| Objective: Stem cell-based restorative treatment has become a new trend of the regeneration and recondition of infracted myocardium. The resident cardiac stem cell possesses not only the characteristics of tissue specificity and low immunogenicity,but also can differentiate into myocardial lineage cells, which is considered to be the ideal seed cells for transferring to new myocardium,and it has provided a promising means of reduceing ventricular remodeling and improveing heart function concomitantly. However, the low survival rate and proliferation ability of the transplanted stem cells in the infracted myocardium faces a bad local microenvironment(such as oxidative stress, inflammation, blood anoxic) which is a major obstacle to achieve long-term improvements after stem cell therapy.Therefore, developing novel strategies to boost stem cell survival and proliferation will be highly beneficial to this field. Numerous studies have shown that miR- 21 involved in regulating cell proliferation, differentiation, apoptosis process in tumor cells and cardiovascular lineage cells. Over expression of miR-21 can promote proliferation and resist hydrogen peroxide-induced apoptosis of tumor cells and myocardial cells. It is well known that PI3K/AKT signaling pathway plays an important role in cell proliferation, differentiation and apoptosis. PTEN,one targeted gene of miR-21, a kind of negative regulator of AKT signaling pathway, holds the ability to promotes cell apoptosis. Therefore,this study was to explore the relationship between overexpression of miR-21 and C-kitpos CSCs’ proliferation and its related mechanism in nomoral oxygen state. At the same time,we demonstrated that the relationship between overexpression of miR-21 and C-kitpos CSCs’ apoptosis induced by hydrogen peroxide to simulate the microenvironment of oxidative stress after myocardial infarction and then analyzeed the underlying mechanisms. Methods: Part one1 C-kitpos CSCs was cultured and selected in SD rats by the methods of enzyme digestion and magnetic bead which we have mastered before.Use immunofluorescence and flow cytometry methods to identificate the purity for subsequent experiment.2. The group of miR-21’s negative control and its simulation(50nM) were transferred in C-kitpos CSCs with life2000 TM,a kind of vector. We established a control group of normal cells without any treatment. After transfection of 24 h, 48 h and 72 h, RT-PCR was used to assess the expressive level of mi R-21,so as to determine the efficiency of transfection and its optimal transfer time.3.This experiment was divided into 3 groups: ①. Control Group: C-kitpos CSCs without any treatment. ②.Mimics nagetive control(MNC): the control cells of miRNA-21 mmics negative transfected. ③.Mimics Group(Mimics): cells were transfected with miR-21. RT-PCRwas used to asses the expression of miR-21 in all groups respectively.4.To verify the relationship between miR-21 and cell differentiation in vitro, we adopted RT-PCR and immunofluorescence to detect Nkx2.5,CD31,α-SMA mRNA and its proteins of early stage markers of myocardial cells in each group All the methods above were observed to insure if overexpression of miR-21 will promote the differentiation of C-kitpos CSCs in vitro. Cell scratch test was used to asses the migration of C-kitpos CSCs transfected with miR-21.CCK8 method was used to the detection of cell viability after transfection by 24 h, 48 h, 72 h. On the basis of the results of CCK8, selecting the best point of time with cell viability. Edu method and Weston blot were used to determine cell proliferation in corresponding time point.5.The results of this study above eloquently showed that overexpression of miR-21 could promote C-kitpos CSCs’ proliferation at a certain extent. To further investigate whether the PTEN/PI3K/AKT signaling pathway involved this process,, we divided the experiment into five groups : ①Control ② MNC. ③Mimics. ④Phen: pretreatment C-kitpos CSCs with Phen for 1h after transfected miR-21, as a positive control of miR-21;⑤Mimics+LY294002: pretreatment C-kitpos CSCs with PI3K/AKT inhibitor LY294002 for 1h after transfected mi R-21. Using Edu and flow cytometry to detect the proliferation,, Weston blot was used to determine the expression of PCNA, a