[논문]쥐L6 근원세포에서 miR-128의 근육세포 분화와 인슐린신호에서의 역할

오명주; 김소현; 김지현; 전병학

doi:10.5352/jls.2020.30.9.772

쥐L6 근원세포에서 miR-128의 근육세포 분화와 인슐린신호에서의 역할
Roles of miR-128 in Myogenic Differentiation and Insulin Signaling in Rat L6 Myoblasts 원문보기

생명과학회지 = Journal of life science, v.30 no.9, 2020년, pp.772 - 782

오명주 (식품안전처) , 김소현 (부산대학교 인제메카트로닉스공학과) , 김지현 (부산대학교 인제메카트로닉스공학과) , 전병학 (부산대학교 인제메카트로닉스공학과)

초록
AI-Helper

골격근의 분화 또는 근육 분화는 근육량과 신진대사 항상성을 유지하기 위해 중요하다. 근육 특이적 microRNAs (miRNAs)는 골격근 분화에 중요한 역할을 한다. 본 연구에서는 rat miRNAs 마이크로어레이를 사용하여 rat L6 근아세포의 근육 분화 과정에서의 miRNAs 발현 양상을 조사했다. 우리는 miR-128의 발현 증가를 발견했고, 동시에 이미 알려진 근육 분화 조절 miRNAs인 miR-1, miR-133b와 mi-206의 발현 증가를 확인했다. 이 microarray 결과를 확인하기위해 우리는 Quantitative RT-PCR 기술을 사용하였고, microarray 결과와 유사하게 발현 초기 mRNAs와 발현 후 성숙 miRNAs에서 모두 miR-128의 발현 증가를 확인했다. 또한 Rat L6 근아세포로의 miR-128 발현 향상은 muscle creatine kinase (MCK), myogenin, myosin heavy chain (MHC)와 같은 근육분화 표지 유전자 발현을 유발했고, 또한 MHC의 단백질 발현을 증가시켰다. 억제 PNAs를 사용한 miR-128의 작용 억제는 이러한 근육 분화 표지 유전자들의 발현을 차단했다. 또한, miR-128 발현 향상은 Erk와 Akt 단백질의 인슐린 자극에 의한 인산화를 증가시켰고, 고인슐린혈증과 고혈당증으로 인해 유도된 인슐린 저항성으로 인한 Erk와 Akt의 억제된 인산화를 회복했다. 이러한 발견은 miR-128이 근육분화와 인슐린 작용에 중요한 역할을 할 수 있다는 것을 시사한다.

Abstract ▼ AI-Helper

Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.

주제어

표/그림 (6)

