BMSCs-Exosomal MiRNA-214 Suppresses Oxidative Stress-induced Ca²⁺ Overload And Apoptosis Of C-kit^posCSCs

Background and Objective: The strategy of stem cell therapy is a promising approach to the treatment of myocardial infarction.Endogenous c-kit^poscardiac stem cells（CSCs）originate from the heart itself and may be the optimal progenitor cell for cardiac regeneration.However,in the microenvironment of oxidative stress,the poor ability of engraftment and lower survival rate after transplantation of stem cell can not play an effective role in it’s regenerative repair.Therefore,it is important that enhance the stem cell transplantation or increase the survival rate of endogenous stem cell.Recently such researches have demonstrated that exosomes derived from bone marrow mesenchymal stem cells（BMSCs-Exosomes）can promote the repair process of variety of tissues and cell regeneration by transfer to recipient cells with non-coding RNAs,proteins,cytokines and so on.Accumulating evidence indicates that mi RNA-214 plays important roles in apoptosis,proliferation,and differentiation by targeting Ca MK2 d m RNA.Here are some question worth considering that under certain pathological conditions such as oxidative stress,what happens to the expression quantity of BMSCs-Exosomal mi RNA-214,and whether the c-kit+CSCs of BMSCs-Exosomes preconditioning can increase the cell viability and the moderating effect of homeostatic regulation of Ca²⁺ under oxidative stress by regulate Ca MK2 d via mi RNA-214? These problems have still not been reported now.Methods: Part one 1.c-kit^pos CSCs was isolated and cultured SD rat with the method of enzyme digestion and magnetic bead,and identification of CSCs phenotype;Using immunofluorescence and flow cytometry to identify the positive rate of c-kit on CSCs.Interfere with c-kit^pos CSCs by different concentration hydrogen peroxide thus simulation oxidative stress micro-environment,flow cytometry was used to detect cell apoptosis and death,we also explore the optimum concentration which can induce an oxidative stress cell model of c-kit^pos CSCs,and prepare for subsequent experiments.2.Bone marrow mesenchymal stem cells were isolated from SD rats with whole bone marrow cells.Using flow cytometry to identify P3-P5 BMSCs’ biomaker and cell purity,prepare for subsequent experiments;we imitate the same pathological conditions of c-kit^pos CSCs by different concentrations of hydrogen peroxide impact to BMSCs,and detect MSCs activity with CCK-8.3.We extracted exosomes from the conditioned medium of P3-P5 BMSCs by the combination of step-by-step centrifugation and ultracentrifugation.To identify the Exosomes with Transmission Electron Microscopy,Nanoparticle Tracking Analysis and Western Blotting,and then we added the exosomes labeled with Di I,absorb of Exosomes by CSCs will be observed under fluorescence microscope;Cell Counting Kit-8 method was applied to try to find the BMSCs-Exosomes concentration to prepare for subsequent experiments;4.In order to verify whether Exosomes can play a role in paracrine effects,we designed the following experiment groups:Normal Group（Nor）: c-kit^pos CSCs without any treatment;Control group（Ctrl）: c-kit^pos CSCs under 100μM H2O2 to induce apoptosis;Exosomes-Free group（Exo-F）: c-kit^pos CSCs pretreated with BMSCs’ Exosomes-Free conditioned medium for 24 h,then adding 100μM H2O2 for 2h;Normal Exosomes group（Exo-N）: Exosomes was extracted from the conditioned medium of normal BMSCs,then c-kit^pos CSCs pretreated with BMSCs-Exosomes,then adding 100μM H2O2 for 2h to make sure c-kit^posCSCs under oxidative stress.Using flow cytometry to detect the rate of apoptosis of all groups.Western Blotting detecting the relative expression of cleaved caspase-3（cleaved CASP3）and caspase-3（CASP3）.5.In order to explore whether the function of H2O2 can urge the functional variation of exosome,and then play a more effective