proliferation-associated protein; The PTEN mRNA’s expression in each group were dedected by RT-PCR. Meanwhile Weston blot was used to determine the expression of PTEN, P-AKT, T-AKT genes. Part two:6. Processed C-kitpos CSCs with different concentration of H2O2(200, and 100, and 50 μMol/L) for 2h to simulate hypoxia micro-environment of different degrees.The control group was established with normal cells without H2O2. Detect C-kitpos CSCs’ survival rate, early apoptosis rate and necrosis rate with flow cytometry. Finally, make sure that the best reasonable concentration of H2O2 induced C-kitpos CSCs’ early apoptosis to simulate the hypoxia micro-environment.7. The part of experiment was divided into six groups in order to explore over-expression of miR-21 on apoptosis of C-kitpos CSCs under oxidative stress status.: ①Control. ②MNC③Mimics, ④Control+ H2O2, ⑤MNC+ H2O2 group ⑥Mimics+ H2O2. Mimics and negative for miR-2l was established and transferred into the C-kitpos CSCs for 48 h,then added H2O2(100 μm) for 2h. RT-PCR was used to detect the relative expression levels of miR-21. Flow cytometry was used to detect the apoptosis of C-kitpos CSCs. Western blot and Immunofluorescence was used to detect the expression of Caspase-3, Bax and Bcl-2, in each group.8. This part of experiment was divided into 5 groups for further investigate whether the PTEN/PI3K/AKT signaling pathway plays an important role in the territory to inhibite the apoptosis of C-kitposCSCs in oxidative stress by overexpression of miRNA-21: ① Control, ②Control+H2O2, ③Control+H2O2+Phen, ④Mimics +H2O2 ⑤ Mimics + H2O2 + LY294002; H2O2 group only adde the optimal concentration of H2O2 to treat for 2h. Using flow cytometry to detect the rate of apoptosis of all groups. Using Immunofluorescence to detect the expression of Caspase-3. Micro R-21 and PTENmRNA were examined by RT-PCR.Intracellular signal molecules or apoptosis-related proteins,such as the expression of PTEN,P-AKT,T-AKT,Caspase-3, Bcl-2 and Bax Using Western blot to the detection of in each group such as. were detected by Western bloL expression of pathway related protein PTEN, P-AKT and T-AKT. Results: Part one :1. Primary(Primaryly) cultured CSCs were adhered to the wall within 48 hours, cell morphology were round(rounded) highlights and stretched into polygon or triangles after 4-5days, then the cell proliferation rate was gradually accelerated through the form of logarithm. It reached the period of plateau around 7-8 days. Immunofluorescence detected that c-kit was positive after the separation by the method of magnetic bead.What’s more,The signs of C-kitpos cells were detected by the glow(flow) cytometry, and the results showed that the expression of c-kit were 90.2%, CD45 1.8% and CD34 1.0%.2.C-kitposCSCs were transfected with miR-21 mimics(50nM)by liposome Lipofec-tamineTM2000, RT-PCR was used to detect the expression of miR-21 in each group after transfecting of 24 h, 48 h and 72 h. Compared with the control group, the expression level of miR-21 was significantly higher at each time point in mimics group(P < 0.05), The phenomenon was more obvious after transfection of 48 h. Compared with the control,the expression level of mi R-21 in MNC group was not obviously changed(P >0.05), while the Mimics group was significantly higher after transfecting(of) 48h(P <0.05). Therefore all the transfected treatment time of cells in the following experiment was 48h3.Immunofluorescence assays detected that compared with the Control Group, there were no significant differences in the expression of CD31,α-SMA and Nkx2.5 in Mimics or MNC group.We have not found out any difference in Nkx2.5 and CD31, α-SMA at mRNA levels in these three groups by RT-PCR(P >0.05). So as to its obvious effects on its migration ability(P >0.05).4.CCK8 detection showed that the viability of C-kitposCSCs which compared with the Control group, there was a significant increase in the transfected with miR-21 mimics after 24, 48, 72 hours(P < 0.05), and the cell activity reached a peak at the time point of 48 h after transfected(P < 0.05), but the MNC group did not show much difference significantly(P > 0.05). The cell proliferation ability was detected by Edu. The results showed that after 48 h transfected, the cell proliferation was significantly