ncRNA links copy number variation to a polycomb/trithorax epigenetic switch in FSHD muscular dystrophy.Cell.2012, 149 (4): 819-31, prompting DBE-T participates in developing of FSHD.

These result of study promptings lncRNAs participates in the regulation and control of Skeletal Muscle Cell propagation, differentiation and skeletal development, and plays a significant role in the pathology generating process of muscle disease.MyoD is important transcription factor during flesh generates, it regulates a series of fleshes such as Myogenin to generate the expression of related gene, there is defect in the mouse muscle injury repairing that MyoD knocks out, referring to Yablonka-Reuveni Z, Rudnicki MA, Rivera AJ, Primig M, Anderson JE, Natanson P.The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD.Dev Biol.1999,210 (2): 440-55; And ectopic expression MyoD can change fibroblast into sarcoplast, referring to Tapscott SJ, Davis RL, Thayer MJ, Cheng PF, Weintraub H, Lassar AB.MyoD1:a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts.Science.1988,242 (4877): 405-11.Thereby find be subject to MyoD regulation and control and in Skeletal Muscle Cell specifically expressing lncRNAs may flesh generate and the pathology generating process of muscle disease in play an important role.

Summary of the invention

The inventor is subject to long-chain non-coding RNA--the 2310015B20Rik of MyoD regulation and control by one of series of experiments first identified, according to RACE experimental result, its 5 ' end increases by four base sequences (CAGT), then total length becomes 447bp, because of its specifically expressing in Skeletal Muscle Cell, its expression raises gradually in the primary myoblastic atomization of mice skeletal growth, C2C12 cell and separation, and in genome between the gene of two encoding proteins, called after Linc-RAM (linc-RNA Activator of Myogenesis).The inventor finds first, Linc-RAM is regulated and controled by transcription factor MyoD, significantly promote Skeletal Muscle Cell differentiation, in the acute muscle injury repairing model of CTX induction, significantly promote Skeletal muscle injury to repair process, Linc-RAM separately or to assist MyoD be sarcoplast by fibroblast transdifferentiation.By the Linc-RAM of mice ectopic expression in embryonal rhabdomyosarcoma cell line RD cell, Linc-RAM significantly suppresses RD cell proliferation, and promotes differentiation.And people-Linc-RAM is down-regulated expression in mankind's muscular dystrophy disease, prompting may play a role in muscle disease.

Study on Molecular Mechanism finds that Linc-RAM regulates the expression of muscle-derived gene as co-activation of MyoD, further research finds that Linc-RAM is by being combined with MyoD, the formation of mediation MyoD-Baf60c-Brg1 complex, thereby make chromatin remodeling, regulate the expression of downstream muscle-derived gene, and this effect is that p38a relies on.Molecular signal conduction studies finds that bFGF regulates the expression of Linc-RAM by RAS/Raf/Mek/Erk1/2/MyoD signal path.

Our research prompting Linc-RAM promotes Skeletal Muscle Cell differentiation, and independent or assistance MyoD is sarcoplast by fibroblast transdifferentiation, promotes RD cell differentiation, in the treatment of mankind's muscle disease, has latent effect.

Therefore, the invention provides the purposes of a kind of long-chain non-coding RNA molecule in the medicine of preparation treatment muscle disease, described long-chain non-coding RNA molecule is selected from Linc-RAM.

In a preferred embodiment, described muscle disease is selected from muscle injury or rhabdomyosarcoma.

In another preferred embodiment, described muscle injury is acute muscle injury.

The present invention provides the purposes of a kind of long-chain non-coding RNA molecule in the medicine of the differentiation of preparation promotion muscle stem cell, fibroblast transdifferentiation, the differentiation of promotion human rhabdomyosarcoma cells on the other hand, and described long-chain non-coding RNA molecule is selected from Linc-RAM.

The carrier that the present invention further provides a kind of nucleotide that contains the Linc-RAM that encodes is used for the treatment of muscle disease, particularly muscle injury and rhabdomyosarcoma purposes.Preferably, described muscle injury is acute muscle injury.

Term " muscle injury " refers in the contusion of direct external force or the muscle that indirectly causes under the effect of external force, pulls etc., and acute muscle injury refers to the acute injury of muscle.

Accompanying drawing explanation

Figure 1A shows the expression of Linc-RAM in different tissues of mice; Figure 1B shows that Linc-RAM is at the mice embryonic expression of 11.5 days; Fig. 1 C shows the expression of Linc-RAM in mice skeletal different developmental phases; Fig. 1 D shows the expression of Linc-RAM in C2C12 cell differentiation procedure; Fig. 1 E shows that Linc-RAM is at the myoblastic expression of mouse primary; Fig. 1 F shows that ChIP-PCR detects MyoD and has binding site in the promoter region of Linc-RAM; Fig. 1 G shows that luciferase reporter gene detects MyoD and by the E-Box in conjunction with Linc-RAM promoter region, regulates the expression of Linc-RAM; 1H shows that, in the fibroblast of ectopic expression MyoD, Linc-RAM is induced expression; Expression in the mice skeletal that Fig. 1 I demonstration Linc-RAM knocks out at MyoD; Fig. 1 J shows the distribution of Linc-RAM in Cytoplasm and nucleus; Fig. 1 K shows that FISH detects the distribution of Linc-RAM in Cytoplasm and nucleus.

