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

This study was supported by the Student Research Grant (13-14) of University of Ulsan College of Medicine, Seoul, Korea and a grant (2014-464) from the Asan Institute for Life Sciences, Seoul, Korea.

1. Cell Line
<ol>
	<li>Culture 106 telomerase-immortalized human corneal epithelial cells (HCECs) in a 6-well plate (density: 1039.9 cell/mm2) in a 37 &#176;C incubator with a 5% CO2 atmosphere using bronchial epithelium growth medium (BEGM) until they reach 95% confluence.</li>
</ol>
2. Western Blot Analysis after Creating Epithelial Scratch Wounds
<ol>
	<li>Streak a sterile 200 &#181;l pipette tip across the surface of a well of confluent cultured HCECs four ...

<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...

Corneal epithelial cells (CECs) are continuously shed into tear film, while they are simultaneously replaced by cells from the limbus and corneal epithelial basal layers.1 Various extrinsic stressors can induce the apoptosis and desquamation of CECs.2 The heat shock proteins (HSPs) are highly conserved and can be divided into two families according to molecular size.3 The largest HSP family includes HSP90, HSP70, and HSP60, and the smaller family includes HSP27.4 The phosphorylation of HSP27 is known to play an important role in cell survival and is required for cell migration because of the role of this protein in actin remodeling.5-7 Therefore, we attempted to test the potential role of HSP27 phosphorylation in CEC migration and apoptosis in an in vitro model of epithelial wound healing.
RNA interference (RNAi) using either small or short interfering RNAs (siRNA) has generated interest in both basic and applied biology, as it potentially allows the expression of any gene of interest to be knocked-down.8 Herein, we used HSP27-specific siRNA to assess the contribution of HSP27 to CEC wound healing and apoptosis. Traditional RNAi methods for gene knock-down in cells use synthetic RNA duplexes, including two unmodified 21-mer oligonucleotides that can be assembled to create siRNAs. The RNAi siRNA that we used in this present study is a simple and highly efficient method to transfect cells, and this reagent works with various immortalized cell lines. In this present study, we demonstrate the methods used for this analysis, including a scratch-induced directional wound assay, western blotting, siRNA transfection assay, immunofluorescence assay, and flow cytometry.