cell protection effect,this part of experiment was divided into 4 groups:Normal Group（Nor）,Control group（Ctrl）,Normal Exosomes group（Exo-N）,Exosomes derived from BMSCspretreated with H2O2（Exo-H）: Exosomes was extracted from the conditioned medium of H2O2-preconditioned BMSCs for 2h,then c-kit^posCSCs were pretreated with BMSCs-Exosomes for 24 h,next undergoing 100μM H2O2 stimulated oxidative stress for 2h.Using flow cytometry to detect the rate of c-kit^posCSCs apoptosis of all groups.Detecting the relative expression of cleavedcaspase-3（cleaved CASP3）,caspase-3（CASP3）.Part two 1.To explore the potential mechanism of regulating calcium homeostasis and anti-apoptosis of exosomes,we using Exo Carta database and the research advances in mi RNAs in Ca²⁺-mediated apoptosis of cardiomyogenic lineage,and we select some candidated Exosomal mi RNAs.We use RT-PCR to preliminary verificate prediction results,we first detection of Exosomal mi RNA-214 expression in normal and oxidative stress environment to make sure that whether mi RNA-214 exists in BMSCs-Exosomes,and whether exosomal mi RNA-214 occur differential expression under pathophysiological conditions;we follow detect the expression of mi RNA-214 from groups Nor,Ctrl,Exo-N and Exo-H of the Part 1 to make clear whether the expression levels of mi RNA-214 in c-kit^pos CSCs produce change after experience different treatment.2.In order to verify whether the mi RNA-214 participate in effect of anti CSCs apoptosis,we divided the experiment into 6 groups,Nor group: CSCs without any treatment;Control group: CSCs with 100μM H2O2 treatment for 2h;CSCs-M group: CSCs transfected with mi RNA-214 Mimics for 48h;CSCs-I group: CSCs transfected with mi RNA-214 Inhibitor for 48h;CSCs-M+H2O2 group: CSCs transfected with mi RNA-214 Mimics for 48 h and meanwhile treatment with H2O2 for 2h;CSCs-I+ H2O2 group: CSCs transfected with mi RNA-214 Inhibitor for 48 h and meanwhile treatment with H2O2 for 2h.Flow cytometry was used to detect the apoptosis rate of CSCs in each group.3.Next we go to verify whether BMSCs-Exosomal mi RNA-214 participate in the process of anti apoptosis.At first,we overexpress or inhibit mi RNA-214 in BMSC,the expression of mi RNA-214 in each group was detected under oxidative stress by PT-PCR,then extract Exosomes and the expression of mi RNA-214 was detected by PT-PCR.And then the experiment was divided into 8 groups:Nor group: CSCs without any treatment;Control group: CSCs with 100μM H2O2 treatment for 2h;Exo-H group: BMSCs pretreatment with 100μM H2O2 and extract its Exosomes,then with CSCs co-incubation for 24 h,and these CSCs treatment with 100μM H2O2 for 2h subsequently;Exo-M group and Exo-I group:after overexpression or inhibition of mi RNA-214 in BMSCs,pretreatment ofBMSCs with H2O2 and then extract exosomes,co-cultured with CSCs for 24 h,and these CSCs treatment with 100μM H2O2 for 2h subsequently;Exo-H+CSC-I group,Exo-M+CSC-I group and Exo-I+CSC-I group:in the Exo-H group,Exo-M group and Exo-I group,the expression of mi RNA-214 in CSCs was inhibited.RT-PCR was used to examine the expression of mi RNA-214 in each group;Flow cytometry was used to detect the apoptosis of each group.The above results confirmed that mi RNA-214 play the anti-apoptotic effect in oxidative stress environment,the experiment was divided into four groups,Nor group,Control group,Exo-H group,Exo-H+CSC-I group,Western blotting was used to further detect the expression of apoptosis related protein CASP3 and Cleaved CASP3.And the intracellular calcium load was measured by confocal laser scanning microscope.4.This part of experiment in order to further verify whether Ca MK2 d is the target gene of Exosomal mi RNA-214.We divide the experiment into 6 groups: Nor Group（the same as above）,Ctrl Group（the same as above）,Exo-H Group（the same as above）,Exo-M Group（the same as above）,Exo-I Group（the same as above）,Exo-I+CSC-I Group（the