표 Table 1. Primers used for qRT-PCR
그림 Fig. 1. Expression of microRNAs during myogenic differentiation in L6 cells. (A) L6 myoblasts were induced to differentiate for the indicated days. The differentiated myocytes were identified by immunostaining myosin heavy chain (MHC) (green) as a differentiation marker. Nuclei (blue) were stained with Hoechst 33342. (B) Heat map of miRNA expression during L6 differentiation. Total RNAs from differentiated L6 myoblasts on day 0, 1, 3, 6 were prepared and subjected to miRNA microarray analysis on the Agilent rat miRNA array. Differentially-expressed miRNAs expressed in undifferentiated (GM) and differentiated (DM) L6 cells are shown in a heat map representation. Upregulated miRNAs and downregulated miRNAs were labeled with red and green, respectively. Expression patterns of miR-11, miR-128, miR-206 and miR-133b were compared at the bottom (three independent replicates, *p<0.05). Results are obtained from three independent microarray experiments. Values from undifferentiated L6 cells are set to 1. Bars, the mean result ± SD. *p<0.05 relative to control undifferentiated L6 cells. (C) qRT-PCR analysis of the upregulated miRNAs. To validate the result of miRNA microarray, qRT-PCR analysis of mature miRNAs (upper panel) and primary miRNAs (lower panel) was performed with control undifferentiated and differentiated L6 cells on day 6. Rat U6 small nuclear RNA was used for normalization. Results are obtained from three independent experiments and are expressed as the fold increase related to control undifferentiated L6 cells. Bars, the mean result ± SD. *p<0.05 relative to control undifferentiated L6 cells.
그림 Fig. 2. Induction of myogenic differentiation by miR-128 in rat L6 myoblasts. L6 myoblasts were transfected with double-stranded mature miRNAs of either miR-128, miR-1 or control miR-125b-5p. The cells were further incubated for 3 days. (A) Total RNA was prepared from the transfected cells and RT-PCR was performed using primers specific for muscle creatine kinase (MCK), myogenin, myosin heavy chain (MHC) and GAPDH. Band intensities were quantified using ImageJ software, and the relative intensities of three independent experiments are presented as mean ± SD. Values from L6 cells transfected with control miR-125b-5p are set to 1. *p<0.05 relative to control miRNA-transfected L6 cells. (B) The cells were lysed and immunoblotted with anti-MHC antibody. Band intensities were quantified as in (A). Results are obtained from three independent experiments. Values from L6 cells transfected with control miR-125b-5p are set to 1. (C) The cells transfected with miRNAs were fixed on day 0 and day 3, and immunostained with anti-MHC antibodies (green) and Hoechst dye (blue). The 1,000 L6 cells stained with Hoechst were counted and in the same field of the microscope, the number of cells immunostained with anti-MHC antibody were also counted. Values of cells immunostained with anti-MHC antibody are obtained from three independent experiments. Bars, the mean result ± SD. *p<0.05 relative to control cells transfected with miR-125b-5p.
그림 Fig. 3. Inhibition of myogenic differentiation by inhibitory PNA-128 in rat L6 myoblasts. Undifferentiated L6 myoblasts in 50% confluence were transfected with inhibitory PNA-128, PNA-1 and control PNA-125b-5p for 1 day, and then the cells were induced to differentiate by differentiation medium for 3 days. The effects of inhibitory PNA-128 and miR-1 were examined by RT-PCR (A), immunoblotting (B) and immunostaining (C) as in Fig. 2. Values are obtained from three independent experiments. Bars, the mean result ± SD. *p<0.05 relative to control cells transfected with PNA-125b-5p.
그림 Fig. 4. Expression of miR-128 regulates insulin signaling and insulin resistance in L6 cells. (A) L6 myoblasts were transfected with miR-128, miR-1 and control miR-125b-5p, and then differentiated for 3 days. The cells were serum-starved for 4 hr and stimulated with 100 ng/ml of insulin for 5 min. The cells were then lysed and immunoblotted with antibodies against p-Akt, Akt, p-Erk, Erk and GAPDH. Band intensities were quantified using ImageJ software, and the relative intensities of three independent experiments are presented as mean ± SD. Values from L6 cells transfected with control miR-125b-5p are set to 1. *p<0.05 relative to control miR-125b-5p-transfected and insulin-stimulated L6 cells. (B) To examine a role of miR-128 in insulin resistance, L6 myoblasts were transfected with miR-128, miR-1 and control miR-125b-5p, and then differentiated for 3 days in the presence of high glucose (25 mM) and high insulin (1 μg/ml). The serum-starved cells were treated with insulin, immunoblotted and analyzed as above. Values from L6 cells transfected with control miR-125b-5p are set to 1. *p<0.05 relative to control miR-125b-5p-transfected and insulin-stimulated L6 cells. **p<0.05 relative to control miR-125b-5p-transfected L6 cells stimulated with insulin in the presence of high glucose and high insulin.
그림 Fig. 5. Transfection of miR-128 suppresses expression of IRS-1 protein in L6 cells. L6 myoblasts were transfected with miR-128, miR-1 and control miR-125b-5p, and then differentiated for 3 days. The cells were lysed and immunoblotted with antibodies against IRS-1, IRS-1 and GAPDH. Band intensities were quantified using ImageJ software, and the relative intensities of three independent experiments are presented as mean ± SD. Values from L6 cells transfected with control miR-125b-5p are set to 1. *p<0.05 relative to control microRNA-transfected L6 cells.