Fig. 2 A demonstration utilizes slow-virus transfection C2C12 cell construction Linc-RAM to strike low cell line, and real-time PCR detects the low multiple that strikes of Linc-RAM; Fig. 2 B shows that C2C12-NC and C2C12-Linc-RAM strike low cell differentiation MHC (green) immunofluorescence dyeing result after 60 hours, DAPI dyeing (redness) positioning cells core; Fig. 2 C shows the statistical result of MHC positive cell quantity; Fig. 2 D shows the testing result that C2C12-NC and C2C12-Linc-RAM strike low cell differentiation MHC protein level after 60 hours; Fig. 2 E shows the cell line of utilizing slow-virus transfection C2C12 cell construction Linc-RAM high expressed, and real-time PCR detects the high expressed multiple of Linc-RAM; The cell differentiation of Fig. 2 F demonstration C2C12-NC and C2C12-Linc-RAM high expressed MHC (green) immunofluorescence dyeing result after 60 hours, DAPI dyeing (redness) positioning cells core; Fig. 2 G shows the statistical result of MHC positive cell quantity; Fig. 2 H shows the testing result of cell differentiation MHC protein level after 60 hours of C2C12-NC and C2C12-Linc-RAM high expressed; Fig. 2 I demonstration utilizes slow-virus transfection C2C12 cell construction MyoD to strike low cell line, and real-time PCR detects MyoD mRNA and strikes low multiple; Fig. 2 J shows that Linc-RAM high-expression plasmid transfection MyoD strikes after low C2C12 cell differentiation 24h, Myogenin (green) immunofluorescence dyeing result, DAPI dyeing (redness) positioning cells core; Fig. 2 K shows the statistical result of Myogenin positive cell quantity; Fig. 2 L shows that Linc-RAM high-expression plasmid transfection MyoD strikes after low C2C12 cell differentiation 24h, detects the expression of Myogenin mRNA.

Fig. 3 A-1 and Fig. 3 A-2 show respectively the cell line of utilizing slow-virus transfection C3H10T1/2 cell construction Linc-RAM high expressed and MyoD high expressed, and real-time PCR detects the high expressed multiple of Linc-RAM and MyoD; The C3H10T1/2 cell differentiation of Fig. 3 B demonstration Linc-RAM high expressed and MyoD high expressed MHC (green) immunofluorescence dyeing result after 3 days, DAPI dyeing (blueness) positioning cells core; The C3H10T1/2 cell differentiation of Fig. 3 C-1 to Fig. 3 C-3 demonstration real-time PCR detection MyoD high expressed Linc-RAM after 3 days, the expression of MyoG and MHC mRNA; The C3H10T1/2 cell differentiation of Fig. 3 D-1 to Fig. 3 D-3 demonstration real-time PCR detection Linc-RAM high expressed Linc-RAM after 3 days, the expression of MyoG and MHC mRNA; The C3H10T1/2 cell that Fig. 3 E shows MyoD high expressed respectively high expressed and strike low Linc-RAM after break up 48 hours, the expression of MHC mRNA; The C3H10T1/2 cell that Fig. 3 F shows MyoD high expressed respectively high expressed and strike low Linc-RAM after break up 48 hours, MHC immunofluorescence dyeing result; Fig. 3 G shows that striking low Linc-RAM broke up after 48 hours, the statistical result of MHC positive cell quantity; Fig. 3 H shows that striking low Linc-RAM breaks up the positive multinuclear of MHC (>2) cell quantity statistical result after 48 hours; Fig. 3 I shows the C3H10T1/2 cell collect in 48 hours Linc-RAM of proliferation period and differentiation and MyoD high expressed, the gene that RNA-seq checkout discrepancy is expressed, and Heat map shows the gene of differential expression (fold>2); Fig. 3 J shows that RT-PCR is detected as fibrocyte pedigree and the expression of sarcoplast pedigree related gene in the C3H10T1/2 cell differentiation procedure of Linc-RAM high expressed and MyoD high expressed changes.

Fig. 4 A-1 and Fig. 4 A-2 show that the CTX damage tibialis anterior electricity of 24 hours turns after Linc-RAM high-expression plasmid, and RT-PCR detects the expression of Linc-RAM; Fig. 4 B shows that the tibialis anterior electricity of CTX damage turns Linc-RAM high-expression plasmid 5.5 and after 7.5 days, tibialis anterior cross section HE coloration result; Fig. 4 C shows 7.5 days tibialis anterior muscle fiber cross-sectional area statistics of CTX damage; Fig. 4 D-1 to Fig. 4 D-3 shows CTX damage 3.5 days, and real-time PCR detects MyoD, the expression of MyoG and MHC mRNA.

Fig. 5 A shows the cell line of utilizing slow-virus transfection RD cell construction mice-Linc-RAM high expressed, and RT-PCR detects the high expressed multiple of Linc-RAM; The RD cell differentiation of Fig. 5 B demonstration Linc-RAM high expressed MHC (green) immunofluorescence dyeing result after 72 hours, DAPI dyeing (redness) positioning cells core; Fig. 5 C shows the statistical result of MHC positive cell quantity; Fig. 5 D-1 and Fig. 5 D-2 show the expression of RD cell differentiation that real-timePCR detects Linc-RAM high expressed MyoD and MyoG mRNA after 3 days; Fig. 5 E shows according to mice Linc-RAM the position in genome, by the homology range of linearity, analyzes human-Linc-RAM in human genome, comprises two kinds of variants; In the sample of Fig. 5 F-1 to Fig. 5 F-4 demonstration mankind muscular dystrophy, detect the expression of human-Linc-RAM, and the expression of MyoD and MyoG mRNA, wherein human-Linc-RAM comprises two variants, A represents normal skeletal muscle tissue, and B represents muscular dystrophy tissue, 3,5,6,7,8 represent the patient of 5 muscular dystrophy.

