The Mechanism Study Of Myocardial Fibrosis Induced By MiR-223-3p In Rats

At present,cardiovascular diseases are still the major diseases threatening human health,Pathological repair after myocardial injury is the main abnomal pathophysiological process.Mammalian heart is an organ of terminal differentiation,which cannot regenerate after myocardial necrosis.The necrotic myocardium will undergo inflammatory reactions,extracellular matrix deposition,collagen formation,and finally myocardial fibrosis（MF）with increased myocardial stiffness and decreased compliance under the internal and external pathological factors.However,the factors and mechanisms affecting MF are still unknown.On the one hand,it is committed that the solution of myocardial regeneration can provide a broad prospect for myocardial diseases in the clinic.Previous studies have shown that the heart of the lower vertebrates,such as adult zebrafish,can completely regenerate after resecting≈20%heart tissue.Subsequent studies found that newborn mice had the ability of temporary heart regeneration,which disappeared one week later.But none of these findings had been translated into clinical practice;Stem cell differentiation,once a sensation in the treatment of myocardial infarction diseases,had failed to solve the problem of myocardial regeneration.On the other hand,after cardiac injury and other stimulation,cardiac fibroblasts（CF）are activated to proliferate largely,and transdifferentiate into myofibroblasts（MyoFB）with stronger secretory activity and character of smooth muscle cells,which is the most important pathological process of MF.Both CF and MyoFB can secrete abundant extracellular matrix（ECM）.The occurence of MF is mainly caused by excessive accumulation and/or reduction of ECM degradation.Therefore,any factor that can inhibit the activation and transdifferentiation of CF into MyoFB,as well as that can promote the degradation of ECM may inhibit MF;So relevant influencing factors and regulatory genes need to research further during MF development.If the abnormal pathological processes the above mentioned can be effectively controlled or inhibited,MF may be inhibited or reversed,which will be of great benefit to the prevention and treatment of such diseases.A large number of studies on the function of miRNA had found that miRNA is widely involved in the pathophysiological process of cardiovascular diseases,including MF.It has been reported that mir-130a,mir-29,mir-21,mir-22,mir-133,mir-101a,mir-208a,mir-15,mir-489,etc.were related to MF,and mir-29of them had been studied thoroughly.Animal experiments have shown that mir-29 inhibitor was effective for skin and lung fibrosis and is expected to be applied in clinical practice.The latest miRBase data shows that there are more than 2000 kinds of miRNAs in humans,so the role and regulatory mechanism of miRNA in related cardiovascular diseases,including MF,are far from clarified.At the same time,miRNA showed dynamic expression changes in the process of disease,and it was reported that dynamic expression and change indicated stronger biological significance.Mir-223 is involved in many important biological activities.It was related to the occurrence and development of tumor.It could promote the progress of lung cancer、the proliferation and invasion of bladder cancer、increase the malignance of liver cancer.It has become one of the important markers for early diagnosis and prognosis of tumor diseases.Meanwhile,mir-223 expression was also associated with liver fibrosis,pulmonary fibrosis,abdominal aortic aneurysm,etc.In addition,mir-223 also regulates cell biological activities such as apoptosis of liver cells,proliferation and differentiation of CF,invasion and migration of tumor cells.Further studies showed that mir-223 achieved the above biological behaviors by activating TGFβ,smad2/3,p38 and other signaling pathways.The expression and role of mir-223 in cardiovascular diseases has also attracted extensive attention.Recent literature has reported that mir-223 participated in myocardial remodeling after myocardial infarction by regulating RASA1 in mice.Overexpression of mir-223 activated both pro-hypertrophic and anti-hypertrophic signaling pathways,while the terminal effect tended to develop physiological hypertrophy.According to the latest biological information network from targetscan.org,mir-223 includes 256 conservative transcripts,266 conservative and 102 poorly conservative binding sites.Mir-223 can regulate genes in post-transcriptional level by binding different transcripts and conservative or non-conservative sites and affect the biological processes such as myocardial remodeling,cell proliferation,differentiation and apoptosis.In