Jiang, Tao
(Department of Vascular Surgery, China-Japan Union Hospital of Jilin University)
,
Wang, Xingang
(Department of Burns and Plastic Surgery, China-Japan Union Hospital of Jilin University)
,
Wu, Weiwei
(Department of Burns Surgery, the First Bethune Hospital of Jilin University)
,
Zhang, Fan
(Center of Tuberculous Meningitis, Changchun City Hospital for Infectious Diseases)
,
Wu, Shifeng
(Department of Burns and Plastic Surgery, China-Japan Union Hospital of Jilin University)
Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured...
Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured fibroblasts and that inhibiting let-7c expression leads to the increased proliferation and migration of dermal fibroblasts, whereas the overexpression of let-7c exerts an opposite effect. Further investigation has identified heat shock protein 70 as a direct target of let-7c and has demonstrated that the expression of HSP70 in fibroblasts is negatively correlated with let-7c levels. Moreover, down-regulation of let-7c expression is accompanied by up-regulation of Bcl-2 expression and down-regulation of Bax expression, both of which are the downstream genes of HSP70. Notably, the knockdown of HSP70 by HSP70 siRNA apparently abrogates the stimulatory effect of let-7c inhibitor on heat-denatured fibroblasts proliferation and migration. Overall, we have identified let-7c as a key regulator that inhibits fibroblasts proliferation and migration during wound healing.
Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured fibroblasts and that inhibiting let-7c expression leads to the increased proliferation and migration of dermal fibroblasts, whereas the overexpression of let-7c exerts an opposite effect. Further investigation has identified heat shock protein 70 as a direct target of let-7c and has demonstrated that the expression of HSP70 in fibroblasts is negatively correlated with let-7c levels. Moreover, down-regulation of let-7c expression is accompanied by up-regulation of Bcl-2 expression and down-regulation of Bax expression, both of which are the downstream genes of HSP70. Notably, the knockdown of HSP70 by HSP70 siRNA apparently abrogates the stimulatory effect of let-7c inhibitor on heat-denatured fibroblasts proliferation and migration. Overall, we have identified let-7c as a key regulator that inhibits fibroblasts proliferation and migration during wound healing.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
, 2015). In this study, we illustrate for the first time that HSP70 is a direct target gene of let-7c as the mediator of fibroblasts proliferation and migration. Among the more than 100 putative targets of let-7c known from bioinformatic analysis, we noticed that HSP70 was the special heat stimuli stress-associated gene that was predicted by miRanda and TargetScan (data not shown).
가설 설정
In this study, we characterized let-7c as a negative regulator of proliferation and migration in heat-denatured dermal fibroblasts. First, we identified dynamic expression of let-7c during recovery from wound healing in heat-denatured dermal fibroblasts.
After undergoing heat denaturation, fibroblasts are activated and become major cells at the wound site, which can secret collagen fibers and several growth factors (Martin, 1997). In this study, we examined the role of let-7c in heat-denatured dermal fibroblasts. The results demonstrated a down-regulation of let7c expression in the heat-denatured dermal fibroblasts, consistent with the miRNA array results described earlier (Liang et al.
제안 방법
To sum up, previous work has suggested that let-7 may be involved in wound healing, but little evidence is available to support this conjecture. In this study, we confirm that let-7c is involved in wound healing through down-regulating the proliferation and migration of heat-denatured dermal fibroblasts via inhibiting HSP70, and that inhibition of let-7c promotes cell survival, providing a putative therapeutic target for wound healing.
To further analyze and thus identify any link between the dynamically altered levels of let-7c and HSP70 expression, the total protein levels of HSP70 at different time points post heat damage were determined. Compared with the untreated dermal fibroblasts, HSP70 expression was significantly increased after 12 h and peaked at 24 h post heat damage, then gradually decreased after 48 h (Figs.
To further validate that HSP70 was a direct target of let-7c, we analyzed the expression of HSP70 in heat-denatured dermal fibroblasts transfected with let-7c mimics or inhibitor. As shown in Figs.
3A). To validate whether HSP70 was a direct target gene of let-7c, we performed a dual luciferase reporter assay. As shown in Fig.