Fig. 6 A shows Linc-RAM high expressed and strikes low C2C12 cell differentiation 12 hours, the gene that RNA-seq checkout discrepancy is expressed, and Heat map shows the gene of differential expression (fold>2); Fig. 6 B shows the result of GO gene annotation; Fig. 6 C shows MyoD and the common gene regulating of Linc-RAM; Fig. 6 D shows MyoD and the common adjusting of Linc-RAM and muscle cell multiplication, breaks up the gene relevant with structure; Fig. 6 E demonstration RT-PCR further determines the common gene regulating of MyoD and Linc-RAM at Linc-RAM high expressed and strikes the expression variation of low C2C12 cell differentiation after 12 hours; Fig. 6 F shows various dose (0,0.6ug, Linc-RAM high-expression plasmid 1.0ug) and 0.4ug MyoD high-expression plasmid cotransfection C3H10T1/2 cell, luciferase reporter gene detects MyoG promoter (MyoG genetic transcription initiation site upstream-470bp～+ 103bp) activity; Fig. 6 G shows that C3H10T1/2 cell difference transfection MyoD and the differentiation of Linc-RAM high-expression plasmid are after 48 hours, and real-time PCR detects the expression of MyoG; Fig. 6 H-1 to Fig. 6 H-3 shows formaldehyde fixed L inc-RAM high expressed and strikes low C2C12 cell, after ultrasonication, add respectively MyoD, H3K4Me3 and Pol II antibody carry out the experiment of chromatin co-immunoprecipitation, and real-time detects respectively these three kinds of albumen at the enrichment degree of MyoG promoter region; Fig. 6 I-1 to Fig. 6 I-3 shows formaldehyde fixed L inc-RAM high expressed and strikes low C2C12 cell, after ultrasonication, use respectively MyoD, H3K4Me3 and Pol II antibody staining matter co-immunoprecipitation, real-time detects respectively these three kinds of albumen at the enrichment degree of miR-206 (far-end) promoter region.

Fig. 7 A shows the C2C12 cell pyrolysis liquid that extracts RNase-free, utilizes the co-precipitation of MyoD antibody mediated immunity, and Western Blot detects MyoD protein level, and RT-PCR detects Linc-RAM level; Fig. 7 B shows the C2C12 cell pyrolysis liquid that extracts RNase-free, utilizes respectively MyoD, Baf60c and the co-precipitation of Brg1 antibody mediated immunity, and RT-PCR detects Linc-RAM level; Fig. 7 C-1 and Fig. 7 C-2 show that real-time PCR detects Linc-RAM high expressed and strikes the expression of Baf60c and Brg1 mRNA in low C2C12 cell; Fig. 7 D demonstration is extracted respectively Linc-RAM high expressed and strikes low C2C12 cell RNase-free lysate, utilizes the co-precipitation of MyoD antibody mediated immunity, and Western Blot detects MyoD, Baf60c and Brg1 protein level; Fig. 7 E-1 to Fig. 7 E-4 shows formaldehyde fixed L inc-RAM high expressed and strikes low C2C12 cell, after ultrasonication, use respectively MyoD, Baf60c, Brg1 and H3K9ac antibody staining matter co-immunoprecipitation, real-time PCR detects respectively MyoD, Baf60c, Brg1 and H3K9ac albumen are at the enrichment degree of MyoG promoter region; The C2C12 cell differentiation of Fig. 7 F demonstration 10uM SB203580 processing Linc-RAM high expressed 36 hours, MHC (green) immunofluorescence dyeing result, DAPI dyeing (blueness) positioning cells core; Fig. 7 G shows the statistical result of MHC positive cell quantity; The C2C12 cell differentiation of Fig. 7 H demonstration 10uM SB203580 processing Linc-RAM high expressed 36 hours, the testing result of MHC mRNA level; The C2C12 cell differentiation of Fig. 7 I demonstration 10uM SB203580 processing Linc-RAM high expressed 18 hours, the expression of results of Linc-RAM; The C2C12 cell differentiation of Fig. 7 J demonstration 10uM SB203580 processing Linc-RAM high expressed 18 hours, the C2C12 cell of formaldehyde fixed L inc-RAM high expressed, after the fragmentation of excusing from death sound, use Brg1 antibody staining matter co-immunoprecipitation, real-time detects respectively Brg1 at the enrichment degree of MyoG promoter region.

Fig. 8 A1 to Fig. 8 A3 shows that 10ng/mL bFGF processes C2C12 cell differentiation and after 6,12,24 hours, detects the expression of Linc-RAM; Fig. 8 B shows C2C12 cell transfection Linc-RAM wild type and saltant type promoter respectively, be Linc-RAM-WT (390bp～+ 80bp) and Linc-RAM-Mutant (87 and-88 bit base sudden change), 10ng/mL bFGF processes C2C12 cell differentiation 24 hours, luciferase reporter gene detects the activity of Linc-RAM promoter, and MyoD (1kb) and MyoG (GBBS) promoter are as positive control; Fig. 8 C shows that 10uM PD98059 pretreatment C2C12 cell detects p-Erk, t-Erk, p-MEK, the expression of t-MEK albumen for 24 hours with bFGF processing C2C12 cell differentiation after 1 hour again; Fig. 8 D-1 to Fig. 8 D-3 shows that PD98059 pretreatment C2C12 cell detects MyoD, the expression of Linc-RAM and MyoG for 24 hours with bFGF processing C2C12 cell differentiation after 1 hour again; Fig. 8 E demonstration 10uM PD98059 pretreatment C2C12 cell is processed C2C12 cell differentiation 24 hours with bFGF after 1 hour again, and luciferase reporter gene detects the activity of Linc-RAM-WT and Linc-RAM-Mutant promoter; Fig. 8 F-1 to Fig. 8 F-3 demonstration 5uM FTA pretreatment C2C12 cell is processed C2C12 cell differentiation 24 hours with bFGF after 30 minutes again, detects MyoD, the expression of Linc-RAM and MyoG; Fig. 8 G-1 to Fig. 8 G-3 shows that 2.5uM GW pretreatment C2C12 cell after 24 hours, detects MyoD, the expression of Linc-RAM and MyoG with bFGF processing C2C12 cell differentiation after 30 minutes again; Fig. 8 H demonstration bFGF processing C2C12-NC and C2C12-Linc-RAM-OE cell differentiation be Myogenin and MHC (green) immunofluorescence dyeing result after 24,36 hours, DAPI dyeing (redness) positioning cells core; Fig. 8 I shows the statistical result of Myogenin positive cell quantity; Fig. 8 J shows the statistical result of MHC positive cell quantity.