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

제안 방법

In order to confirm the upregulated expression of miR-1, miR-128, miR-133b and miR-206, we examined the expression level of both primary miRNA and mature miRNAs of these miRNAs on day 0 and day 6 after differentiation by qRT-PCR (Fig. 1C). We used miR-125b-5p as the control miRNA, as it showed constant expression on the array during differentiation.
Next, we examined the expression level of miRNAs during differentiation of rat L6 myoblasts at day 0, 1, 3 and 6 using a rat miRNA array. We found that some of miRNAs were upregulated or downregulated during differentiated L6 myoblasts (Fig.
For analysis of primary miRNAs expression, 1 μg of total RNA was used as the template for first-strand cDNA synthesis with Superscript III and random primer according to the manufacturer’s protocol. Then, qRT-PCR was performed with the 7500 Real-Time PCR System (Applied Biosystems) with Platunum SYBR Green qPCR SuperMix-UDG. Rat U6 was used for normalization, and relative amounts of the target were calculated using the comparative Ct method (2^-DDCt).
Previous studies reported upregulation of miR-1, miR-133 and miR-206 during skeletal muscle development [5, 13], but not that of miR-128. These studies utilized mouse-originated C2C12 myoblasts in order to investigate the expression profiling and functional roles of miRNAs. We made use of rat-originated L6 myoblasts for the study of miRNA array and additionally found the induction of miR-128 during differentiation.
(B) Heat map of miRNA expression during L6 differentiation. Total RNAs from differentiated L6 myoblasts on day 0, 1, 3, 6 were prepared and subjected to miRNA microarray analysis on the Agilent rat miRNA array. Differentially-expressed miRNAs expressed in undifferentiated (GM) and differentiated (DM) L6 cells are shown in a heat map representation.

데이터처리

The results were statistically analyzed by one-way ANOVA method and the statistical significance was determined by the Fischer’s Protected LSD post-hoc test.

이론/모형

Then, qRT-PCR was performed with the 7500 Real-Time PCR System (Applied Biosystems) with Platunum SYBR Green qPCR SuperMix-UDG. Rat U6 was used for normalization, and relative amounts of the target were calculated using the comparative Ct method (2^-DDCt). For analysis of mature miRNAs expression, qRT-PCR was performed using NCode miRNA first-strand cDNA synthesis and qRT-PCR Kits according to the manufacture’s protocol.

성능/효과

Similar to array results, we observed the upregulation of the expression of these four miRNAs in the level of both primary miRNA and mature miRNA during differentiation. The array and qRT-PCR results showed that the expression of miR-1, miR-128, miR-206 and miR-133b was significantly increased during the myogenic differ-entiation of rat L6 myoblasts. It has been reported that miR-1, miR-133b and miR-206 were increased during differentiation of mouse C2C12 myoblasts [5].

후속연구

Furthermore, we demonstrated that miR-128 plays a role in insulin signaling and insulin resistance. Future research is needed to further characterize the molecular mechanisms underlying the role of miR-128 in myogenic differentiation and insulin actions.

참고문헌 (34)

Abdelmoez, A. M., Sardon Puig, L., Smith, J. A., Gabriel, B. M., Savikj, M., Dollet, L., Chibalin, A. V., Krook, A., Zierath, J. R. and Pillon, N. J. 2020. Comparative profiling of skeletal muscle models reveals heterogeneity of transcriptome and metabolism. Am. J. Physiol. Cell Physiol. 318, C615-C626.

상세보기
Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297.

상세보기
Cervelli, M., Leonetti, A., Duranti, G., Sabatini, S., Ceci, R. and Mariottini, P. 2018. Skeletal muscle pathophysiology: The emerging role of spermine oxidase and spermidine. Med. Sci. 6, 14.
Chal, J. and Pourquie, O. 2017. Making muscle: skeletal myogenesis in vivo and in vitro. Development 144, 2104-2122.