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			<td height="38" style="height:38px;width:212px;">Biological safety cabinet</td>
			<td style="width:247px;">CHC LAB Co.Ltd,&#160; Daejeon, Republic of Korea&#160;</td>
			<td style="width:109px;">CHC-777A2-06</td>
			<td style="width:199px;">Class II, Type A2&#160;</td>
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			<td height="58" style="height:58px;width:212px;">Stealth RNAi&#8482; siRNA</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
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			<td style="width:199px;">RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td>
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			<td height="38" style="height:38px;width:212px;">BEGMTM</td>
			<td style="width:247px;">Lonza, Inc., Walkersville, MD</td>
			<td style="width:109px;">CC-3171, CC4175</td>
			<td style="width:199px;">Bronchial epithelium growth medium&#160;</td>
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			<td height="38" style="height:38px;width:212px;">Protease inhibitor&#160;</td>
			<td style="width:247px;">Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td style="width:109px;">P8340 ,P7626</td>
			<td style="width:199px;">1 uM Pepstatin A, 1 uM Leupetin, 0.1 uM Aprotin</td>
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			<td height="20" style="height:20px;width:212px;">Bradford protein assay&#160;</td>
			<td style="width:247px;">Bio-Rad Laboratories, Hercules, CA</td>
			<td style="width:109px;">#500-0001</td>
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			<td style="width:247px;">Amersham, Little Chalfont, UK</td>
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			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
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			<td style="width:199px;">1:1000 dilution (Total HSP27)</td>
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			<td height="38" style="height:38px;width:212px;">Phosphorylated HSP27 (Ser85)</td>
			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
			<td style="width:109px;">ab5594</td>
			<td style="width:199px;">1: 1000 dilution HSP27 was phosphorylated at Ser85</td>
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			<td height="42" style="height:42px;width:212px;">Lipofectamine&#174; RNAiMAX reagent&#160;</td>
			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:109px;">13-778-075</td>
			<td style="width:199px;">Transfection reagent</td>
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			<td height="57" style="height:57px;width:212px;">Phosphorylated Akt (Ser473)</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4060</td>
			<td style="width:199px;">1: 1000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td>
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			<td height="38" style="height:38px;width:212px;">Non-phosphorylated Akt&#160;</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4061</td>
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			<td height="38" style="height:38px;width:212px;">Bcl-2-associated X protein&#160;</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4062</td>
			<td style="width:199px;">1: 1000 (anti-apoptotic protein marker)</td>
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			<td height="57" style="height:57px;width:212px;">GAPDH</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">No. 4063</td>
			<td style="width:199px;">1:1000 loading control&#160; marker (house keeping gene)</td>
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			<td height="43" style="height:43px;width:212px;">Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">NCI1460KR</td>
			<td style="width:199px;">1:10000 dilution</td>
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			<td height="38" style="height:38px;width:212px;">OPTI-MEM</td>
			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
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			<td style="width:199px;">reduced serum medium for transfection</td>
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			<td height="40" style="height:40px;width:212px;">Image analysis software</td>
			<td style="width:247px;">Olympus, Inc., Tokyo, Japan</td>
			<td style="width:109px;">Image-Pro Plus 5.0</td>
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			<td height="44" style="height:44px;width:212px;">Skimed milk powder&#160;</td>
			<td style="width:247px;">Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td>
			<td style="width:109px;">T145.2</td>
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			<td height="20" style="height:20px;width:212px;">Tris&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0497</td>
			<td style="width:199px;"></td>
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			<td height="20" style="height:20px;width:212px;">Sodium Chloride&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0241</td>
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			<td height="38" style="height:38px;width:212px;">Six well culture plate</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">140675</td>
			<td style="width:199px;">35.00 mm diameter / well</td>
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			<td height="38" style="height:38px;width:212px;">24-well culuture dish</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">142475</td>
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			<td height="20" style="height:20px;width:212px;">Orbital shaker</td>
			<td style="width:247px;">N-Bioteck, Inc., Seoul, South Korea</td>
			<td style="width:109px;">NB1015</td>
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			<td height="38" style="height:38px;width:212px;">Bovine serum albumin</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2323&#160;</td>
			<td style="width:199px;"></td>
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			<td height="23" style="height:23px;width:212px;">BDFACSCantoTM II</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Flow cytometry</td>
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			<td height="38" style="height:38px;width:212px;">X-Ray Film</td>
			<td style="width:247px;">Kodak, Rochester, NY</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Medical X-Ray Cassette with Green 400 Screen&#160;</td>
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			<td height="38" style="height:38px;width:212px;">western blotting luminol reagent</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2048&#160;</td>
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			<td height="38" style="height:38px;width:212px;">FITC Annexin V Apoptosis Detection Kit I</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;">556547</td>
			<td style="width:199px;"></td>
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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.

The expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding compared with unwounded HCECs13. Western blot analysis revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced, whereas the expression of Bax was significantly increased in HSP27-specific siRNA-transfected HCECs (Figure 1A-E). The phosphorylated HSP27 expression was reduced by 30% and 40% in 10 nM and 50 ...

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.

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 ...