The specific embodiment

Below in conjunction with specific embodiment, describe the present invention in detail, these embodiment do not limit the present invention in any way for illustrating the present invention.

Linc-RAM used in the present invention is obtained by bioinformatic analysis, and RT-PCR detects the expression of Linc-RAM.

Embodiment 1 find one be subject to MyoD regulation and control and in Skeletal Muscle Cell the long-chain non-coding RNA of specifically expressing, called after Linc-RAM

According to two sets of data storehouses in publishing an article, referring to Cao Y, Yao Z, Sarkar D, Lawrence M, Sanchez GJ, Parker MH, MacQuarrie KL, Davison J, Morgan MT, Ruzzo WL, Gentleman RC, Tapscott SJ.Genome-wide MyoD binding in skeletal muscle cells:a potential for broad cellular reprogramming.Dev Cell.2010,18 (4): 662-74, Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.2010, 28 (5): 511-5, utilize bioinformatic analysis to obtain the lncRNAs of a series of MyoD of being subject to regulation and control, RT-PCR detects the expression map of these lncRNAs in flesh generative process, testing result is found the lncRNA--2310015B20Rik of a specifically expressing in skeletal muscle, in body early embryo (E11.5) body segment, its expression also detected simultaneously, 2310015B20Rik is positioned at No. 10 chromosomes of mice, between ccdc6 and two encoding egg white genes of slc16a9, 2310015B20Rik all has expression in caryoplasm, and conservative is lower, the expression of 2310015B20Rik is along with flesh generative process raises gradually, called after Linc-RAM.

By Northern Blot, detect the distribution of Linc-RAM in each tissue of mice; By in situ hybridization, detect Linc-RAM at brephic expression.According to routine operation, carry out separation and the cultivation of the primary sarcoplast of skeletal muscle (primary myoblasts); Extract total RNA in C2C12 cell and skeletal muscle tissue, RT-PCR detects the expression of Linc-RAM.

Result: as shown in Figure 1A to 1E, Linc-RAM is specifically expressing in Skeletal Muscle Cell, mice embryonic detects Linc-RAM for 11.5 days and expresses.Its expression is at skeletal development, in C2C12 cell differentiation and primary sarcoplast, along with flesh generative process raises gradually.Results suggest Linc-RAM plays a role in myocyte's atomization.

MyoD regulates Linc-RAM to express at transcriptional level

ChIP-PCR detects MyoD and regulates Linc-RAM to express at transcriptional level

The many groups of binding site (E-Box) design primer according to the MyoD of prediction at Linc-RAM promoter region, RT-PCR detects the promoter region whether MyoD is combined in Linc-RAM.

Result: as shown in Fig. 1 F, MyoD has binding site at the promoter region of Linc-RAM, results suggest MyoD regulates the expression of Linc-RAM.

In addition, the inventor is inserted into pGL3-Basic carrier by the promoter region of Linc-RAM (390bp～+ 80bp), and the MyoD binding site (E-Box) of Linc-RAM promoter region is carried out being inserted into pGL3-Basic carrier after point mutation (87 and-88 bit base sudden change), luciferase reporter gene experimental result (as Fig. 1 G) shows that MyoD regulates Linc-RAM to transcribe by being combined in the E-Box of Linc-RAM promoter region.In the fibroblast of dystopy transfection MyoD high-expression plasmid, the expression (as Fig. 1 H) of MyoD induction Linc-RAM.In the mice skeletal that MyoD knocks out, (as Fig. 1 I) significantly lowered in the expression of Linc-RAM.All results show that MyoD regulates the expression of Linc-RAM above.

Detect the distribution of Linc-RAM in cell

According to the step of caryoplasm separating kit (Ambion company), extract respectively Cytoplasm and cell nRNA, RT-PCR detects the distribution of Linc-RAM in cell.FISH technology further detects the distribution of Linc-RAM in cell.

Result: as shown in Fig. 1 J and 1K, Linc-RAM all has expression in caryoplasm, and in Cytoplasm expression more than nucleus.Results suggest Linc-RAM may bring into play different effects in Cytoplasm and nucleus.

The effect of embodiment 2 Linc-RAM to the differentiation of skeletal muscle stem Cells system

Build Linc-RAM high expressed and strike low cell line

Utilize BLOCK-It Lentiviral RNAi Expression System (Invitrogen) to build sh-Linc-RAM and strike low plasmid, with viral packaging plasmid pVSVG, △ 08.91 coinfection 293T cell, collect respectively 24 hours and 48 hours supernatants, 1100g removes cell debris for centrifugal 3.5 minutes, 0.45um filter filters, the centrifugal 2.4h of 24000rpm/min, 100ul PBS lytic virus granule, infect C2C12 (purchased from ATCC) cell after 48 hours, blasticidin S (Invitrogen) screening obtains Linc-RAM (sh-Linc-RAM) and strikes to cell line.Same method obtains control plasmid cellular control unit system.

The pCMV promoter expression region that is pEGFP-N1 by the pENTRTM/U6 Entry Construct pU6 promoter expression regional replacement in BLOCK-It Lentiviral RNAi Expression System, obtain plasmid called after pvirus (obtaining from teacher Wu Zhenguo of Hong Kong University of Science and Thchnology), Linc-RAM fragment is inserted to this plasmid, after the restructuring of pLenti6/BLOCK-iT-DEST plasmid, packaging virus, infects the C2C12 cell line that C2C12 cell obtains Linc-RAM high expressed (Linc-RAM OE).

Real-time RT-PCR detects Linc-RAM and expresses

Result: as shown in Fig. 2 A and 2E, Linc-RAM is respectively by high expressed with strike lowly in high expressed and the cell line that knocks out, and results suggest cell line successfully constructs.

