| PART I: Changes of lncRNA-MALAT1 and miR-211-5p in peripheral blood of patients with ischemic strokeObjective: To clarify the expression changes of LncRNA-MALAT1 and miR-211-5p in peripheral blood of patients with ischemic stroke.Method: Peripheral venous blood samples of healthy subjects and patients with ischemic stroke were collected in February 2020 to June 2020 in Chongqing Dianjiang county people’s hospital.q RT-PCR was used to observe the expressions changes of LncRNA-MALAT1 and miR-211-5p.Results: According to the diagnostic criteria of ischemic stroke,a total of 40 healthy subjects and 41 ischemic stroke patients were included in our study.1.There were no significant differences in age,sex,diabetes mellitus,total cholesterol(TC),triglyceride(TG),and low density lipoprotein(LDL-C)between the two groups.However,significant differences in hypertension and high-density lipoprotein(HDL-C)levels were found in the ischemic stroke group.2.The NIHSS scores of the ischemic stroke group were mainly between mild and moderate severe.3.Compared with healthy subjects,LncRNA-MALAT1 expression was significantly increased and the expression of miR-211-5p was significantly decreased,there was a negative correlation between LncRNA-MALAT1 and miR-211-5p.4.There was no clearly linear relationship between LncRNAMALAT1,miR-211-5p and NIHSS scores.Conclusion: LncRNA-MALAT1/miR-211-5p may be involved in the occurrence and development of ischemic stroke.Part II: Effect of lncRNA-Malat1 /miR-211-5p axis on neuronal injury in rats subjected to cerebral ischemia reperfusionObjective: To observe the expressions of LncRNA-Malat1 and miR-211-5p in brain tissue of MCAO/R-induced rats.Our study adopted that intervention the LncRNA-Malat1 and miR-211-5p invided and combinated to explore the role of LncRNA-Malat1/miR-211-5p axis in neuronal injury of rats with cerebral ischemia reperfusionMethods:1.MCAO/R modelMale SD rats weight 260-280 g were used to build MCAO/R model,intraperitoneal injection with 1% sodium pentobarbital(45 mg/kg)depth of anesthesia for rats,fully exposed CCA,ligation near heart of CCA and ECA,arterial clip the ICA,at the intersection of the ICA and ECA,between CCA ligation cut mouth,nylon suture along the incision into until the initial period of middle cerebral artery(depth of about 18 mm),draw after 1.5h to restore blood flow perfusion,24 h reperfusion.2.Experimental groupAnimals were divided into three groups: Normal group(Sham),model group(MCAO/R),n=8.To observe the interference efficiency of LV-Malat1-RNAi,animals were divided into four groups: Sham,model(MCAO/R),MCAO/R+ noload virus group(LV-NC)and MCAO/R+ lentivirus interference group(LV-Malat1-RNAi),n=3.Combined intervention of lncRNA-Malat1 +miR-211-5p on cerebral ischemia reperfusion injury in rats,the animals were divided into seven groups: Sham operation group(Sham),model group(MCAO/R),MCAO/R +empty vector virus group(LV-NC),MCAO/R +virus group(LV-Malat1-RNAi),MCAO/R+control group(Antagomir-NC+CSF),MCAO/R+Antagomir-211-5p(Antagomir-211-5p),MCAO/R+LVMalat1-RNAi + Antagomir-211-5p(LV-Malat1-RNAi + Antagomir-211-5p),n=12.3.Detection methods and observation indexesThe change of blood in brain during the modeling process was observed by Doppler blood flow meter.Zea-Longa score was used to evaluate the neurological function deficit of MCAO/R-treated rats.TTC staining was used to detect the cerebral infarction volume.The pathological changes of cortex were detected by HE staining.The m RNA expressions of LncRNA-Malat1,miR-211-5p and COX-2 were detected by q RT-PCR.ELISA kit for IL-10 and PGE2 to test their expressions.The localization of COX-2 in neurons was labeled by immunofluorescence.The expressions of proteins such as COX-2,BCL-2 and BAX in brain tissue were detected by western blot.Results:1.A drop in blood flow to the brain of more than 70 percent when the cord was inserted,which was observed by the Doppler flow meter.2.LncRNA-Malat1 was dramatically up-regulated,the expression of miR-211-5p was significantly decreased after MCAO/R.3.Compared with the Sham group,the neurological deficit and cerebral infarction in MCAO/R group were significantly increased.COX-2m RNA and protein were significantly up-regulated,and BAX protein was significantly up-regulated.miR-211-5p was significantly down-regulated;Nuclear hyperchromatism,nuclear pyknosis and vacuoles increased obviously.The content of PGE2 was significantly increased and IL-10 was significantly decreased.4.Compared with LV-NC,knockdown of LncRNA-Malat1 expression significantly improved the neurological deficit score of rats and significantly reduced the cerebral infarction volume;HE staining showed that knockdown of LncRNA-Malat1 significantly reduced the nuclear