the early stage of this study,it was found that the heart injury was mainly repaired with MF in heart injury mode of neonatal 2-day SD rats,and mir-223-3p expression showed dynamic changes and specific increase during the damage-repair process,which was consistent with the time window of the repair process.It suggested strongly that the mir-223-3p expression might be related to MF,but no systematic study has been reported so far.Therefore,this study includes the following contents:1)To observe repair modes and explore the relationship between morphological changes and mir-223-3p expression after heart injury of the 2-day neonatal rats model;2)To study effect of mir-223-3p on proliferation,collagen synthesis and transdifferentiation of primary CF by vitro experiments;3)Prediction and validation of mir-223-3p target genes.Part I:The correlation about repair modes and the changes ofmir-223-3p expression after heart injury in neonatal SD rats1.Objective:To observe the main cardiac repair modes and screen probably related mirnas with major repair modes after heart injury in neonatal SD rats.2.Methods:50 2-day newborn SD rats for the subjects,the numbers were produced according to random method by computer（n=25/group）,dividng into sham（S）and model（M）group,carrying out heart damage operation（resection of a little heart apex tissue in M group,avoiding heart rupture,the rest operations were both the same）;At the 7 days after postoperation（dap）and 14 dap,the experimental animals（n=3/group）were dislocated and killed,then heart were extracted to detect morphology change by H&E,Masson and picric acid-Sirius red（PSR）staining;The properties of collagen deposition in group M were observed by polarized light after PSR staining at the 7dap and 14dap.At the same time,at7dap,3 hearts of the two groups were sent to high-throughput miRNA microarray detection for screening differentially expressed mirnas.Combined with references,Ten differentially expressed miRNAs（mir-133b,mir-429,mir-412-5p,let-7g-3p,mir-34a,mir-223,mir-299b,mir-3558,mir-222,mir-488）were selected as the experimental subjects of RT-qPCR in vitro.4 cases of heart tissues were collected and detected the relative expression levels of10 miRNAs screened with apical tissues at each time point of 7,14 and 21dap（note:the expression levels of miRNAs in 4 heart tissues were retained for baseline control at 0dap after surgery,and the fold changes of each miRNA at each time point compared with the baseline control were known as the relative expression levels of corresponding miRNAs.）The relative expression levels of miRNAs at different time points were compared and the differentially expressed miRNAs were screened between groups.The dynamic change trend of miRNAs screened in the S and M groups were analyzed,and the miRNAs consistent with the histomorphological change time were identified.3.Results:（1）Morphological changes:7dap:H&E and Masson staining of heart tissue structure were normal in group S.The myocardialcyte（CM）in group M showed obvious degeneration and necrosis,some were fused into slices.In the case of no obvious necrotic tissue,a large number of inflammatory cells were observed in the vicinity of apical injury.The cases with the least injury in group M still indicated obvious collagen deposition in the apex of the heart by Masson staining.Quantitative calculation of the average collagen deposition area under high microscope was significantly greater than that of group S and showed statistical significance（5.38±1.05 vs 0.31±0.09mm²,p<0.01）.14dap:H&E and Masson staining of heart tissue structure were still normal in group S.While H&E staining indicated that the injuried heart tissue was replaced by scar without inflammatory reaction around it in group M;Masson staining showed that the repaired scar was collagen fiber,namely as MF,which was clearly separated from normal myocardial tissue,indicating the completion of repair process.Polarized light was used to observe collagen component in group M at 7 and 14dap,respectively:at 7dap:a small amount of scattered collagen deposition was observed,mainly typeⅢcollagen;while at 14dap,there were more collagen fibers mainly composed of typeⅠcollagen,and some typeⅢcollagen still existed,indicating that most of typeⅢcollagen had been converted into typeⅠcollagen,and the repaired pathological myocardium was mainly composed of typeⅠcollagen.（2）Results of high-throughput miRNA assays:A total of 584 mirnas were detected,with 160 up-regulated,95 down-regulated and 329 unchanged.There were 45 mirnas with more than 2 times,including 26 up-regulation and 19 down-regulation.Ten differentially expressed miRNAs（mir-133b,mir-429,mir-412-5p,let-7g-3p,mir-34a,mir-223,mir-299b,mir-3558,mir-222,mir-488）were selected as the next RT-qPCR experimental subjects in vitro.