Immunofluorescence detects the impact of Linc-RAM on C2C12 cell differentiation

Respectively by 1.2 * 10 ⁴individual Linc-RAM high expressed and strike low C2C12 cell culture (DMEM culture medium in 12 orifice plates, comprise 10% hyclone, 1% penicillin, 1% streptomycin, 1% glutamine), cultivate 24h and change division culture medium (DMEM culture medium, comprise 2% horse serum, 1% penicillin, 1% streptomycin, 1% glutamine), after 60 hours, collect cell.

Result: as shown in Fig. 2 B, 2C, 2F, 2G, during differentiation 60h, the cell number of the MHC positive of Linc-RAM high expressed group is than the remarkable increase of matched group, and Linc-RAM strikes the cell number of the MHC positive of low group than the remarkable minimizing of matched group, results suggest Linc-RAM significantly promotes C2C12 cell differentiation.(MHC is C2C12 cell differentiation marker gene).

Western Blot detects protein expression

Result: Western Blot result shows (as Fig. 2 D, 2H), breaks up after 60 hours, and the MHC expressing quantity of Linc-RAM high expressed group is than the remarkable increase of matched group, and Linc-RAM strikes the MHC expressing quantity of low group than the remarkable minimizing of matched group.Results suggest Linc-RAM significantly promotes C2C12 cell differentiation.

Linc-RAM can partly make up because MyoD strikes the low C2C12 cell differentiation defect causing

BLOCK-It Lentiviral RNAi Expression System, builds MyoD and strikes low C2C12 cell line as described in Example 1.MyoD strikes low C2C12 cell transfecting Linc-RAM high-expression plasmid, detects cell differentiation situation.

Result: real-time PCR result shows that MyoD mrna expression amount significantly reduces (as Fig. 2 I), break up after 24 hours, transfection the MyoD of Linc-RAM high-expression plasmid strike low C2C12 cell, MyoG positive cell number significantly strikes low group (as Fig. 2 J more than MyoD, 2K), the expression of MyoG mRNA also significantly knocks out group (as Fig. 2 L) more than MyoD, and results suggest Linc-RAM can partly make up by MyoD and strike the low C2C12 cell differentiation defect causing.

Embodiment 3 Linc-RAM change fibroblast into myoblastic effect

BLOCK-It Lentiviral RNAi Expression System as described in Example 1, builds the C3H10T1/2 cell line of MyoD high expressed, is labeled as MyoD OE C3H10T1/2 cell.Utilize Lenti-XTM Tet-on 3G Inducible Expression System (Clontech Laboratories) to build the C3H10T1/2 cell line of Linc-RAM high expressed, be labeled as pLVX-Linc-RAM OE C3H10T1/2 cell, build its control plasmid simultaneously and be labeled as pLVX-TRE C3H10T1/2 cell.Wherein C3H10T1/2 cell line is purchased from Chinese Academy of Medical Sciences's cell centre.

Linc-RAM changes fibroblast into sarcoplast

As embodiment 2, respectively by 1.5 * 10 ⁴individual pLVX-Linc-RAM OE, pLVX-TRE and MyoD OE C3H10T1/2 cell culture are in 12 orifice plates, cultivation 12h changes division culture medium, and (DMEM culture medium, comprises 10ug/mL insulin, 5ug/mL transferrins, 1% penicillin, 1% streptomycin, 1% glutamine), after differentiation 72h, collect respectively cell, immunofluorescence, the specific experiment operations such as real-time RT-PCR are as embodiment 2.By pLVX-Linc-RAM OE, pLVX-TRE and MyoD OE C3H10T1/2 cell culture are in 10 centimetres of culture plates, break up after 48 hours, collect total RNA, send in Bei Rui and Kanggong department, carry out after RNA-seq order-checking, the gene of bioinformatic analysis differential expression, RT-PCR is detected as the expression of fibrocyte and sarcoplast pedigree related gene.

Result: as shown in Fig. 3 A-1 and 3A-2, the C3H10T1/2 of Linc-RAM and MyoD high expressed system all successfully constructs.Break up after three days, in pLVX-Linc-RAM OE and MyoD OE C3H10T1/2 cell, have the cell of the MHC positive, and in MyoD OE C3H10T1/2 cell the cell of the MHC positive more than the C3H10T1/2 cell (as Fig. 3 B) of Linc-RAM high expressed; MyoG in the C3H10T1/2 cell of real-time RT-PCR result demonstration Linc-RAM high expressed, MHC mrna expression amount lower than MyoD OE C3H10T1/2 cell (as Fig. 3 C-1 to 3C-3,3D-1 to 3D-3), results suggest Linc-RAM has and changes fibroblast into myoblastic ability, but this ability is lower than MyoD.As shown in Fig. 3 I, heap map diagram RNA-seq result, shows with respect to matched group, the fibroblast of pLVX-Linc-RAM OE is raised in 1247 gene expressions of proliferation period, 613 down regulation of gene expression; Break up after 48 hours, 1265 gene expressions are raised, 968 down regulation of gene expression; Shown in Fig. 3 J, RT-PCR is further detected as the expression of fibrocyte and sarcoplast pedigree related gene.

Embodiment 4 Linc-RAM assist MyoD to change fibroblast into sarcoplast

Linc-RAM assists MyoD to change fibroblast into sarcoplast

By 1.2 * 10 ⁴individual MyoD OE C3H10T1/2 cell culture is in 12 orifice plates, the plasmid of difference transient transfection Linc-RAM high expressed and shRNA after 12h, (DMEM culture medium, comprises 10ug/mL insulin, 5ug/mL transferrins after 24h, to change division culture medium, 1% penicillin, 1% streptomycin, 1% glutamine), after differentiation 48h, collect respectively cell, immunofluorescence, the specific experiment operations such as Western Blot and real-time RT-PCR are as embodiment 2.Wherein insulin is purchased from Ruo Henuo moral company, and transferrins is purchased from Sigma company.