higher in Mimics group compared with Control group(P <0.05), while no significant difference was found in MNC group(P >0.05). Weston blot also showed the expression of PCNA in Mimics group was significantly increased(P < 0.05), but not in the MNC group(P > 0.05).5.To further investigate whether PTEN/PI3K/AKT signaling pathway play an important role in proliferation by over-expressing miR-21 in the C-kitposCSCs. We divided this experiment into 5 groups.Edu test results showed while compared with the Control Group, proliferation capability of C-kitposCSCs in Mimics and Phen Group significantly increased(P <0.05),but not the MNC group(P >0.05). Compared with Mimics group, the cell proliferation ability was obviously inhibited in Mimics+LY294002 group(P <0.05). FCM was used to detect the cell cycle.We found that compared with the Control group, the number of cells in MNC group in each phase has no significant difference(P > 0.05). But the proliferation of C-kitposCSCs break through the G1/S restriction point into S phase,and the cell proliferation ability was enhanced in Mimics group and Phen group(P < 0.05),while compared with the Mimics group, the number of Mimics+LY294002 cells in S phase cells decreased significantly(P < 0.05). PCNA was determined by Weston blot, The results accord with the each group of Edu and FCM outcomes. RT-PCR results displayed that compared with the Control group, mRNA of PTEN in the Phen group significantly decreased(P <0.05), while in the Mimics group, MNC group and Mimics+LY294002 group did not change obviously(P >0.05). Weston blot results demonstrated that compared with the Control group, the expression of PTEN and P-Akt protein had no significant difference in MNC group(P > 0.05), while the expression of PTEN protein was obviously decreased and the expression of phosphorylated protein Akt increased significantly in both Mimics group and Phen group(P < 0.05).And we also found that the expression of PTEN protein had no significant difference in Mimics+LY294002 group compared with Mimics group(P > 0.05), but the Phosphorylated Akt was lower than Mimics group(P < 0.05). There was no significant difference in the expression of total AKT(P>0.05) in C-kitpos CSCs in each group. Part two :6. FCM was applied to observe C-kitposCSCs apotosis rates. Compared with the Control、100、50μM group, the early apoptosis rates were significantly higher while the cell death rates had no significant change(P >0.05) after treatment H2O2(100μmol/L)for 2 hours. Therefore the oxidative stress microenvironment after myocardial infarction in vitro was simulated by100μmol/L H2O2, for 2h of C-kitposCSCs in the following experiment7. RT-PCR was used to detect the expression level of miR-21.Compared with the Control group, miR-21 up-regulated obviously in Mimics group(P<0.05),While there were no significant difference(p >0.05)in MNC group.After treatment of C-kitposCSCs with H2O2(100μmol/L)for 2 hours,both Control groups and MNC groups showed miR-21 down-regulate(P<0.05).Compared with Control+H2O2 groups, the level of miR-21 was significantly increased(P<0.05) in Mimics+ H2O2 groups.8. Apoptosis rates in C-kitposCSCs H2O2-induced was detected by FMC. Compared with Control groups, The level of apoptosis was no no statistically significant difference(P >0.05)in Mimics group and MNC group. C-kitposCSCs were processed with 100 uM H2O2 for 2h to simulate the microenvironment of oxidative stress after myocardial infarction. Compared with Control groups, apoptosis rate in Control+ H2O2 group and MNC+ H2O2 group was significantly reduced(P<0.05),but survival difference between later two groups were small(p >0.05).Compared with control groups, Mimics+ H2O2 groups were significantly increased the apoptosis rate(P<0.05).but compared with the Control+H2O2 group, the apoptosis rate significantly decreased(P<0.05). Immunofluorescence showed that compared with Control group, the expression of Caspase-3 had no change in Mimics groups, while it was significantly increased both in Control+ H2O2 group and MNC+ H2O2 group. Compared with Control+ H2O2 group, Caspase-3 significantly suppressed in Mimics+ H2O2 group.Western blot analysis revealed that the expression of the Caspase-3,Bcl-2,Bax protein in C-kitposCSCs had no significant