1. Overexpression and inhibition of miR155 in the cerebral of model rats
To test the efficiency of the re-combined LV- miR155 and LV- miR155 sponge,which intending to make the miR155 over-expressed or under-expressed after stereotaxtic intracranial injection distinguishly,we tested the miR155 levels by qRT-PCR in different groups,including sham group, blank control group(MCAO/R group),over-expression group(MCAO/R+LV-miR155 group),down-regulated group(MCAO/R+ LV- miR155 sponge) and negative control group(MCAO/R+scrambled LV- miR155 group) (Figure 1).As we expected,compared to the sham group,the miR155 levels increased(&, p<0.05),meanwhile,compared to the MCAO/R group, miR155 level in the MCAO/R+LV- miR155 group was up-regulated(*, p<0.05) and in the MCAO/R+ LV- miR155 sponge was down-regulated(*, p<0.05).Moreover, miR155 level had no change in the MCAO/R+scrambled LV- miR155 group(p>0.05).
2. MiR155 negatively influence the survival rate of the model rats
In order to estimate the influence of miR155 on the survival rate of model rats, the survival rates of different groups were calculated (Table 1).All 20 rats in the sham group were survived,that is,the survival rate in the sham group is 100%.While in the MCAO/R group,only 26 out of 53 rats survived,thus,the survival rate was 49%.Interestingly,only 14 out of 42 rats survived in the miR155 over-expressed group,nevertheless,12 out of 20 rats survived in the miR155 inhibited group,with the survival rate was 33% and 60% respectively.In the negative control group,11 out of 21 rats were survived,meaning the survival rate was 52%.In summary, the data above showed that knocking down of miR155 increased the survival rate of model rats,while over-expressed miR155 leading to the survival rate declined.In one word, miR155 negatively influences the survival rate of the model rats.
3. MiR155 positively influence the infarct volume of the cerebra of the rats enduring MCAO/R.
To estimate the influence of miR155 on infarct volume of the cerebra of the rats enduring MCAO/R, the infarct volume and its percentage of different groups were calculated.The infarct volume of the cerebra was estimated by TTC staining of the brain slices of rats in different groups (Figure 2).The pictures in panel A were the results of the TTC staining,the red part of every slice represents normal brain tissue while the white part represents the infarcted brain tissue. The panel B showed the infarct percentages of different groups.Compared with the sham group,the infarct percentages of other groups increased(*, p<0.05). Compared with the blank control group(MCAO/R group),the infarct percentage increased in the miR155 over-expressed group (#, p<0.05),while the infarct percentage declined in the miR155 inhibited group(#, p<0.05).In conclusion,the results described above showed that knocking down of miR155 decreased the infarct volume of model rats,while over-expressed miR155 leading to the survival rates increased,that is, miR155 positively influences the infarct percentage of the model rats,the differences has statistical significance.
4. MiR155 positively influence the TUNNEL-positive cells of the cerebra of the rats enduring MCAO/R.
To estimate the influence of miR155 on cell apoptosis of the cerebra of the rats enduring MCAO/R, the TUNNEL staining of the brain slices of different groups were conducted and the percentage of TUNNEL-positive cells in each group were calculated (Figure 3).Panel A showed the results of TUNNEL staining of different groups,in which the blue fluorescence signal represent the cell nucleus and the green fluorescence signal represent the cells undergoing apoptosis.Panel B is the analysis of the percentage of TUNNEL-positive cells in different groups.It revealed that compared to the sham group,all the other groups had more TUNNEL-positive cells,and compared to the blank control group(MCAO/R group), the percentage of TUNNEL-positive cells increased in the miR155 over-expressed group (#, p<0.05) but decreased in the miR155 inhibited group(#, p<0.05).Moreover,there was no difference between the blank control group and the negative control group(p>0.05). In summary,the results described above showed that knocking down of miR155 decreased the percentage of TUNNEL-positive cells of model rats,while over-expressed miR155 led to the percentage of TUNNEL-positive cells increased,that is, miR155 positively influences the TUNNEL-positive cells of the cerebra of the rats enduring MCAO/R.
5. Expression of miR155 increased but the mRNA levels of Rheb,mTOR,4EBP1 and S6K1 decreased after cerebral ischemia.
In order to observe the change of the expression of Rheb/mTOR and miR155 after cerebral ischemia,qRT-PCR was conducted (Figure 4).The results showed that miR155 levels increased in the MCAO/R group compared to the sham group(&, p<0.05).In contrast,the mRNA levels of Rheb,mTOR,4EBP1 and S6K1 decreased in the MCAO/R group compared to the sham group(&, p<0.05).