상세보기
Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E., Hammond, S. M., Conlon, F. L. and Wang, D. Z. 2006. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat. Genet. 38, 228-233.

상세보기
Cui, J., Zhao, Y., Sethi, P., Li, Y., Mahta, A., Culicchia, F. and Lukiw, W. 2010. Micro-RNA-128 (miRNA-128) downregulation in glioblastoma targets ARP5 (ANGPTL6), Bmi-1 and E2F-3a, key regulators of brain cell proliferation. J. Neurooncol. 98, 297-304.

상세보기
Ebert, M. S. and Sharp, P. A. 2012. Roles for microRNAs in conferring robustness to biological processes. Cell 149, 515-524.

상세보기
Filipowicz, W., Bhattacharyya, S. N. and Sonenberg, N. 2008. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102-114.

상세보기
Ge, Y. and Chen, J. 2011. MicroRNAs in skeletal myogenesis. Cell Cycle 10, 441-448.

상세보기
Greene, N. P., Brown, J. L., Rosa-Caldwell, M. E., Lee, D. E., Blackwell, T. A. and Washington, T. A. 2018. Skeletal muscle insulin resistance as a precursor to Diabetes: Beyond glucoregulation. Curr. Diabetes Rev. 14, 113-128.

상세보기
Han, H., Wang, L., Xu, J. and Wang, A. 2018. miR-128 induces pancreas cancer cell apoptosis by targeting MDM4. Exp. Ther. Med. 15, 5017-5022.
Hauser, B., Zhao, Y., Pang, X., Ling, Z., Myers, E., Wang, P., Califano, J. and Gu, X. 2015. Functions of miRNA-128 on the regulation of head and neck squamous cell carcinoma growth and apoptosis. PLoS One 10, e0116321.

상세보기
Horak, M., Novak, J. and Bienertova-Vasku, J. 2016. Muscle-specific microRNAs in skeletal muscle development. Dev. Biol. 410, 1-13.

상세보기
Khan, A. P., Poisson, L. M., Bhat, V. B., Fermin, D., Zhao, R., Kalyana-Sundaram, S., Michailidis, G., Nesvizhskii, A. I., Omenn, G. S. and Chinnaiyan, A. M. 2010. Quantitative proteomic profiling of prostate cancer reveals a role for miR-128 in prostate cancer. Mol. Cell. Proteomics 9, 298-312.

상세보기
Kim, S. H., Yi, S. J., Lee, H., Kim, J. H., Oh, M. J., Song, E. J., Kim, K. and Jhun, B. H. 2019. ${\beta}2$ -Adrenergic receptor ( ${\beta}2$ -AR) agonist formoterol suppresses differentiation of L6 myogenic cells by blocking PI3K-AKT pathway. Anim. Cells Syst. 23, 18-25.

상세보기
Lian, B., Yang, D., Liu, Y., Shi, G., Li, J., Yan, X., Jin, K., Liu, X., Zhao, J. and Shang, W. 2018. miR-128 targets the SIRT1/ROS/DR5 pathway to sensitize colorectal cancer to TRAIL-induced apoptosis. Cell. Physiol. Biochem. 49, 2151-2162.

상세보기
Liang, R. F., Li, M., Yang, Y., Wang, X., Mao, Q. and Liu, Y. H. 2017. Circulating miR-128 as a potential diagnostic biomarker for glioma. Clin. Neurol. Neurosurg. 160, 88-91.

상세보기
Nigi, L., Grieco, G. E., Ventriglia, G., Brusco, N., Mancarella, F., Formichi, C., Dotta, F. and Sebastiani, G. 2018. Micro RNAs as regulators of insulin signaling: research updates and potential therapeutic perspectives in type 2 diabetes. Int. J. Mol. Sci. 19, 3705.

상세보기
Oh, M. J., Yi, S. J., Kim, H. S., Kim, J. H., Jeong, Y. H., van Agthoven, T. and Jhun, B. H. 2013. Functional roles of BCAR3 in the signaling pathways of insulin leading to DNA synthesis, membrane ruffling and GLUT4 translocation. Biochem. Biophys. Res. Commun. 441, 911-916.