데이터처리
For statistical analysis, one-way analysis of variance (ANOVA) among multiple groups, and two-tailed Student’s t-test were performed between two groups.
이론/모형
Cell migration was performed using the transwell migration assay. Briefly, the transfected dermal fibroblasts were subjected to heat treatment and cultured in serum-free DMEM for 24 h prior to the experiment.
Cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, dermal fibroblasts were cultured in 96-well plates and transfected with let-7c mimics, let-7c mimics control, let-7c inhibitor, and let-7c inhibitor control, respectively.
성능/효과
To verify this hypothesis, we analyzed the effect of let-7c mimics and inhibitor on Bax and Bcl-2 mRNA and protein levels by qRT-PCR and Western blotting, respectively. The results demonstrated that, compared with the control group, knockdown of let-7c decreased the expression of Bax (Figs. 4A-4C), while it increased the expression of Bcl-2 (Figs. 4D-4F). In contrast, let-7c overexpression exerted the opposite effects (Figs.
후속연구
, 2003), implying that HSP70 may play an important role in resisting heat stimuli. However, further investigation and elucidation of the precise mechanism and function of HSP70 in heat stimuli are needed.
The inhibition of let-7c expression promotes HSP70 expression which contributes to increasing cell proliferation and migration. Overall, this study indicates that let-7c could be a promising therapeutic target for the treatment of burn diseases, although further investigation is needed to reveal the underlying mechanism.
참고문헌 (37)
Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M 2008 Growth factors and cytokines in wound healing Wound Repair Regen. 16 585 601 19128254
Bartel D.P 2004 MicroRNAs: genomics, biogenesis, mechanism, and function Cell 116 281 297 14744438
Bjorner S. Fitzpatrick P.A. Li Y. Allred C. Howell A. Ringberg A. Olsson H. Miller C.J. Axelson H. Landberg G 2014 Epithelial and stromal microRNA signatures of columnar cell hyperplasia linking Let-7c to precancerous and cancerous breast cancer cell proliferation PLoS One 9 e105099 25122196
Calin G.A. Sevignani C. Dumitru C.D. Hyslop T. Noch E. Yendamuri S. Shimizu M. Rattan S. Bullrich F. Negrini M. 2004 Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers Proc. Natl. Acad. Sci. USA 101 2999 3004 14973191
Chen P.Y. Qin L. Barnes C. Charisse K. Yi T. Zhang X. Ali R. Medina P.P. Yu J. Slack F.J. 2012 FGF regulates TGF-beta signaling and endothelial-to-mesenchymal transition via control of let-7 miRNA expression Cell Rep. 2 1684 1696 23200853
De-quan L. Xiao-yuan H. Xing-hua Y 2008 Morphology of human skin fibroblasts after heat injury in vitro J. Clin. Rehabilitative Tissue Engineering Res. 12 2056
Felicetti F. Errico M.C. Bottero L. Segnalini P. Stoppacciaro A. Biffoni M. Felli N. Mattia G. Petrini M. Colombo M.P. 2008 The promyelocytic leukemia zinc finger-microRNA-221/-222 pathway controls melanoma progression through multiple oncogenic mechanisms Cancer Res. 68 2745 2754 18417445
Huang X. Yang X. Lei S. Xiao M. Zhang M. Zeng J 2001 With the preservation of denatured dermis and autoskin grafting to repair of deeply burned hands Zhonghua Shao Shang Za Zhi 17 60 61
Jego G. Hazoume A. Seigneuric R. Garrido C 2013 Targeting heat shock proteins in cancer Cancer Lett. 332 275 285 21078542
Johnson S.M. Grosshans H. Shingara J. Byrom M. Jarvis R. Cheng A. Labourier E. Reinert K.L. Brown D. Slack F.J 2005 RAS is regulated by the let-7 microRNA family Cell 120 635 647 15766527