（3）Comparison of the relative expression levels of mirnas between groups in 7,14and 21dap:7dap:In group M,mir-223-3p and mir-429 were significantly increased,mir-223-3p 404.28 and mir-429 42.74 times respectively.The other miRNAs and all miRNAs in the group S showed increasing trend.Only mir-223-3p expression was significantly different and statistically significant between groups（404.28±302.73 vs6.17±4.61,p<0.05）.At 14、21 dap there were no statistically significant differences in the relative expression levels of miRNAs between groups（p>0.05）.At the same time,mir-223-3p and mir-429 significantly decreased at 14dap,especially mir-223-3p.The dynamic change trend of all miRNAs in the S and M groups was indicated as follows:Mir-223-3p increased most significantly in the first week after cardiac injury,and decreased sharply at 14 days,which was consistent with the repair time after heart injury.It was speculated that mir-223-3p is the main miRNA responsible for collagen deposition and MF after cardiac injury.4:Conclusion:（1）The heart injury of SD rats in newborn 2 days could not be completely regenerated,and MF was the main repair mode.（2）Mir-223-3p showed the most obvious changes in the heart injury model,and showed consistent dynamic changes with the repair time.It was speculated that mir-223-3p may be the main miRNA that causes collagen deposition and MF after cardiac injury.Part II:To study effect of miR-223-3p on the mechanism of myocardiac fibrosis in vitro1.Objective:To explore the gene regulation,molecular and cellular biological mechanisms of MF induced by mir-223-3p in vitro.2.Methods:1)Expression difference of mir-223-3p was detected and compared in vitro CM and CF;2)CF was isolated and cultured,then transfected with mir-223-3p mimic/mimic negative control（NC）/inhibitor/inhibitor NC;3)Target genes have different P_CT（probability of conserved targeting）and TCS（Total context++score）values on www.targetscan.org website,Whether they can provide guidance for the prediction of target genes needs to be verified by experiments.4)The target genes of miRNA were predicted through the biological website.According the screening principle of target genes,MMP16 and MEF2C were selected as the target objectivs of mir-223-3p.Target genes expression of MEF2C,MMP16 andⅠ/Ⅲtype collagen gene expression regulated by miR-223-3p were validated by RT-qPCR.5)MMP16,typeⅠ/Ⅲcollagen protein expression were detected by Western Blot（WB）.6)The vimentin and a-smooth muscle action（a-SMA）protein expression were detected by immunofluorescence induced by mir-223-3p in cultured CF.3.Results:1)Under physiological conditions,the expression of mir-223-3p in CM was significantly lower than that in CF,and the difference was statistically significant（1.00±0.06 vs 30.97±0.98,p<0.05）.2)Target genes MMP16 and MEF2C are both regulated by mir-223-3p.The P_CTT and TCS values of MMP16 and MEF2C are 0.85 and-0.96,0.37 and-0.37,respectively.MMP16 has higher P_CT and TCS values（the absolute value is more closer to 1）than MEF2C.3)Confirmation of screened target genes MMP16 and MEF2C:RT-qPCR verified that mir-223-3p had a negative regulatory relationship with MMP16,while no such correlation with MEF2C,indicating that the closer the absolute value of P_CTT and TCS was to 1,the more likely it was to be a candidate target gene of miRNA.4)Target genes MMP16,MEF2C,typeⅠ/Ⅲcollagen gene expression effected by miR-223-3p were detected in mRNA level:Mir-223-3p mimic significantly inhibited the expression of MMP16,which was significantly different from the NC group（0.32±0.01 vs 1.00±0.03,p<0.001）.The results were the opposite to the former after mir-223-3p imhibitor intervention,MMP16expression was significantly up-regulated,which was significantly different from that of inhibitor NC group（1.60±0.04 vs 1.00±0.02,p<0.001）.The inhibition effect of mir-223-3p mimic on MEF2C expression was not significant,and had no significant difference with NC group（1.06±0.07 vs 1.01±0.17,p>0.05）.No significant difference was observed between groups with mir-223-3p imhibitor intervention（0.82±0.55 vs 1.00±0.10,p>0.05）.MiR-223-3p mimic promoted typeⅠcollagen gene expression,which was significant difference compared with NC group（2.12±0.03 vs 1.00±0.04,p<0.001）,and inhibited typeⅢcollagen gene expression,which was significant difference compared with NC group（0.56±0.02 vs 1.00±0.02,p<0.001）;Contrary to the above results,mir-223-3p inhibitor inhibited typeⅠcollagen gene expression and promoted typeⅢcollagen gene expression,which were significant differences compared with NC group respectively（0.64±0.02 vs.1.01±0.07,p<0.01;1.87±0.04 