Result: as Fig. 3 E, 3F, 3G, shown in 3H, in the MyoD OE C3H10T1/2 cell of the plasmid of transfection Linc-RAM high expressed, MHC mRNA and expressing quantity are significantly more than matched group, and the apocyte of the MHC positive is significantly more than matched group; And in the MyoD OE C3H10T1/2 cell of transfection Linc-RAM shRNA plasmid, MHC mRNA and expressing quantity are significantly less than matched group, and the cell of the MHC positive is significantly less than matched group, results suggest Linc-RAM assists MyoD to change fibroblast into sarcoplast.

The effect of embodiment 5 Linc-RAM to muscle injury regeneration

1, animal is processed

From dimension tonneau China company, buy the male C57BL/6 mice about 8 week age, ((being diluted to final concentration is 10 μ M to be dissolved in PBS for CTX, cardiotoxin to the left lower limb of its hind leg and right lower limb tibialis anterior, to inject 20 microlitre snake venom cytotoxins; Sigma) cause muscle acute injury-Regeneration and Repair model, after 22 hours, inject 20ul 5U hyaluronic acid, after two hours, lower limb tibialis anterior is injected 20ul 25ug Linc-RAM high-expression plasmid (pvirus-Linc-RAM) to the right again, left lower limb injection control plasmid as a control group, reject the hair of mice shank, with electrode (BTX, 10mm) 15v/mm voltage, 8 pulses, 20 milliseconds of electric shock tibialis anterior place muscle of each pulse.

2, muscular tissue is collected

Muscle injury 3.5,5.5 and the neck that breaks afterwards for 7.5 days are put to death mice, with shears, hind leg left and right two lower limb furs are cut off, and find calf shank, tear the sarolemma on lateral of tibia tibialis anterior surface with blunt tweezers, from tendon, cut off and take off monoblock tibialis anterior.

3, paraffin embedding and haematoxylin and Yihong (HE) dyeing

1) tibialis anterior taking off is fixed on to 24-48 hour in 10% formalin (formalin 1:10 is dissolved in PBS (preparation as mentioned above));

2) fix by 70%, 80%, 95%, 100% ethanol and respectively soak 1 hour to dehydration completely;

3) after dehydration, be transferred to dimethylbenzene transparent twice each half an hour;

4) in the saturating wax of 50-60 degree and embedding (the full-automatic embedding machine of Leica EG1150);

5) finishing wax stone sample, section 3-4 micron (Leica RM2255 fully-automatic rotation microtome);

6) section is separated, put into and in the tepidarium of 40 degree, open up sheet (Leica HI1220 stand sheet machine) bak stay and, to microscope slide, upper 60 ℃ of boiling hot plate (Leica HI1210 bakes sheet machine), dry;

7) tissue slice is placed in to dimethylbenzene and soaks 10 minutes, after replacing dimethylbenzene, soak again 10 minutes.In dehydrated alcohol, soak 5 minutes; In 95% ethanol, soak 5 minutes; In 70% ethanol, soak 5 minutes; Finally enter distilled water immersion 2 minutes;

8) by entering section after distilled water, put into hematoxylin aqueous solution and dye 5 minutes.Color separation in sour water and ammonia, each 5-10 second.Flowing water rinses after 5 minutes and enters distilled water for a moment.Enter in 70% and 90% ethanol and dewater each 5 minutes.Enter ethanol Yihong dyeing liquor dyeing 2～3 minutes.

9) dyeing is dewatered by absolute alcohol, then it is transparent through dimethylbenzene, to make to cut into slices, and uses neutral gum mounting.

4, photograph and data statistics

Just put photo under microscope (Olypums BX 53) * 20 times of mirrors, manually utilizing the core of new formation on Image-Pro Plus 5.1 software counting damage fields at central area of muscle fiber.

Result: as shown in Fig. 4 A-1 and 4A-2, RT-PCR detects Linc-RAM high expressed in tibialis anterior; 7.5 days HE coloration results of CTX damage show that the core newly forming in the tibialis anterior of injection Linc-RAM group is significantly greater than matched group (as Fig. 4 B, 4C) at central area of muscle fiber; The expression of 3.5 days real-time RT-PCR detection injury repairing related genes of CTX damage, as shown in Fig. 4 D-1 to 4D-3, inject MyoD in the tibialis anterior of Linc-RAM group, MyoG, the expression of eMHC mRNA is more than matched group, and results suggest Linc-RAM promotes skeletal muscle regeneration.

Embodiment 6 Linc-RAM promote the effect of RD cells (a kind of human rhabdomyosarcoma cells system) differentiation

BLOCK-It Lentiviral RNAi Expression System as described in Example 1, builds the RD cell line of Linc-RAM high expressed, and its RD cell line is purchased from Chinese Academy of Medical Sciences's cell centre.

Linc-RAM promotes RD cell differentiation

As embodiment 2, respectively by 2 * 10 ⁴the RD cell culture of individual Linc-RAM high expressed is in 12 orifice plates, cultivate 12h and change division culture medium (DMEM culture medium, comprise 10ug/mL insulin, 10ug/mL transferrins, 1% penicillin, 1% streptomycin, 1% glutamine), after differentiation 48h, collect cell, immunofluorescence, the specific experiment operations such as RT-PCR are as embodiment 1,2.

Result: system successfully constructs as Fig. 5 A showed cell.5B, in the RD cell of 5C demonstration Linc-RAM high expressed, MHC positive cell number is significantly more than matched group.MyoD in the RD cell of the high expressed of Linc-RAM shown in 5D-1 to 5D-2, the expression of MyoG mRNA is more than matched group, and results suggest Linc-RAM promotes RD cell differentiation.

Embodiment 7 Linc-RAM may participate in developing of mankind's muscular dystrophy disease

Take from muscular dystrophy patient's tissue sample, extract total RNA, real-time RT-PCR detects human-Linc-RAM-variant 1, the expression of human-Linc-RAM-variant2, and MyoD, MyoG mrna expression amount.

