Name: rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing
Uploaded: 2016-09-27T12:30:00
Description: Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

Denna studie stöddes av studentbidrag (13-14) vid University of Ulsan College of Medicine, Seoul, Korea och ett bidrag (2014-464) från Asan Institutet för Life Sciences, Seoul, Korea.

1. Cellinje <ol><li> Kultur 10 6 telomeras-immortaliserade humana korneala epitelceller (HCECs) i en 6-brunnsplatta (densitet: 1039,9 cell / mm 2) i en 37 ° C inkubator med en 5% CO2 atmosfär med användning av bronkial epitel tillväxtmedium (BEGM) tills de når 95% konfluens. </li></ol> 2. Western blot-analys efter att skapa epitelceller Scratch Sår <ol><li> Spruta på en steril 200 mikroliter pipettspets över ytan av en brunn i sammanflytande odlade HCE...

<ol>
	<li>Dua, H. S., Gomes, J. A., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing.">Corneal epithelial wound healing.</a> Br. J. Ophthalmol. 78 (5), 401-408 (1994).</li><li>Estil, S., Primo, E. J., Wilson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+shed+human+corneal+cells.">Apoptosis in shed human corneal cells.</a> Invest. Ophthalmol. Vis. Sci. 41 (11), 3360-3364 (2000).</li><li>Guay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+actin+filament+dynamics+by+p38+map+kinase-mediated+phosphorylation+of+heat+shock+protein+27.">Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.</a> J. cell. Sci. 110, 357-368 (1997).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+heat+shock+protein+mRNAs+under+in+vivo+glutathione+depletion+in+the+mouse+retina.">Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina.</a> Neurosci. Lett. 413 (3), 260-264 (2007).</li><li>Rane, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+controls+apoptosis+by+regulating+Akt+activation.">Heat shock protein 27 controls apoptosis by regulating Akt activation.</a> J. Biol. Chem. 278 (30), 27828-27835 (2003).</li><li>Shin, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+tumor+cell+migration+and+invasion+with+biphenyl+isoxazole+derivative+KRIBB3,+a+synthetic+molecule+that+inhibits+Hsp27+phosphorylation.">Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.</a> J. Biol. Chem. 280 (50), 41439-41448 (2005).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+phosphorylated+heat+shock+protein+27+during+corneal+epithelial+wound+healing.">Expression of phosphorylated heat shock protein 27 during corneal epithelial wound healing.</a> Cornea. 31 (7), 820-827 (2012).</li><li>Alekseev, O. M., Richardson, R. T., Alekseev, O., O'Rand, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+profiles+in+HeLa+cells+in+response+to+overexpression+or+siRNA-mediated+depletion+of+NASP.">Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP.</a> Reprod. Biol. Endocrinol. 7, 45 (2009).</li><li>Park, H. Y., Kim, J. H., Lee, K. M., Park, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+prostaglandin+analogues+on+tear+proteomics+and+expression+of+cytokines+and+matrix+metalloproteinases+in+the+conjunctiva+and+corea.">Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and corea.</a> Exp. Eye. Res. 94 (1), 13-21 (2012).</li><li>Voegeli, T. S., Currie, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+knocks+down+Hsp27+and+increases+angiotensin+II-induced+phosphorylated+NF-kappaB+p65+levels+in+aortic+smooth+muscle+cells.">siRNA knocks down Hsp27 and increases angiotensin II-induced phosphorylated NF-kappaB p65 levels in aortic smooth muscle cells.</a> Inflamm. Res. 58 (6), 336-343 (2009).</li><li>Shi, B., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenite+pre-conditioning+reduces+UVB-induced+apoptosis+in+corneal+epithelial+cells+through+the+anti-apoptotic+activity+of+27+kDa+heat+shock+protein+(HSP27).">Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27).</a> J. Cell. Physiol. 206 (2), 301-308 (2006).</li><li>Shen, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+corneal+epitheliotrophic+capacity+among+different+human+blood-derived+preparations.">Comparison of corneal epitheliotrophic capacity among different human blood-derived preparations.</a> Cornea. 30 (2), 208-214 (2011).</li><li>Song, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+phosphorylation+is+involved+in+epithelial+cell+apoptosis+as+well+as+epithelial+migration+during+corneal+epithelial+wound+healing.">Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.</a> Exp Eye Res. 118 (1), 36-41 (2014).</li></ol>

In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we us...

Korneaepitelialceller (länderna i Centraleuropa) kontinuerligt utsöndras i tårfilmen, medan de samtidigt ersättas med celler från limbus och hornhinneepitelceller basala skikt. 1 Olika yttre faktorer kan inducera apoptos och fjällning av östeuropeiska länderna. 2 värmechockproteiner (HSP) är mycket konserverade och kan delas in i två familjer efter molekylstorlek. 3 den största HSP-familjen omfattar HSP90, HSP70 och HSP60, och den mindre familjen inkluderar Hsp27. 4 fosforyleringen av Hsp27 är känd för att spela en viktig roll i cellöverlevnad och krävs för cellmigration på grund av den roll av detta protein i aktin ombyggnad. 5-7 Därför försökte vi att testa den potentiella rollen av Hsp27 fosforylering i CEC migration och apoptos i en modell av epitel sårläkning in vitro. RNA-interferens (RNAi) med hjälp av antingen små eller korta störande RNA (siRNA) har GEnerated intresse i både grundläggande och tillämpad biologi, eftersom det potentiellt kan uttrycket av varje gen av intresse som ska slås ner. 8 Häri använde vi Hsp27 specifika siRNA att bedöma bidrag Hsp27 CEC sårläkning och apoptos. Traditionella RNAi metoder för gen knock-down i celler använder syntetiska RNA-duplex, inklusive två omodifierade 21-mer oligonukleotider som kan sättas samman för att skapa siRNA. RNAi siRNA som vi använde i denna studie är en enkel och effektiv metod för att transfektera celler, och detta reagens arbetar med olika odödliggjorda cellinjer. I den nu aktuella studien visar vi de metoder som används för denna analys, bland annat en repa-inducerad riktnings sår analys, western blotting, siRNA transfektionsanalysen, immunofluorescensanalys och flödescytometri.

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rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Uttrycket av fosforylerat Hsp27 ökade signifikant vid 5, 10, och 30 min efter scratch såra jämfört med oskadad HCECs 13. Western blot-analys avslöjade att uttrycket av fosforylerat Hsp27 och fosforylerades Akt var båda signifikant, medan uttrycket av Bax ökade signifikant hos Hsp27-specifika siRNA-transfekterade HCECs (Figur 1A-E). Den fosforylerade Hsp27 uttryck minskades med 30% och 40% av 10 nM och 50 nM Hsp27-specifik siRNA-transfekterade celler, j?...

Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

RNA-interferens-baserad undersökning av funktionen av värmechockprotein 27 under korneaepitelet Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

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