same as above）.RT-PCR was used to detect the relative expression of mi RNA-214 and Ca MK2 d m RNA;Western Blotting to detect the protein level of Ca MK2 d in each group;5.Next,we investigated whether Ca MK2 d plays a critical role in the regulation of Exosomal mi RNA-214 against apoptosis and calcium homeostasis.We divide the experiment into the following groups:（1）Ctrl group,（2）Exo-H group,（3）Exo-H+Ca MK2 d group: overexpression of Ca MK2 d in CSCs and treatment with Exo-H for 24h;（4）Exo-I+CSC-I+si R-Ca MK2 d group:suppress the expression of Ca MK2 d in CSCs and transfection of mi RNA-214 Inhibitors for 24 h,then co-cultured with Exo-I for 24h;group of（1）⁴was establishment of oxidative stress cell model.RT-PCR was used to detect the relative expression of mi RNA-214 and Ca MK2 d m RNA in each group;Western Blotting was used to detect the expression of Ca MK2 d protein in each group;flow cytometry was used to detect the apoptosis rate of CSCs in each group;and the expression of apoptosis related protein was detected by Western blotting;the intracellular calcium load was measured by confocal laser scanning microscope.Results:Part one 1.CSCs was isolated and cultured in vitro in primary SD rat,CSCs covered the bottom of the culture bottle which were polygon or triangles after 1 week;identification of CSCs phenotypeby immunofluorescence and flow cytometry: the results show that the expression of CSCs surface antigen c-kit was over 90%;but it did not express CD45 and CD34.The results of flow cytometry suggest that after treating 100μM H2O2 for 2 hours,early apoptosis rate of c-kit^posCSCs reached 70.68%（P<0.05）,and there was no significant change in mortality（P>0.05）,therefore,this experiment intends to use 100μM H2O2 intervene c-kit^posCSCs for 2h to simulate oxidative stress environment,establishment of oxidative stress cell model.2.By whole bone marrow culture BMSCs,heterozygous cells gradually decreased with the passage of cells,we identificated the phenotype of BMSCs by flow cytometry,the results show that there express CD29 and CD90,but did not express CD45;CCK-8 detect the activity of BMSCs at the same pathological condition,the results showed that compared with 0μM group,the activity of BMSCs reached the maximum value at 100μM for 2 hours（P<0.05）.3.BMSCs-Exosomes were extracted from the conditioned medium of BMSCs by the combination of step-by-step centrifugation and ultracentrifugation.Identification of exosomes by TEM: the sizes were homogeneous,average diameter of 100nm;the appearance was round or Tea-tray,and light in the low density area.Identification of exosomes’ diameter by NTA: the average diameter of 111 nm.Identification of Western Blotting showed that BMSCs-Exosomes express CD9,CD63,Alix.What’s more,CSCs surface appeared red Di I fluorescent after Exosomes-Di I added into culture medium.CCK-8 detect CSCs activity after pretreatment with different concentrations of Exosomes,the results suggest that CSCs activity enhancement with increased Exosomes concentration,and there was no significant difference in CSCs activity after intervene with high concentration（400、800μg/ml）of Exosomes,therefore the concentration of exosomes is 400μg/ml in the following experiment;4.Using flow cytometry to observe c-kit^posCSCs apotosis rates of each group after CSCs with different conditions.The results suggest that compared with Normal groups,the level of early apoptosis were significantly increased in Controlgroups（P<0.05）;compared with Control groups,in both Exo-N groups and Exo-F groups the level of early apoptosis rate were significantly reduced,and the apoptosis rate of Exo-N group was lower than that of group Exo-F（P<0.05）.Moreover,Western Blotting showed that Cleaved CASP3/CASP3 in c-kit^posCSCs,processed with 100μM H2O2 for 2h,was significantly lower than without treatment（P<0.05）.Compared with Control groups,Cleaved CASP3/CASP3 in Exo-N groups