The overall goal of this RNA Transfection assay is to use HSP27 specific small interfering RNA to assess the potential role of HSP27 in corneal epithelial wound healing. This method can help answer key questions regarding the the function of Heat Shock Protein 27 during epithelial wound healing process. The main advantage of this technique is that small interfering RNA is sensitive RNA sucrose created to specifically target a messenger RNA transcript to induce it's degradation.It has been used to identify novel pest space various cellular processes. Though this technique can provide the insight into the corneal epithelial wound healing, it may also apply to another system such as skin and mucosa. Demonstrating the procedure will be Soon-Suk Kang, a technician from my laboratory.To begin this procedure, culture human corneal epithelial cells in a six-well plate with with bronchial epithelium growth medium at 37 degree Celsius and five percent carbon dioxide until they 95 percent confluence. Next, dilute 2.5 micro liters, or 7.5 micro liters of the transfection re-agent with 100 micro liters of reduced serum medium for transfection. Then, dissolve HSP27 specific siRNA and scrambled controlled siRNA in 100 micro liters or reduced serum medium to create 10 nanomolar of HSP27 specific siRNA and 15 nanomolar of scrambled control siRNA respectively.Following that, mix 100 micro liters of siRNA solution with 100 micro liters of diluted transfection reagent and incubate the mixture for 15 minutes at room temperature. Subsequently, add siRNA lipid complexes to the cells. After a four hour incubation, change the medium to the complete bronchial epithelium growth medium, and incubate the cells two days at 37 degrees Celsius before western blot analysis.In this step, extract HSP27 specific and scrambled control siRNA transfected HCECs in a biological safety cabinet using 100 micro liters of ice cold lysis buffer. Then incubate the cells for 30 minutes on ice to induce cell lysis. Next, centrifuge the lysates at 10, 000 times G for 15 minutes.Subsequently, transfer the supernatants to fresh 1.5 milliliter tubes and store them at minus 80 degrees Celsius. After that, determine the protein concentrations of the cell lytates using the Bradford protein assay. Then load the samples with equal amounts of total cell proteins on a 10 percent or 12 percent acrylamide gel.Subject the gel to SDS page, and electrophoretically transfer the separated protein bands to the nitrocellulose filters with 200 milliamps of current for one hour and four degrees Celsius. Next, block the nitrocellulose filter membranes with five percent skimmed milk in TBST for one hour. Then add primary anti-bodies and incubate the membranes overnight at four degrees Celsius on a shaker.The next day, wash the membranes three times with TBST allowing ten minutes for each wash. Then detect the immunolreactive bands using horseradish peroxidase conjugated goat anti-rabbit antibodies in five percent BSA. Subsequently, incubate the membranes in the western blotting luminol reagents for one minute at room temperature.Remove the membrane from the reagent solution. Then, remove excess liquid with an absorbent towel and place them in a plastic sheet protector. Afterward, place the covered membrane in a film cassette with the protein side facing up.Place an x-ray film on top of the membranes and expose it for one minute. In a biological safety cabinet, make a wound by dragging a sterile pipette tip across the confluent cultures of HSP27 specific or scrambled control siRNA transfected HCECs. Immediately after wounding wash the cells twice with 1XPBS and maintain them in bronchial epithelium growth medium at 37 degrees Celsius and five percent carbon dioxide for 24 hours.24 hours after wounding take the HCECs image using an upright microscope at 100x and perform background flattening using the filter command in image analysis software. Using the select measurements command, define the area of interest with the same sized polygonal shape that can cover the initial wound perpendicularly from end to end. Then, determine three different area of interest in the wounded area of each sample.Automatically count the cell number in each field using the count size measure menu option. Western blot analysis shown here, revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced. Where as the expression of backs was significantly increased in HSP27 specific siRNA transfected HCECs.The phosphorylated HSP27 expression was reduced by 30 percent and 40 percent in 10 nanomolar and 15 nanomolar of HSP27-specific siRNA transfected cells respectively, compared with control siRNA transfected cells. Moreover, the nonphosphorylated HSP27 expression was reduced by 20 percent and 30 percent in ten nanomolar and 15 nanomolar of HSP27 specific siRNA transfected cells respectively. The scratch induced directional wound assay indicated that 24 hours after wounding, HSP27-specific siRNA transfected cells at 10 and 15 nanomolar exibited reduced migration.In addition HSP27-specific siRNA transfected HCEC's underwent more apoptotic and necrotic cell death compared with the scrambled control siRNA transfected cells shown by flow cytometry. Once mastered RNA transfection technique can be done in 24 hours if it is performed properly. Following these procedures an episode like lock mouth can be generated in order to answer additional question like the initial function of Heat Shock Protein 27 in cornea epithelial wound healing.After watching this video, you should have a good understanding of how to use Heat Shock Protein 27 specific small interfering RNA to assess the potential role of Heat Shock Protein 27 in cornea epithelial wound healing. Don't forget that working with small interfering RNA and reagents can be hazardous and precursors such as a biological safety cabinet and protective mask should always be used while performing these procedures.

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