vs 1.00±0.02,p<0.001）.MMP16,typeⅠ/Ⅲcollagen protein expression were detected by WB in post-transcription level:Mir-223-3p mimic inhibited the expression of MMP16 protein,while mir-223-3p inhibitor had the reverse effect on MMP16 protein expression.The gray value of MMP16protein in mir-223-3p mimic was significant lower than that in mimic NC（0.50±0.03 vs1.00±0.04,p<0.001）.Protein expression of MMP16 was up-regulated by mir-223-3p inhibitor,and the grey value of mir-223-3p inhibitor was significantly higher than that in inhibitor NC（1.52±0.13 vs 1.04±0.03,p<0.05）.Compared with mimic NC,miR-223-3p mimic promoted typeⅠcollagen,inhibited typeⅢcollagen protein expression;while effect of miR-223-3p inhibitor on typeⅠandⅢcollagen protein expression was the reverse results compared with miR-223-3p mimic.The gray value analysis:miR-223-3p mimic promoted typeⅠcollagen protein expression and existed significant difference compared with mimic NC（1.60±0.07 vs0.98±0.04,p<0.01）;miR-223-3p mimic inhibits typeⅢcollagen protein expression and the difference was also significant compared with mimic NC（0.38±0.03 vs.1.32±0.32,p<0.05）;Mir-223-3p inhibitor inhibited typeⅠcollagen protein expression,which was significant difference compared with inhibitor NC（0.45±0.16 vs.0.99±0.01,p<0.05）;TypeⅢcollagen protein expression regulated by miR-223-3p inhibitor was the same as inhibitor NC and no significant difference between groups（1.86±0.43 vs 1.96±0.52,p=NS）.5)Immunofluorescence results:miR-223-3p promoted the expression of vimentin protein,while the expression of a-SMA protein was not significantly changed in cultured CF transfected with the miR-223-3p mimic/mimic NC/inhibitor/inhibitor NC.4.Conclusions:1),The research results showed that miR-223-3p influenced the occurrence of MF by affecting typeⅠ/Ⅲcollagen protein expression and or inhibiting MMP16 target genes expression.2)mir-223-3p promoted MF development probably mainly by influencing the CF proliferation.The partⅢ:the dual-luciferase reporter gene system verified the targeted regulatory relationship between mir-223-3p and MMP161、Objective:To verify the regulatory relationship further between mir-223-3p and the target gene MMP16 by dual-luciferin reporter gene detection system which has the specificity and sensitivity.2、Methods:The binding sites and sequences of miR-223-3p and MMP16 3’UTR were searched through the biological information network.The experiment was divided into three groups:psicheck2-MMP16-Wt+mimics NC;PsiCheck2 MMP16-Wt+rno-mir-223-3p mimics;Psicheck2-MMP16-Mut+rno-mir-223-3p mimics;The wild type and Mutant type（with four bases mutations）of 3’UTR sequence in purpose gene MMP16were inserted into the plasmid attached to the back of the report vector together;then 293T cells were cultivated and contransfected with the plasmid and miR-223-3p,next step was to crack cells 24 hours later.According to dual luciferase application kits instructions,relative luciferase activities were detected.The inhibitory effect of mir-223-3p on the target gene MMP16 was determined by comparing the relative activities among groups.3、Results:mir-223-3p mimics significantly inhibited the luciferase activity of the target gene MMP16-Wt.The activity expression in PsiCheck2 MMP16-Wt+rno-mir-223-3p mimics was obviously weak and statistically significant differences compared with it in psicheck2-mmp16-Wt+mimics NC（0.55±0.03 vs 1.00±0.09,p<0.01）and in psicheck2-mmp16-Mut+rno-mir-223-3p mimics group（0.55±0.03 vs0.79±0.06,p<0.01）.4、Conclusion:The dual-luciferase gene reporting system verified further that MMP16 was a downstream target gene regulated by miR-223-3p.MMP16 affected the synthesis and degradation of ECM and the occurrence of MF.Therefore,mir-223-3p-MMP16-ECM axis may be a potential important therapeutic target for MF.All conclusions:（1）The heart in 2-day newborn SD rats could not be completely regenerated after injury.MF was the main repair mode.Mir-223-3p showed the most obvious changes in the model,and had consistent dynamic changes with the repair period.Mir-223-3p was speculated to be the main miRNA that caused collagen deposition and MF occurrence after cardiac injury.（2）Mir-223-3p may promote MF directly or indirectly by inhibiting the target genes MMP16 mRNA and protein expression,increasing the typeⅠcollagen and vimentin protein expression,reducing typeⅢcollagen protein expression,promoting the CF proliferation.（3）The dual-luciferase assay confirmed that MMP16 was the target gene regulated by mir-223-3p.It suggested that mir-223-3p-MMP16 axis may be a potentially important regulatory pathway for MF.（4）This study indicated that mir-223-3p may be an important regulatory gene for MF after heart injury in rats.