and Exo-F groups was lower,but was still higher than Normal groups（P<0.05）.5.Furthermore,we explore the anti apoptotic effect of Exosomes with functional variant which induced by H2O2.The result of flow cytometry showed that both early apoptosis and later apoptosis in Exo-H groups were reduced significantly than Exo-N groups（P<0.05）.What’s more,Compared with Exo-N groups,Cleaved CASP3/CASP3 in Exo-H groups were significantly reduced（P<0.05）.Part two 1.With the development of mi RNAs-related research and Exo Carta database,we screened mi RNA-214 as a candidate Exosomal mi RNAs.We treatment BMSCs with 100μM H2O2 and then extracting BMSCs-Exosomes,RT-PCR was used to detect the expression level of mi RNA-214.The results suggest that Compared with the Normal group,mi RNA-214 up-regulated obviously in Control group（BMSCs-Exosomal treatment with H2O2）（P<0.05）;besides,in order to understand the effect of different treatment conditions on the level of mi RNA-214 in CSCs,the result of RT-PCR show that both in Exo-N group and Exo-H group,the level of mi RNA-214 in c-kit^posCSCs were significantly higher than Control group,and the expression level of mi RNA-214 in Exo-H group was up-regulated more obviously（P<0.05）.2.Whether exosomal mi RNA-214 is involved in the anti apoptotic process?（1）We divide the experiment into 6 groups,the results showed that if without H2O2,there was no obvious apoptosis in CSCs-M group and CSCs-I group,and there was no difference with Nor group（P>0.05）,after treatment with H2O2,compared with Nor group,the apoptosis of Ctrl group increased significantly（P<0.05）,compared with Ctrl,the apoptosis of CSCs-M+H2O2 group was decreased（P<0.05）,the apoptosis of CSCs-I+H2O2 group was up-regulated（P<0.05）.And then overexpression or inhibition of mi RNA-214 inBMSCs,the results show that under the situation of oxidative stress,the expression of mi RNA-214 in exosomes which released from BMSCs was up-regulated or down regulated,extracted exocrine for subsequent trials.（2）The experiment was divided into 8 groups,mi RNA-214 expression in each group was examined by RT-PCR,the results show that compared with Nor,the expression of mi RNA-214 was up-regulated in Ctrl group,after internalization of Exosomes,compared with Crtl,the expression of mi RNA-214 in Exo-H group and Exo-M group increased,the expression of mi RNA-214 was significantly higher in group Exo-M（P<0.05）,and there was no significant difference in mi RNA-214 expression between Exo-I group and Ctrl group.After inhibiting the expression of mi RNA-214 in CSCs,compared with Ctrl,the expression of mi RNA-214 in Exo-H+CSC-I group and Exo-M+CSC-I group was still high level（P<0.05）,the expression of mi RNA-214 was significantly decreased in Exo-I+CSC-I group.Flow cytometry was used to detect the apoptosis of each group,we can find that compared with Ctrl group,Exo-H group,Exo-M group and Exo-I group apoptosis decreased significantly,while the Exo-M group decreased most significantly,followed by Exo-H group（P<0.05）.After inhibition of mi RNA-214 expression in CSCs,we can found that compared with Ctrl group,in group Exo-H+CSC-I and group Exo-M+CSC-I,the apoptosis was still decreased（P<0.05）,however,the apoptosis of the Exo-H group and Exo-M group increased（P<0.05）.While compared with Ctrl group,there was no significant difference in Exo-I+CSC-I group（P>0.05）.3.The above results confirmed the anti-apoptotic effect of BMSCs-Exosomal mi RNA-214,the experiment was divided into 4 groups,Western blotting results show that the expression of Cleaved CASP3 in cell was up-regulated in Ctrl group（P<0.05）,compared with Ctrl group,the expression of Cleaved CASP3 was down regulated in Exo-H group（P<0.05）,while there was no significant change of Cleaved CASP3 expression in Exo-H+CSC-I group（P>0.05）.So whether BMSCs-Exosomal mi RNA-214 was involved in the regulation of calcium homeostasis?The results show that after