hyperchromatism and nuclear pyknosis of cortical neurons,and reduced the formation of vacuoles.The expressions of COX-2 and BAX proteins were down-regulated,while the expression of BCL-2 was up-regulated.The number of COX-2 positive neurons was decreased which was detected by Immunofluorescence staining.The expression of IL-10 was significantly up-regulated,and PGE2 was significantly down-regulated.q RT-q PCR results showed that the COX-2 m RNA significantly downregulated after knocking down the LncRNA-Malat1,and miR-211-5p expression was significantly up-regulated.5.Compared with the antagomir-NC +CSF group,the antagomir-211-5p group,the neurobehavioral score and cerebral infarction volume were significantly increased in the Antagomir-211-5p group.neuronal nuclei hyperchromaticity and nuclear pyknosis increased,the number of vacuoles increased,and the pathological injury aggravated.COX-2 protein was significantly up-regulated,BAX protein expression was up-regulated,and BCL-2 was down-regulated.The immunofluorescence showed that the COX-2 positive neurons obviously up-regulated.The IL-10 was downregulated and the up-regulate of PGE2 was pretty obvious.q RT-PCR results showed that compared with the antagomir-NC +CSF group,the expression of COX-2 m RNA was significantly increased in the antagomir-211-5p group,and the expression level of miR-211-5p was significantly decreased in the Antagomir-211-5p group,6.Compared with the LV-Malat1-RNAi group,the cerebral infarction volume and neurobehavioral score were significantly increased in the combined intervention group.HE staining showed that the combined intervention group could reverse the protective effect of knockdown of LncRNA-Malat1,with increased vacuoles,nuclear hyperchromia and nuclear pyknosis.The expressions of COX-2 and BAX proteins were increased,while the expression of BCL-2 was decreased.Immunofluorescence staining indicated that the number of primary neurons with COX-2 positive in brain tissue significantly upregulated.ELISA results suggested that IL-10 was significantly up-regulated and PGE2 expression was increased,but there was no significant between the groups.q RT-PCR results showed that COX-2 m RNA was up-regulated,but there was no significant difference.miR-211-5p was significantly downregulated.Conclusion:1.The expression of LncRNA-Malat1 was up-regulated and miR-211-5p was down-regulated in the MCAO/R-induced rats,their expression were negatively correlated.2.LncRNA-Malat1 knockdown significantly alleviated cerebral ischemia reperfusion injury.Meanwhile,the expression of COX-2 and BAX protein was dramaticly reduced,An increase in BCL-2,COX-2m RNA was down-regulated,and miR-211-5p was up-regulated,the level of IL-10 was significantly increased,PGE2 was reduced.3.Antagomir-211-5p induced more severe injury in rats.the expression of COX-2 and BAX protein was increased,down-regulation of BCL-2 and PGE2,the expression of COX-2m RNA was significantly increased,miR-211-5p was decreased.4.The effect of LV-Malat1-RNAi on cerebral CIRI in rats can be reversed by Antagomir-211-5p5.Down-regulation of lncRNA-Malat1 upregulates the expression of miR-211-5p,which associated with COX-2-related inflammatory response and apoptosis,contribute to a significant protective effect on CIRI in rats.Part III: Down-regulation of LncRNA-Malat1 protected PC12 cells from OGD/R-induced injuryObjective: To investigate the effect of lncRNA-Malat1 on OGD/Rinduced PC12 injury.Methods:1.OGD/R modelPC12 cells were cultured in vitro,replaced with DMEM glucose-free medium,and cultured in a three-atmosphere incubator for 2h,the conditions of the three-atmosphere incubator were:37℃,95%N2 and5%CO2,and then replaced with DMEM high glucose complete medium,and cultured for 24 h in a 37℃ ordinary cell incubator.2.Experimental groupsTo observe the change of LncRNA-Malat1 in OGD/R-induced PC12,cells were divided into two groups: Normal and OGD/R,n=3.To observe the role of LncRNA-Malat1 in OGD/R-treated PC12 cells,experimental groups were: Normal group and OGD/R group;In order to investigate the role of lncRNA-Malat1 in PC12 injury induced by OGD/R,LncRNA-Malat1 si RNAs were transfected into PC12 cells 24 h before OGD/R.PC12 cells were divided into four groups: Normal group,OGD/R group,OGD/R+si-NC group(si-NC),OGD/R+LncRNA-Malat1 small interfering RNA group(si RNA),n=3.3.Experimental methods and observation indexesqRT-PCR detected LncRNA-Malat1 expression,PC12 cell vitality was determined by MTT experiment testing,flow cytometry was used to measure the rate of PC12 cells apoptosis,Western blot analyzed the proteins expressions of COX-2,BAX,BCL-2,ELISA kit was used to detect the content of IL-10,PGE2 and TNF-α:compared with normal group,OGD/R group resulted in higher