Johnson C.D. Esquela-Kerscher A. Stefani G. Byrom M. Kelnar K. Ovcharenko D. Wilson M. Wang X. Shelton J. Shingara J 2007 The let-7 microRNA represses cell proliferation pathways in human cells Cancer Res. 67 7713 7722 17699775
Kariya A. Furusawa Y. Yunoki T. Kondo T. Tabuchi Y 2014 A microRNA-27a mimic sensitizes human oral squamous cell carcinoma HSC-4 cells to hyperthermia through downregulation of Hsp110 and Hsp90 Int. J. Mol. Med. 34 334 340 24789751
Koba S. Jinnin M. Inoue K. Nakayama W. Honda N. Makino K. Kajihara I. Makino T. Fukushima S. Ihn H 2013 Expression analysis of multiple microRNAs in each patient with scleroderma Exp. Dermatol. 22 489 491 23800063
Li D. Wang A. Liu X. Meisgen F. Grunler J. Botusan I.R. Narayanan S. Erikci E. Li X. Blomqvist L. 2015 MicroRNA-132 enhances transition from inflammation to proliferation during wound healing J. Clin. Invest 125 3008 3026 26121747
Liang P. Lv C. Jiang B. Long X. Zhang P. Zhang M. Xie T. Huang X 2012 MicroRNA profiling in denatured dermis of deep burn patients Burns 38 534 540 22360957
Motoyama K. Inoue H. Nakamura Y. Uetake H. Sugihara K. Mori M 2008 Clinical significance of high mobility group A2 in human gastric cancer and its relationship to let-7 microRNA family Clin. Cancer Res. 14 2334 2340 18413822
Nadiminty N. Tummala R. Lou W. Zhu Y. Zhang J. Chen X. eVere White R.W. Kung H.J. Evans C.P. Gao A.C 2012 MicroRNA let-7c suppresses androgen receptor expression and activity via regulation of Myc expression in prostate cancer cells J. Biol. Chem. 287 1527 1537 22128178
Nardai G. Vegh E.M. Prohaszka Z. Csermely P 2006 Chaperone-related immune dysfunction: an emergent property of distorted chaperone networks Trends Immunol. 27 74 79 16364688
Pasquinelli A.E. Reinhart B.J. Slack F. Martindale M.Q. Kuroda M.I. Maller B. Hayward D.C. Ball E.E. Degnan B. Muller P. 2000 Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA Nature 408 86 89 11081512
Schmitt E. Parcellier A. Gurbuxani S. Cande C. Hammann A. Morales M.C. Hunt C.R. Dix D.J. Kroemer R.T. Giordanetto F. 2003 Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant Cancer Res. 63 8233 8240 14678980
Schultz J. Lorenz P. Gross G. Ibrahim S. Kunz M 2008 MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth Cell Res. 18 549 557 18379589
Shin J.U. Lee W.J. Tran T.N. Jung I. Lee J.H 2015 Hsp70 knockdown by siRNA decreased collagen production in keloid fibroblasts Yonsei Med. J. 56 1619 1626 26446645
Singh S. Suri A 2014 Targeting the testis-specific heat-shock protein 70-2 (HSP70-2) reduces cellular growth, migration, and invasion in renal cell carcinoma cells Tumour Biol. 35 12695 12706 25213699
Yi R. O’Carroll D. Pasolli H.A. Zhang Z. Dietrich F.S. Tarakhovsky A. Fuchs E 2006 Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs Nat. Genet. 38 356 362 16462742
Yu F. Yao H. Zhu P. Zhang X. Pan Q. Gong C. Huang Y. Hu X. Su F. Lieberman J 2007 let-7 regulates self renewal and tumorigenicity of breast cancer cells Cell 131 1109 1123 18083101
Zhao B. Han H. Chen J. Zhang Z. Li S. Fang F. Zheng Q. Ma Y. Zhang J. Wu N. Yang Y 2014 MicroRNA let-7c inhibits migration and invasion of human non-small cell lung cancer by targeting ITGB3 and MAP4K3 Cancer Lett. 342 43 51 23981581
Zhu X. Wu L. Yao J. Jiang H. Wang Q. Yang Z. Wu F 2015 MicroRNA let-7c inhibits cell proliferation and induces cell cycle arrest by targeting CDC25A in human hepatocellular carcinoma PLoS One 10 e0124266 25909324
Zylicz M. King F.W. Wawrzynow A 2001 Hsp70 interactions with the p53 tumour suppressor protein EMBO J. 20 4634 4638 11532927
※ AI-Helper는 부적절한 답변을 할 수 있습니다.