joining H2O2,the fluorescence intensity of Ca²⁺ in CSCs was significantly increased;comparedwith H2O2 treatment group,the fluorescence intensity of Ca²⁺ in CSCs group decreased significantly in Exo-H group,while there was no significant difference in the Exo-I+CSCs-I intervention group of the fluorescence intensity of Ca²⁺.4.We use Target Scan database,GCBI data platform and KEGG Pathway and other bioinformatics analysis platform to predict mi RNA-214 may be involved in the regulation of BMSCs-Exosomes in the process of oxidative stress and calcium homeostasis by acting on Ca MK2 d m RNA 3 ’UTR.RT-PCR results suggest that compared with Ctrl group,mi RNA-214 in Exo-H and Exo-M group was increased significantly,and the latter is obviously higher than the former;the mi RNA-214 in Exo-I and Exo-I+CSC-I group was significantly lower,and the latter was significantly lower than the former;compared with Ctrl group,there was no significant difference in Ca MK2 d m RNA expression between the five groups（P>0.05）.Western Blotting results show that compared with Ctrl group,the levels of Ca MK2 d protein in Exo-H and Exo-M group were significantly lower,and the Exo-M group decreased significantly（P<0.05）,the levels of Ca MK2 d protein in Exo-I and Exo-I+CSC-I group were significantly increased,and the Exo-I+CSC-I group was significantly lower（P<0.05）;5.To further investigate whether Ca MK2 d is involved in the anti apoptotic process of Exosomal mi RNA-214,the experiment was divided into 4 groups.RT-PCR results showed that compared with Ctrl group,the expression of mi RNA-214 in Exo-H group and Exo-H+Ca MK2 d group increased（P<0.05）;the expression of mi RNA-214 in Exo-I+CSC-I+si RNA-Ca MK2 d group was significantly lower than that in Ctrl group,the difference was statistically significant（P<0.05）,compared with Ctrl group,the expression of Ca MK2 d m RNA in the Exo-H group had no significant change,while the expression of Ca MK2 d m RNA was up-regulated in Exo-H+Ca MK2 d group（P<0.05）,the expression of Ca MK2 d m RNA in Exo-I+CSC-I+si RNA-Ca MK2 d group was decreased significantly,difference was statistically significant（P<0.05）;after Exo-H intervention,the level of Ca MK2 d protein in CSCs was significantly down regulated,while the expression of Ca MK2 d protein was up-regulated in Exo-H+ group Ca MK2d（P<0.05）,the expression of Ca MK2 d protein in Exo-I+CSC-I+si RNA-Ca MK2 d group decreased significantly,thedifference was statistically significant.The results of flow cytometry showed that compared with Ctrl group,the apoptosis of Exo-H+ Ca MK2 d group was significantly h i g h e r t h a n t h a t o f E x o-H g r o u p（P < 0.0 5）,t h e a p o p t o s i s o f Exo-I+CSC-I+si RNA-Ca MK2 d group was decreased,the difference was statistically significant（P<0.05）.The intracellular calcium homeostasis was observed under confocal laser scanning microscope: compared with Ctrl group,the fluorescence intensity of calcium ion in c-kit^posCSCs group decreased significantly in Exo-H group;while the difference of calcium ion fluorescence in Exo-H+Ca MK2 d group was significantly higher（P>0.05）.After inhibition of Exosomes and mi RNA-214 in cells,inhibit Ca MK2 d expression at meanwhile,the difference of Exo-I+CSC-I+si RNA-Ca MK2 d calcium ion fluorescence was decreased obviously,the difference was statistically significant.Conclusion: 1.Using step-by-step centrifugation and ultracentrifugation to successfully extracted exosomes from the conditioned medium of BMSCs.2.H2O2-preconditioning BMSCs-Exosomes existence functional variationmay,and it may regulate intracellular calcium homeostasis in c-kit^pos CSCs cells under oxidative stress and to further regulate the process of apoptosis;3.H2O2-preconditioning induced functional variation of BMSCs-Exosomes,which inhibit Ca MK2 d levels,reduction ox-Ca MK2 level in c-kit^pos CSCs cells,and suppressed H2O2-induced c-kit^posCSCs apoptosis.