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Objective To explore the effects of miR -30c over -expression on early cardiac development of zebrafish.Methods The single -cell -stage zebrafish embryos were micro -injected with 3 different concentrations of miR -30c mimics in order to model the miR -30c over -expressed animal.Besides,the effects of miR -30c overex-pression on cardiac development were investigated.The mortality rate and the heart rate were assessed.Beside,the gross morphology and heart morphology of the zebrafish were observed.Moreover,the molecular mechanisms underlying miR -30c function in zebrafish cardiac development was explored,and in -situ hybridization was performed.Results Compared with the wild -type embryos,the mortality rate of zebrafish was increased with the rising miR -30c concen-tration.Furthermore,when the miR -30c mimic concentration was up to 8 μmol/L,the mortality rate reached 80% at 96 h post -fertilization.Simultaneously,the heart rate,which was viewed as an important index to cardiac function,was decreased.Moreover,the pericardial edema was getting worse over time with increasing concentration of overexpression. Then,the cardiac specific gene expression on the zebrafish embryos by whole -mount in situ hybridization was ex-plored.The area of the cardiac chamber was extended and the heart tube looping was negatively affected.Besides,the expression of atrioventricular canal marker genes was absent in zebrafish embryos from miR -30c over -expression groups.Conclusion miR -30c can impact the early cardiac development of zebrafish.
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Objective To observe the expression changes in microRNA (miR)-379 in the developmental process of the mouse heart and during the differentiation of P19 cells into cardiac myocytes,and to explore the possible relationship between miR-379 and the differentiation of cardiacmyocytes.Methods Heart tissues were collected from fetal mice in pregnant ones at their gestational age (8.5,11.5,14.5 and 18.5 days) respectively.Heart tissue sections of the fatal mice were obtained to observe the heart development process.Then total RNA was isolated from heart tissues by using the TRIzol method.Complementary DNA was synthesized from 1 μg total RNA by using a Reverse Transcriptase Kit.Finally,real-time PCR (RT-PCR) was employed to detect the expression of miR-379.At the same time,P19 cells were cultured with 10 mL/L Dimethyl sulfoxide in suspension for 4 days to form cell aggregation,and these aggregations were transferred into 6-wells plate for culturing by adherence.Beating cells were detected with microscopy on the 10th day after induction.Afterwards,total RNA was extracted from cultured P19 cells at different time points.Reverse transcription was executed to get DNA.At last,RT-PCR was used to explore the expression of miR-379 on 0,4,6,10 days after aggregation.Results The expression level of miR-379 was down-regulated gradually in the developing heart (at gestational age of 8.5,11.5,14.5,16.5 days,respectively),and there were significant differences on the different days (F =21.13,P ＜ 0.05).On the other hand,myocardial markers of troponin T represented an increasing trend during the process of P19 cells induction,which demonstrated that P19 cells were successfully induced into cardiomyocyte-like cells.Meanwhile,miR-379 showed a low expression on day 0 of P19 cells aggregation.On day 4,miR-379 demonstrated a higher level.Afterwards,miR-379 proved to be down-regulated gradually.Conclusions miR-379 plays a role in the process of the heart development,but the specific mechanisms need further research.
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Objective To explore the effect of shRNA silenced aromatic hydrocarbon receptor (AHR) on WNT signaling path-way during the differentiation of P19 cells into cardiac myocytes. Methods The eukaryotic expression vector of mouse AHR gene was designed and constructed. The interference plasmid was transfected into P19 cell and the positive stains to AHR gene silencing were screened by G418. The mRNA expression of important genes GSK3βandβ-catenin were evaluated by real-time fluorescent quantita-tive PCR during the differentiation of P19 cells. Results The constructed AHR-shRNA plasmid significantly inhibited the expression of AHR gene. Along with the differentiation of P19 cell into cardiac myocytes, in the interference group the expression ofβ-catenin gene was lower whereas the expression of GSK3βgene was elevated than those of control group with significant differences (all P<0.01). Conclusions The interference of AHR gene expression can regulate WNT signaling pathway in the development of heart.