LncRNA-Malat1 expression,cell vitality significantly reduced,cell apoptosis rate increased significantly;Compare with OGD/R group,the viability of PC12 cells was significantly increased after si-Malat1 administration,the apoptosis rate was significantly decreased,the inflammatory cytokines PGE2 and TNF-α were decreased,and the expression of IL-10 was increased.Conclusion:1.The expression of LncRNA-Malat1 significant increase in OGD/R group.2.Downregulation of LncRNA-Malat1 could reduce the OGD/Rinduced injury in PC12 cells,the mechanism might be related to the reduction of COX-2-mediated inflammatory response and reduce cell apoptosis and damage.Part IV: Effect of LncRNA-Malat1 /miR-211-5p axis on primary neuron injury induced by OGD/RObjective: To observe the changes of LncRNA-Malat1 and miR-211-5p in OGD/R-induced neurons,and to clarify the mechanism of LncRNAMalat1 /miR-211-5p axis in OGD/R-induced neuron injury by intervention of LncRNA-Malat1/miR-211-5p.Methods:1.The OGD/R injury model established in primary neurons.The primary neurons were extracted and cultured to maturity.abandon themaintain medium,orifice plate replacement for glucose-free medium DMEM,in 37 ℃,95% N2 and 5% CO2 incubator of three gas,oxygen deprivation of glucose cultivation,the length of 30 min,after keep glucose-free culture medium for culture medium,is due to return to 95% after processing neurons air,5% CO2 incubator double sugar after oxygen,time control for 24 h.2.Experimental groupsTo observe the expression changes of LncRNA-Malat1 and miR-211-5p,primary neurons were divided into two groups: Normal group(Normal),model group(OGD/R),n=3.To observe the interference efficiency of LV-Malat1-RNAi,neurons were individe in to four groups: Normal,model(OGD/R),OGD/R + noload virus group(LV-NC)and OGD/R + lentivirus interference group(LVMalat1-RNAi),n=3.Combined intervention of lncRNA-Malat1 +miR-211-5p on OGD/Rinduced neurons,there was seven groups: Normal,model(OGD/R),OGD/R+empty vector virus group(LV-NC),OGD/R+virus group LVMalat1-RNAi(LV-Malat1-RNAi),OGD/R+Inhibitor-NC(Inhibitor-NC),OGD/R+Ihibitor-211-5p(Ihibitor-211-5p),OGD/R +LV-Malat1-RNAi +Antagomir-211-5p(LV-Malat1-RNAi + Antagomir-211-5p),n=3.3.The main experimental methods and observation indexesImmunofluorescence staining was used to identify the purity of neurons.Neurons viability was detected by MTT assay.Neurons apoptosis was detected by TUNEL staining.The expressions of LncRNA-Malat1,COX-2 m RNA and miR-211-5p were detected by q RT-PCR.The proteins as COX-2,BCL-2 and BAX were detected by Western blot.ELISA kit was used to detect the levels of IL-10 and PGE2.Results:1.Compared with the Normal group,the expression of LncRNAMalat1 in OGD/R-induced primary neurons was significantly upregulated,miR-211-5p was down-regulated.2.Neuronal viability was dramatically increased in the LV-NC group.TUNEL positive cells were significantly decreased,LncRNA-Malat1 and COX-2 m RNA expressions was down-regulated,miR-211-5p expression was increased,the proteins of COX-2 and BAX expressions was downregulated,BCL-2 was up-regulated,PGE2 expression was decreased.However,the expression of IL-10 did not show differences between the groups.3.Compared with Inhibitor-NC group,there was significantly decreased of the cell viability in the Inhibitor-211-5p group,and the number of TUNEL positive neurons was dramatically increased,the expression of COX-2 m RNA was significantly up-regulated,the expression of miR-211-5p was down-regulated,the expression of COX-2was up-regulated,the expression of BAX protein was up-regulated,and the expression of PGE2 was increased.4.Compared with LV-Malat1-RNAi group,the cell viability of LVMalat1-RNAi +Inhibitor-211-5p group was decreased,the number of TUNEL positive cells was up-regulated,COX-2 m RNA was up-regulated,the expression of miR-211-5p was down-regulated,the COX-2 and BAX proteins were up-regulated,and the content of PGE2 was increased.Conclusion:1.Up-regulation of LncRNA-Malat1 and down-regulation of miR-211-5p in OGD/R-induced neuron injury,showing a negative correlation between the expressions of them.2.Knocking down LncRNA-Malat1 alleviate OGD/R-induced injury,reduce neuronal apoptosis,and enhance neuronal activity,the expression of COX-2m RNA and COX-2,BAX protein were significantly decreased,and BCL-2 was significantly up-regulated.miR-211-5p was up-regulated.3.The effect of LncRNA-Malat1-RNAi may be involved into the upregulation of miR-211-5p,and the effect was reversed by Inhibitor-211-5p.4.The lncRNA-Malat1/miR-211-5p axis is involved in neuronal injury by OGD/R-treated,and miR-211-5p may act as the downstream of lncRNA-Malat1 and target endogenous miR-211-5p. |
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