Result: 5E shows according to mice Linc-RAM the position in genome, analyzes the position of human-Linc-RAM in human genome by the homology range of linearity, comprises two kinds of variants; As shown in Fig. 5 F-1 to 5F-4, with respect to normal muscle tissue, human-Linc-RAM-variant 1 and human-Linc-RAM-variant 2 expression in tissue of patient is lowered, and MyoD, MyoG mrna expression amount is lowered, and what results suggest human-Linc-RAM may participate in muscular dystrophy develops that (A represents normal muscle tissue; B represents muscular dystrophy tissue).

Embodiment 8 Linc-RAM, as co-activation of MyoD, regulate the expression of muscle-derived gene

Respectively by 1.2 * 10 ⁴individual Linc-RAM high expressed, is changed division culture medium and after 12 hours, is collected cell total rna, RNA-seq checkout discrepancy expressing gene with the C2C12 cell culture knocking out in 6 orifice plates; According to the known gene that is subject to MyoD regulation and control, utilize bioinformatic analysis to be subject to MyoD and the common gene regulating of Linc-RAM; The plasmid of MyoD and Linc-RAM high expressed is transfection C3H10T1/2 cell respectively, after 48 hours, collects RNA, detects the expression of MyoG gene; Various dose (0,0.6ug, Linc-RAM high-expression plasmid 1.0ug) and 0.4ug MyoD high-expression plasmid cotransfection C3H10T1/2 cell, luciferase reporter gene detects MyoG promoter (MyoG genetic transcription initiation site upstream-470bp～+ 130bp) activity; As embodiment 1, formaldehyde fixed L inc-RAM high expressed and strike low C2C12 cell, after ultrasonication, use respectively MyoD, H3K4Me3 and Pol II antibody staining matter co-immunoprecipitation, real-time detects respectively these three kinds of albumen at the enrichment degree of MyoG and miR-206 (far-end) promoter region.

Result: as Fig. 6 A shows Linc-RAM high expressed and strikes low C2C12 cell differentiation 12 hours, the gene that RNA-seq checkout discrepancy is expressed, Heat map shows the gene of differential expression (fold>2); 6B shows the result of GO gene annotation, and the gene of differential expression is mainly enriched in the transcriptional regulatory of nucleosome assembling and muscle-derived gene; 6C shows MyoD and the common gene regulating of Linc-RAM; 6D shows MyoD and the common adjusting of Linc-RAM and muscle cell multiplication, breaks up the gene relevant with structure; 6E demonstration RT-PCR further determines the common gene regulating of MyoD and Linc-RAM at Linc-RAM high expressed and strikes the expression variation of low C2C12 cell differentiation after 12 hours; 6F shows various dose (0,0.6ug, Linc-RAM high-expression plasmid 1.0ug) and 0.4ug MyoD high-expression plasmid cotransfection C3H10T1/2 cell, luciferase reporter gene result shows that Linc-RAM is dose dependent and promotes MyoD to regulate the activity of MyoG promoter; 6G shows that the differentiation of C3H10T1/2 cell transfecting MyoD high-expression plasmid is after 48 hours, and real-timePCR result shows the expression of MyoD induction MyoG; 6H-1 to 6H-3 result shows with respect to normal group, in the C2C12 cell of Linc-RAM high expressed, more MyoD, H3K4Me3 and Pol II are enriched in MyoG and miR-206 (far-end) promoter region, and Linc-RAM strikes in low C2C12 cell, the enrichment of these albumen reduces, and results suggest Linc-RAM, as co-activation of MyoD, regulates the expression of muscle-derived gene.

The mode that embodiment 9 Linc-RAM rely on p38a, by interacting with MyoD, mediates the formation of MyoD-Baf60c-Brg1 complex

The C2C12 cell pyrolysis liquid that extracts RNase-free, utilizes the co-precipitation of MyoD antibody mediated immunity, and Western Blot detects MyoD albumen, and RT-PCR detects Linc-RAM; The C2C12 cell pyrolysis liquid that extracts RNase-free, utilizes respectively MyoD, Baf60c and the co-precipitation of Brg1 antibody mediated immunity, and RT-PCR detects Linc-RAM; Real-time PCR detects Linc-RAM high expressed and strikes the expression of Baf60c and Brg1 mRNA in low C2C12 cell; Extract respectively Linc-RAM high expressed and strike low C2C12 cell RNase-free lysate, utilizing the co-precipitation of MyoD antibody mediated immunity, Western Blot detects MyoD, Baf60c and Brg1 albumen; Formaldehyde fixed L inc-RAM high expressed and strike low C2C12 cell, after ultrasonication, use respectively MyoD, Baf60c, Brg1 and H3K9ac antibody staining matter co-immunoprecipitation, real-time PCR detects respectively these four kinds of albumen at the enrichment degree of MyoG promoter region; 10uM SB203580 processed the C2C12 cell differentiation of Linc-RAM high expressed after 36 hours, immunofluorescence, and real-time PCR and ChIP-PCR detect as embodiment 2.