difference between Control,MNCand Mimics group(P >0.05), Compared with the Control groups, caspase-3 and Bax(pro- apoptotic protein) expression was remarkablely increased(P < 0.05) in the Control+ H2O2 group and MNC+ H2O2 group. While the expression of anti-apoptotic protein Bcl-2 was prominently decreased(P < 0.05) In Mimics+ H2O2,the expression of pro-apoptotic protein Bax, Caspase-3 were lower(P < 0.05), the expression of anti-apoptotic protein Bcl-2 were higher(P < 0.05) than Control+ H2O2 group.9. In order to further explore whether the PTEN/PI3K/AKT signaling pathway, which was regulated by miR-21, plays an important role in inhibiting the apoptosis of C-kit+CSCs at the risk of the oxidative stress environment, the experiment was divided into 5 groups. FMC displayed that compared with the Control groups, the cell apoptosis rates were obviously increased(P < 0.05) in Control+ H2O2 group, however, in Control +Phen+ H2O2 group and Mimics+ H2O2 group, the apoptosis rates of cells were reduced significantly(P < 0.05)than Control+ H2O2 group. especaily in Mimics+ H2O2 groups. Compared with Mimics+ H2O2 groups, apoptosis rates in Mimics+ H2O2 +LY294002 group were rised with statistical significance(P < 0.05),but still lower than Control+ H2O2 group(P <0.05).Immunofluorescence showed that the expression of Caspase-3 in Control+ H2O2 group was significantly higher thane Control group. Compared with the Control+ H2O2 group,the expression of Caspase-3 significantly reduced in Control +Phen+ H2O2 group and Mimics+ H2O2 group; the level of Caspase-3 in Mimics+ H2O2 +LY294002 group was much higher than Mimics+ H2O2 group.At the same time, Western blot results showed that compared with the control group, the level of pro-apoptotic protein(Caspase-3 & Bax) were increased in Control+ H2O2,and the anti-apoptotic protein(Bcl-2) was suppressed remarkblely( P < 0.05);but the result opposite in Control +Phen+ H2O2 group and Mimics+ H2O2 group. Compared with Mimics+ H2O2 group the level of Pro-apoptotic protein(caspase-3 & Bax protein)were increased, the anti-apoptosis protein Bcl-2 protein was decreased(P< 0.05) in Mimics+ H2O2 +LY294002 group.10.The RT-PCR results showed that, compared with the Control group, miR-2 1 was significantly down-regulated in the Control+ H2O2 group andControl +Phen+ H2O2 group(P<0.05).However, there was no significant difference between Control+ H2O2 group and Control +Phen+ H2O2 group(P>0.05). The level of miR-21 increased significantly(P <0.01) in the Mimics+ H2O2 group and Mimics+ H2O2 +LY294002 group, there is no difference between the two groups(P>0.05). Compared with the Control group, the level of PTEN mRNA was increased significantly(P<0.05) in the Control+ H2O2 group, Mimics+ H2O2 group and Mimics+ H2O2 +LY294002 group, while Control +Phen+ H2O2 group changed oppositely(P<0.05). Western blot analysis showed that compared with Control group, Protein of PTEN was increased significantly in the Control+ H2O2 group, and the protein level of p-Akt was decreased significantly(P<0.05);However, Control +Phen+ H2O2 group and Mimics+ H2O2 group, the expression of PTEN was suppressed, and the expression of p-Akt was increased with statistical significance(P<0.05). Compared with Mimics+ H2O2 group, not only the expression of PTEN,but also the p-akt were decreased significantly(P< 0.05)in the Mimics+ H2O2 +LY294002 group, The total Akt protein level had no difference in each group(P > 0.05). Conclusion: 1. C-kiposCSCs can be successfully isolated and sorted out,FMC showed that 90.2% ofcells were c-kit positive after the purification which were in good growth state. 2. C-kiposCSCs were incubated with miR-21 mimics or its negative control by usingtransfection reagent lipofectamine 2000,RT-PCR showed a significant increase ofmiR-21 when cells were transfected with miR-21 mimics 48 later. 3. MiR-21 is efficient in promoting proliferation in c-kitposCSCs which is contributed bythe PTEN/PI3K/AKT signal pathway 4. We esterblished an in vitro oxidative stress model with 100 uM H2O2 to stimulated themicroenvironment of infracted myocardium 5. The anti-H2O2-induced apoptpsisi effect of miR-21 in c-kitposCSCs is partiallycontributed by PTEN/PI3K/AKT signal pathway. |
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