Experimental Study On The Protective Role Of MiR-34a Silencing In Blister Agent-damaged Keratinocytes And Its Mechanisms

Background and purpose:MicroRNAs (miRNAs) regulate gene expression by non-exactly pairing to the3’-UTRregion of target mRNAs. MiR-34a is a p53-induced miRNA, which plays an important rolein the development, proliferation, differentiation, DNA damage response, apoptosis andclosely related to many biological events of tumorigenesis.Mustard gas (sulfur mustard, SM) is a skin and DNA damaging agents, causing skininflammation, edema, blisters, and etc. Once ulceration formed, a delayed wound healingusually occurs accompanying with scar and infection. The changes of miR-34a expressionmay play a role in the process of SM-caused response of the skin. Interfering with itsexpression may affect the process of skin damage and healing. Therefore, to evaluate therole of miR-34a silence in mustard simulant (CEES) affected cells in the aspects of cellmigration, adhesion and invasion, we delivered the inhibitor of miR-34a, agomiR (Ago34a), to reduce the miR-34a level in HaCaT cells before the CEES exposure. The resultscan be used for better understanding how miR-34a participate in mustard damaing of theskin.Method:Part One: The changes of miR-34a expression and its mechanism in HaCaT cellsexpousred to CEES1. The establishment of CEES-poisoned HaCaT cell model：To find the optimal doseof CEES used for our further study, we employed MTS to detect the viability of HaCaTcells exposured to0.2,0.5,1.0，2.0mM CEES. Morphological changes were observed bylight microscopy24h after CEES exposure.2. Cell differentiation examination: The protein was harvested from HaCaT cellsexposured to0.5mM CEES. To detect whether HaCaT cells was differentiated after CEES exposure, the antibody of basal cell marker keratin5and differentiation marker keratin10were employed.3. Stem-loop Q-PCR detection of matured miRNANA: Primers for detectingmatured miRNANA is commercially obtained. Primer quality was evaluated accoding tothe dissolution curves before using. To get a better internal reference, small nuclear RNAU6and ribosome5S subunit genes were choosen and their stablities in our CEES poisoningmodel were compared with fluorescence curve.Part Two: The study of protective role of miR-34a silencing in blister agent-damagedkeratinocytes and its mechanisms1. miRNA silence: To silence the expression of the interested miRNA, chemicallysynthesized miR-34a inhibitor agomiR was transfected into the cells by using liposometransfection reagent. The transfection effiency was evaluated by counting the rate of cy3positive cells after the transfection of the cy3-labled agomiR and unlabled agomiR control.2. Western blotting analysis:6groups was designed as follows: the transfection ofagomiR-34a or ago control,0.5mM CEES exposure plus the transfection of agomiR-34a orago control, p38or ERK1/2inhibitor interference in0.5mM CEES exposure plus thetransfection of agomiR-34a or ago control.3. Cell scratch test: Grouping was followed as mentioned above. This test was toevaluated the cells migration ability under various processing factors.4. Cell adhesion experiments: Grouping was followed as mentioned above. This testwas to evaluated the cells adhesion ability under various treatments.5. Cell invasion assay: Grouping was followed as mentioned above. This test was toevaluated the cells invasion ability to pass through the Matrixgel.6. Immunofluorescence staining F-actin: Grouping was followed as mentioned above.FITC labled Phalloidin was employed to tracing F-actin, which was associated with thefunction of cytoskeleton remodeling, migration, and adhesion This test was to observe theF-actin location and the fluorescence intensity in cells under various treatments.7. Flow cytometry detecting F-actin fluorescence intensity: Grouping was followed asmentioned above. This test was to detect the mean fluorescence intensity of F-actin in cellsunder various treatments.8. Western blotting：Grouping was followed as mentioned above. This test was to detect the miR-34a targeting or non-targeting proteins which is related to migration.9. Bioinformatical analysis: HDAC6, a non-reported target protein of miR-34a, wasupregulated after AgomiR-34a transfection. To predict the possible pairing sites of miR-34ain HDAC6, the web of TargetScan and Vector NTI software were employed to find thetarget sequence of miR-34a in the HDAC63’-UTR.Part Three: Factors may affected by miR-34a silence to enhance cell migration---energy metabolism, autophage and Ca2+content.1. Western blotting: Grouping was followed as mentioned above. This test was todetect COX-10expression.2. Flow cytometry detecting the content of Ca2+: Grouping was followed as mentionedabove. By employing the Fluo-3/AM dye, we detected the content of Ca2+in cells undervarious treatments.3. HPLC detecting energy metabolism-related ATP, ADP and AMP, and energy chargewas calculated.Results:1. HaCaT cells exposured to0.5mM CEES showed the reduction of cell adhesion,migration and invasion. Some structural and functional proteins, such as K5, iNOS, MMP-1and Fra-1significantly reduced.2. The content of basal cell marker keratin5was significantlly reduced in HaCaTcells expousred to0.5mM CEES. As the expression of keratin10and the cell morphologydid not changed, it is suggested that CEES exposure may not significantly induce HaCaTcells differentiation.3. Ribosome5S was verified as a suitable internal reference for qRT-PCR aftercomparing the stability of U6and5S in our cell model. By employing5S as internalreference, we found that the expression of miR-34a was increased since12h and reached3-fold increase in HaCaT cells exposured to0.5mM CEES. The elevation of miR-34a maybe partially regulated by the mutant p53of HaCaT cells.4. miR-34a silence can increase cell migration both in cells exposured to CEES or not,but did not increase adhesion in cells exposured to CEES. The migration change could beinhibited partially by the inhibitors of p38and ERK1/2signaling pathways.5. miR-34a silence caused molecular changes in cells exposured to0.5mM CEES: The increase of c-myc and activate p-p38MAPK and p-ERK1/2; The increase of c-Met,ARHGAP1, and HDAC6; The increase of iNOS.6. miR-34a silence did not significantly change the levels of MMP-1, Integrin β1，Ca2+and energy metabolism level in cells exposured to0.5mM CEES.7. miR-34a silence induced ERK1/2activation in HaCaT exposured to0.5mM CEES.By adding a ERK1/2inhibitor, U0126, ERK1/2activation was inhibited, and cellular Ca2+and integrin β1was increased. Cells’ morphology also exhibited a differentiation change.Conclusions and Outlook:miR-34a silencing can significantly increase the cell adhesion, migration and couldpartially reverse the migration reduction caused by CEES, but no significant effect onadhesion. The mechanism of migration enhancement may be explained as:①. The increaseof c-myc may activate p-p38MAPK and p-ERK1/2, both of which were related tomigration and cytoskeleton recognization.②The increase of c-Met may lead to isdownstream pathway activation.③The increase of ARHGAP1may upregulate RhoGTPase, which is also migration-related.④The increase of HDAC6may enhancedmigration and cytoskeleton remodeling by deacetylating proteins like α-tubulin, Hsp90andcortactin.Further study on present topic should be explored in following aspects:1. Our researchwas performed in vitro, to verify the effect of Ago34a on CEES damaging and skin healing,in vivo study is needed.2HaCaT cells is a p53mutant cell line, this is not suitable formechanisms research in cell toxicity and DNA damage.4. The function of target proteinregulated by Ago34a is only deduced from the related literatures rather than an interferingstudy.