Conclusion: Fig. 7 A is presented in the co-immunoprecipitation product of MyoD antibody and Linc-RAM detected, prompting Linc-RAM and MyoD combine; 7B result demonstration Linc-RAM is combined with MyoD, and is combined with Baf60c and Brg1; 7C shows Linc-RAM high expressed and strikes the expression of Baf60c and Brg1 mRNA in low C2C12 cell does not have difference; 7D shows with respect to matched group, in the cell of Linc-RAM high expressed, the enrichment in the co-immunoprecipitation product of MyoD antibody of Baf60c and Brg1 albumen increases, and Linc-RAM strikes in low cell, and the enrichment in the co-immunoprecipitation product of MyoD antibody of Baf60c and Brg1 albumen reduces; 7E-1 to 7E-4 shows with respect to matched group, in the cell of Linc-RAM high expressed, more MyoD, Baf60c, Brg1 and H3K9ac protein enrichment are in MyoG promoter region, and Linc-RAM is high to be struck in low cell, MyoD, Baf60c, Brg1 and H3K9ac albumen reduce in the enrichment of MyoG promoter region; 7F, 7G, 7H shows that the C2C12 cell of 10uM SB203580 processing Linc-RAM high expressed divides 36 hours, the number of MHC positive cell is significantly lower than the Linc-RAM high expressing cell group that does not have to process, and the expression of MHC mRNA reduces; 7I shows that the C2C12 cell differentiation of 10uM SB203580 processing Linc-RAM high expressed is after 18 hours, and the expression of Linc-RAM does not change; 7J shows that the C2C12 cell of SB203580 processing Linc-RAM high expressed is after 18 hours, Brg1 reduces in the enrichment of MyoG promoter region, the mode that results suggest Linc-RAM relies on p38a, by interacting with MyoD, mediates the formation of MyoD-Baf60c-Brg1 complex.

Embodiment 10 bFGF regulate expression and the function of Linc-RAM in myocyte's atomization by Ras/Raf/Mek/Erk1/2/MyoD signal path

10ng/mL bFGF (Sigma) processed C2C12 cell differentiation after 6,12,24 hours, collected RNA, and real-time RT-PCR detects the expression (as shown in Fig. 5-A) of Linc-RAM; 10ng/mL bFGF processes C2C12 cell differentiation 24 hours, and luciferase reporter gene detects the activity of Linc-RAM promoter; 10uM PD98059 pretreatment C2C12 cell is processed C2C12 cell differentiation with bFGF again after 1 hour and within 24 hours, is detected p-Erk, t-Erk, p-MEK, the expression of t-MEK albumen; PD98059 pretreatment C2C12 cell is processed C2C12 cell differentiation with bFGF again after 1 hour and within 24 hours, is detected MyoD, the expression of Linc-RAM and MyoG; 10uM PD98059 pretreatment C2C12 cell is processed C2C12 cell differentiation 24 hours with bFGF after 1 hour again, and luciferase reporter gene detects the activity of Linc-RAM-WT and Linc-RAM-Mutant promoter; 5uM FTA pretreatment C2C12 cell is processed C2C12 cell differentiation 24 hours with bFGF after 30 minutes again, detects MyoD, the expression of Linc-RAM and MyoG; 2.5uM GW pretreatment C2C12 cell is processed C2C12 cell differentiation after 24 hours with bFGF after 30 minutes again, detects MyoD, the expression of Linc-RAM and MyoG; BFGF processing C2C12-NC and C2C12-Linc-RAM-OE cell differentiation be Myogenin and MHC immunofluorescence dyeing after 24,36 hours, and real-time PCR detects as embodiment 2.

Conclusion: 8A shows that bFGF processes C2C12 cell differentiation after 6,12,24 hours, the down-regulated expression of Linc-RAM; 8B shows that bFGF processes C2C12 cell differentiation 24 hours, and luciferase reporter gene result shows the activity decreased of Linc-RAM promoter; 8C shows after PD98059 pretreatment, can partly make up the p-Erk that causes due to bFGF and the increase of p-MEK expressing quantity; 8D-1 to 8D-3,8E shows after PD98059 pretreatment, can partly make up the reduction of the Linc-RAM expression causing due to bFGF; 8F, 8G shows that FTA and GW partly make up respectively the reduction of the Linc-RAM expression being caused by bFGF; 8H, 8I, 8J shows that bFGF has reduced the short differentiation of Linc-RAM to C2C12 cell, prompting bFGF regulates expression and the function of Linc-RAM in myocyte's atomization by Ras/Raf/Mek/Erk1/2/MyoD signal path.

Announcement along with ENCODE plan, on human genome, find at present at least to exist 9640 sites to be transcribed into lncRNAs, these lncRNAs certainly will participate in biological each process, yet only the lncRNAs less than 0.1% has been carried out to functional study at present, thereby in research lncRNAs biological process, function becomes new study hotspot.The inventor finds a lncRNA who is subject to MyoD regulation and control, called after Linc-RAM, Linc-RAM is up-regulated in flesh generative process, significantly promote C2C12 cell differentiation, promote muscle injury reparation, can separately or assist MyoD that fibroblast is converted to sarcoplast, and promote human rhabdomyosarcoma cells differentiation, further experimental result shows that bFGF passes through Ras/Raf/Mek/Erk signal path and in myocyte's atomization, regulates the expression of Linc-RAM.Molecular mechanism of its performance function mode that to be Linc-RAM rely on p38a is by interacting with MyoD, the formation of mediation MyoD-Baf60c-Brg1 complex, thus regulate the expression of muscle-derived gene.These result of study promptings Linc-RAM has important regulating action in skeletal development process, can provide clinical treatment effect for human diseases.

Claims

1. the purposes of long-chain non-coding RNA molecule in the medicine of preparation treatment muscle disease, described long-chain non-coding RNA molecule is selected from Linc-RAM.

2. purposes according to claim 1, wherein said muscle disease is selected from muscle injury or rhabdomyosarcoma.

3. purposes according to claim 1 and 2, wherein said muscle injury is acute muscle injury.

4. long-chain non-coding RNA molecule promotes muscle stem cell differentiation or promotes fibroblast transdifferentiation or promote the purposes in the medicine of human rhabdomyosarcoma cells differentiation in preparation, and described long-chain non-coding RNA molecule is selected from Linc-RAM.

5. the carrier of a nucleotide that contains the Linc-RAM that encodes is used for the treatment of the purposes in the medicine of muscle disease.

6. purposes according to claim 5, wherein said muscle disease is selected from muscle injury or rhabdomyosarcoma.

7. according to the purposes described in claim 5 or 6, wherein said muscle injury is acute muscle injury.