상세보기
Pan, J., Zhou, C., Zhao, X., He, J., Tian, H., Shen, W., Han, Y., Chen, J., Fang, S. and Meng, X. 2018. A two-miRNA signature (miR-33a-5p and miR-128-3p) in whole blood as potential biomarker for early diagnosis of lung cancer. Sci. Rep. 8, 1-12.

상세보기
Petersen, M. C. and Shulman, G. I. 2018. Mechanisms of insulin action and insulin resistance. Physiol. Rev. 98, 2133-2223.

상세보기
Regazzi, R. 2018. MicroRNAs as therapeutic targets for the treatment of diabetes mellitus and its complications. Expert Opin. Ther. Targets 22, 153-160.

상세보기
Rupaimoole, R. and Slack, F. J. 2017. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat. Rev. Drug Discov. 16, 203.

상세보기
Saarbach, J., Sabale, P. M. and Winssinger, N. 2019. Peptide nucleic acid (PNA) and its applications in chemical biology, diagnostics, and therapeutics. Curr. Opin. Chem. Biol. 52, 112-124.

상세보기
Sharma, M., Juvvuna, P. K., Kukreti, H. and McFarlane, C. 2014. Mega roles of microRNAs in regulation of skeletal muscle health and disease. Front. Physiol. 5, 239.

상세보기
Sun, X. J., Wang, L. M., Zhang, Y., Yenush, L., Myers, M. G. Jr., Glasheen, E., Lane, W. S., Pierce, J. H. and White, M. F. 2011. Role of IRS-2 in insulin and cytokine signalling. Nature 377, 173-177.
Shaw, L. M. 2011. The insulin receptor substrate (IRS) proteins: at the intersection of metabolism and cancer. Cell Cycle 10, 1750-1756.

상세보기
Wang, C., Qiao, X., Zhang, Z. and Li, C. 2020. MiR-128 promotes osteogenic differentiation of bone marrow mesenchymal stem cells in rat by targeting DKK2. Biosci. Rep. 40, BSR20182121.

상세보기
Wang, Z., Pang, L., Zhao, H., Song, L., Wang, Y., Sun, Q., Guo, C., Wang, B., Qin, X. and Pan, A. 2016. miR-128 regulates differentiation of hair follicle mesenchymal stem cells into smooth muscle cells by targeting SMAD2. Acta Histochem. 118, 393-400.

상세보기
Wu, L., Shi, B., Huang, K. and Fan, G. 2015. MicroRNA-128 suppresses cell growth and metastasis in colorectal carcinoma by targeting IRS1. Oncol. Rep. 34, 2797-2805.

상세보기
Xia, L., Song, M., Sun, M., Chen, W. and Yang, C. 2019. miR-486 promotes Capan-2 pancreatic cancer cells proliferation by targeting PTEN. Front. Genet. 10, 541.

상세보기
Yu, Y., Du, H., Wei, S., Feng, L., Li, J., Yao, F., Zhang, M., Hatch, G. M. and Chen, L. 2018. Adipocyte-derived exosomal miR-27a induces insulin resistance in skeletal muscle through repression of $PPAR{\gamma}$ . Theranostics 8, 2171.

상세보기
Zhang, W., Kim, P. J., Chen, Z., Lokman, H., Qiu, L., Zhang, K., Rozen, S. G., Tan, E. K., Je, H. S. and Zeng, L. 2016. MiRNA-128 regulates the proliferation and neurogenesis of neural precursors by targeting PCM1 in the developing cortex. Elife 5, e11324.

상세보기
Zhou, T., Meng, X., Che, H., Shen, N., Xiao, D., Song, X., Liang, M., Fu, X., Ju, J. and Li, Y. 2016. Regulation of insulin resistance by multiple MiRNAs via targeting the GLUT4 signalling pathway. Cell. Physiol. Biochem. 38, 2063-2078.

상세보기

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증