6. MiR155 negatively regulated the mRNA levels of Rheb,mTOR, S6K1 and 4EBP1 in the cerebra of the rats enduring MCAO/R.
To estimate the influence of miR155 on the mRNA levels of Rheb,mTOR,S6K1 and4EBP1 in the cerebra of the rats enduring MCAO/R, the qRT-PCR of the brain tissues of different groups were conducted (Figure 5).Panel A showed the qRT-PCR results of Rheb mRNA of different groups,in which reveals that compared to the sham group, Rheb mRNA level decreased(&, p<0.05),however,compared to the MCAO/R group, Rheb mRNA level decreased in the miR155 over-expression group(*, p<0.05) while increased in the hypo-expression group(*, p<0.05),besides,there was no difference in the negative control group(p>0.05).Panel B showed the qRT-PCR results of mTOR mRNA of different groups,in which reveals that compared to the sham group, mTOR mRNA level decreased(&, p<0.05),however,compared to the MCAO/R group, mTOR mRNA level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). Panel C showed the qRT-PCR results of S6K1 mRNA of different groups,in which reveals that compared to the sham group, S6K1 mRNA level decreased(&, p<0.05),however,compared to the MCAO/R group, S6K1 mRNA level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). Panel D showed the qRT-PCR results of 4EBP1 mRNA of different groups,in which reveals that compared to the sham group, 4EBP1 mRNA level decreased(&, p<0.05),however,compared to the MCAO/R group, 4EBP1 mRNA level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). In summary,the results described above showed that knocking down of miR155 increased the mRNA expression of Rheb, mTOR, S6K1 and 4EBP1 in the brain tissue of model rat,while over-expressed miR155 decreased the mRNA expression of Rheb,mTOR, S6K1 and 4EBP1 in the brain tissue of model rat,that is, miR155 negatively influences the mRNA expression of Rheb,mTOR, S6K1 and 4EBP1 in the cerebra of the rats enduring MCAO/R.
7. MiR155 negatively regulated the protein levels of Rheb,mTOR, S6K1 and 4EBP1 in the cerebra of the rats enduring MCAO/R.
To estimate the influence of miR155 on the protein levels of Rheb,mTOR,S6K1and4EBP1 in the cerebra of the rats enduring MCAO/R, the western blot of the brain tissues of different groups were conducted (Figure 6). Panel A showed the western blot results of Rheb,mTOR,S6K1 and 4EBP1 of different groups,in which β-actin was used as inner reference. Panel B was the analysis of the western blot signal of the aforementioned molecules in different groups, reveals that Panel B(a) shows the analysis of Rheb protein in different groups,which reveals that compared to the sham group, Rheb protein level decreased(&, p<0.05),however,compared to the MCAO/R group, Rheb protein level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05),besides,there was no difference in the negative control group(p>0.05).Panel B(b) showed the analysis of mTOR in different groups,which reveals that compared to the sham group, mTOR protein level decreased(&, p<0.05),however,compared to the MCAO/R group, mTOR protein level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). Panel B(c) showed the analysis of S6K1 protein in different groups, which reveals that compared to the sham group, S6K1 protein level decreased(&, p<0.05),however,compared to the MCAO/R group, S6K1 protein level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). Panel B(d) showed the analysis of 4EBP1 protein in different groups,which reveals that compared to the sham group, 4EBP1 protein level decreased(&, p<0.05),however,compared to the MCAO/R group, 4EBP1 protein level decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05) ,besides,there was no difference in the negative control group(p>0.05). In summary,the results described above showed that knocking down of miR155 increased the protein expression of Rheb,mTOR, S6K1 and 4EBP1 in the brain tissue of model rat,while over-expressed miR155 decreased the protein expression of Rheb,mTOR, S6K1 and 4EBP1 in the brain tissue of model rat,that is, miR155 negatively influences the protein expression of Rheb,mTOR, S6K1 and 4EBP1 in the cerebra of the rats enduring MCAO/R.
8. MicroRNA155 negatively regulated the protein levels of p-mTOR, p-S6K1 and p-4EBP1 in the cerebra of the rats enduring MCAO/R.
To estimate the influence of miR155 on the protein levels of p-mTOR, p-S6K1 and p-4EBP1 in the cerebra of the rats enduring MCAO/R, the western blot of the brain tissues of different groups were conducted (Figure 7). Panel A showed the western blot results of p-mTOR, p-S6K1 and p-4EBP1of different groups,in which ß-actin was used as inner reference. Panel B was the analysis of the western blot signal of the aforementioned molecules in different groups, reveals that compared to the sham group, the protein level of p-mTOR, p-S6K1 and p-4EBP decreased(&, p<0.05),however,compared to the MCAO/R group, the protein level of p-mTOR, p-S6K1 and p-4EBP decreased in the miR155 over-expression group(*, p<0.05) while increased in the down-regulated group(*, p<0.05),besides,there was no difference in the negative control group(p>0.05). In summary,the results described above showed that knocking down of miRNA155 increased the protein expression of p-mTOR, p-S6K1 and p-4EBP in the brain tissue of model rat,while over-expressed miRNA155 decreased the protein expression of p-mTOR, p-S6K1 and p-4EBP in the brain tissue of model rat,that is, miRNA155 negatively influences the protein expression of p-mTOR, p-S6K1 and p-4EBP in the cerebra of the rats enduring MCAO/R.