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Application of Linc-RAM for treating muscle diseases

A technology for muscle diseases and uses, which can be used in muscle system diseases, neuromuscular system diseases, gene therapy, etc., and can solve problems such as failure to achieve long-term survival and human harm.

Inactive Publication Date: 2014-12-10
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past few decades, the combination of chemotherapy, radiotherapy and surgery has been used to target different tumor locations and extended ranges, and the survival rate of patients has been significantly improved. Because the treatment of rhabdomyosarcoma has characteristics, that is, The local treatment of surgery and radiation often fails to achieve the goal of long-term survival. Only the application of systemic chemotherapy can significantly improve the survival rate. This kind of systemic chemotherapy will cause great harm to the human body.

Method used

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  • Application of Linc-RAM for treating muscle diseases
  • Application of Linc-RAM for treating muscle diseases
  • Application of Linc-RAM for treating muscle diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 A long non-coding RNA regulated by MyoD and specifically expressed in skeletal muscle cells was found, named Linc-RAM

[0032] Based on two sets of databases in published articles, see 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, using bioinformatics analysis to obtain a series of lncRNAs regulated by MyoD, RT -PCR detection of the expression patterns of these lncRNAs in the process of myogenesis, the detection results found a lncRNA specifically expressed in skeletal muscle --- 2310015B20Rik, and i...

Embodiment 2

[0043] Example 2 The effect of Linc-RAM on the differentiation of skeletal muscle stem cell lines

[0044] Construction of Linc-RAM high expression and knockdown cell lines

[0045] Use BLOCK-It Lentiviral RNAi Expression System (Invitrogen) to construct sh-Linc-RAM knockdown plasmid, and co-infect 293T cells with viral packaging plasmid pVSVG, △08.91, collect supernatants at 24 hours and 48 hours respectively, and centrifuge at 1100g for 3.5 minutes to remove cells Debris, 0.45um filter filtration, 24000rpm / min centrifugation 2.4h, 100ul PBS dissolves virus particle, after infecting C2C12 (purchased from ATCC) cell 48 hours, blasticidin (Invitrogen) screening obtains Linc-RAM (sh-Linc- RAM) knockout cell line. The same method was used to obtain the control plasmid control cell line.

[0046] The pENTRTM / U6 Entry Construct pU6 promoter expression region in the BLOCK-It Lentiviral RNAi Expression System was replaced with the pCMV promoter expression region of pEGFP-N1, and th...

Embodiment 3

[0057] Example 3 The effect of Linc-RAM on transforming fibroblasts into myoblasts

[0058] Using the BLOCK-It Lentiviral RNAi Expression System shown in Example 1, a C3H10T1 / 2 cell line with high expression of MyoD was constructed and labeled as MyoD OE C3H10T1 / 2 cells. Lenti-XTM Tet-on 3G Inducible Expression System (Clontech Laboratories) was used to construct the C3H10T1 / 2 cell line with high expression of Linc-RAM, which was marked as pLVX-Linc-RAM OE C3H10T1 / 2 cells, and its control plasmid was constructed and marked as pLVX- TRE C3H10T1 / 2 cells. The C3H10T1 / 2 cell line was purchased from the Cell Center of the Chinese Academy of Medical Sciences.

[0059] Linc-RAM converts fibroblasts into myoblasts

[0060] As in Example 2, 1.5×10 4 A pLVX-Linc-RAM OE, pLVX-TRE and MyoD OE C3H10T1 / 2 cells were cultured in a 12-well plate, and cultivated for 12 hours to change the differentiation medium (DMEM medium, containing 10ug / mL insulin, 5ug / mL transferrin, 1 % penicillin, 1%...

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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

technical field [0001] Linc-RAM is the inventor's first identification and research on its function and molecular mechanism. The present invention relates to the role of Linc-RAM in treating muscle diseases, especially muscle damage and rhabdomyosarcoma. Background technique [0002] Skeletal muscle is the largest tissue and organ of the human body, accounting for about 40% of body weight, and plays a very important role in the life activities of organisms. Not only does it perform locomotor and support functions, but it is also an important metabolic organ. Most of the sugar and lipids ingested by the body are metabolized in the skeletal muscles to produce energy, which plays a very important role in maintaining the balance of the whole body. The development of skeletal muscle is an orderly and strictly regulated process, which is not only regulated by various muscle-specific transcription factors, but also finely regulated by ncRNAs and epigenetics such as chromatin modif...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P21/00A61P35/00
Inventor 朱大海于潇华张勇李婷婷
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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