The expression of miR-411 and its effects and mechanism research on proliferation and metastasis in breast cancer / 中国医师杂志

Objective To investigate the relationship between miR-411 and breast cancer,and the expression of miR-411 and its effects and mechanism research on proliferation and metastasis in breast cancer.Methods Real-time polymerase chain reaction (RT-PCR) was used to detect the expression of miR411 in breast cancer cells and tissues.Methyl thiazolyl tetrazolium (MTT),clone formation assay and Transwell assay were used to detect the effect of miR-411 expression on the proliferation,migration and invasion in breast cancer cells.The effect of miR-411 on growth factor receptor-bound protein 2 (GRB2) expression in breast cancer cells was detected by RT-PCR and Western blot.The direct effect of miR-411 target on GRB2 was detected by dual luciferase reporter assay.MTT,clone formation assay and Transwell assay detect the effect of GRB2 expression on the proliferation and invasion in breast cancer cells.Detection the effect high expression of miR-411 on GRB2 downstream signaling pathway related molecules expression in breast cancer cell with Western blot.Results The expression of miR-411 in breast cancer tissues was significantly lower than that in adjacent non-cancerous tissues (P ＜ 0.05).The expression of miR-411 in breast cancer cells SK-BR-3,BT-549 and MDA-MB-231 was significantly lower (P ＜ 0.05).Compared to the negative control group,the transfection of miR-411 mimic inhibited the proliferation,migration and invasion of MDA-MB-231 breast cancer cells (P ＜ 0.05).Targetscan showed that miR-411 could bind to GRB2 3'UTR at position 741-747.Compared with the negative control group,GRB2 3'UTR wild-type plasmid and miR-411 co-transfection reduced the fluorescence activity (P ＜ 0.05).Transfection of siGRB2 significantly reduced the expression of GRB2 protein in MDA-MB-231 breast cancer cells (P ＜ 0.05).Compared to the negative control group,the inhibition of GRB2 expression reduced the proliferation and the number of colony formation of MDA-MB-231 breast cancer cells (P ＜ 0.05).Transwell assay showed that transfection of siGRB2 significantly reduced the number of invasive cells in MDA-MB-231 breast cancer cells (P ＜ 0.05).Conclusions miR-411 is related closely to the occurrence and development of breast cancer,miR-411-GRB2-Ras axis is expected to become a new target for biological treatment of breast cancer.

miR-30c regulates proliferation, apoptosis and differentiation via the Shh signaling pathway in P19 cells

MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that suppress protein expression by binding to the 3′ untranslated regions of their target genes. Many studies have shown that miRNAs have important roles in congenital heart diseases (CHDs) by regulating gene expression and signaling pathways. We previously found that miR-30c was highly expressed in the heart tissues of aborted embryos with ventricular septal defects. Therefore, this study aimed to explore the effects of miR-30c in CHDs. miR-30c was overexpressed or knocked down in P19 cells, a myocardial cell model that is widely used to study cardiogenesis. We found that miR-30c overexpression not only increased cell proliferation by promoting cell entry into S phase but also suppressed cell apoptosis. In addition, we found that miR-30c inhibited dimethyl sulfoxide-induced differentiation of P19 cells. miR-30c knockdown, in contrast, inhibited cell proliferation and increased apoptosis and differentiation. The Sonic hedgehog (Shh) signaling pathway is essential for normal embryonic development. Western blotting and luciferase assays revealed that Gli2, a transcriptional factor that has essential roles in the Shh signaling pathway, was a potential target gene of miR-30c. Ptch1, another important player in the Shh signaling pathway and a transcriptional target of Gli2, was downregulated by miR-30c overexpression and upregulated by miR-30c knockdown. Collectively, our study revealed that miR-30c suppressed P19 cell differentiation by inhibiting the Shh signaling pathway and altered the balance between cell proliferation and apoptosis, which may result in embryonic cardiac malfunctions.

Effects of miR-30c over-expression on early cardiac development of zebrafish / 中华实用儿科临床杂志

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.

Effects of microRNA -30c knockdown on proliferation and differentiation of P19 cells / 中华实用儿科临床杂志

Objective To explore the effects of microRNA(miRNA)- 30c knockdown on proliferation,diffe-rentiation of P19 cells. Methods miRNA - 30c knockdown plasmid(miRNA - 30c knockdown group)or no - load vector(negative control group)was transfected into P19 cells by lipo2000 and stable cell lines were selected by Blastici-din;Dual luciferase reporter gene system was used to confirm miRNA - 30c knockdown. Cell counting kit - 8(CCK - 8) assay was adopted to detect cell proliferation activity. An inverted microscope was used to observe morphological chan-ges of P19 cell differentiation. Cells were induced to differentiated to myocardiocyte with dimethyl sulfoxide(DMSO). Differentiation marker genes including cTnT,NKX2. 5,GATA4 relative mRNA expression levels were detected with real - time quantitative polymerase chain reaction,respectively. Results Observation of green fluorescent protein ex-pression under a fluorescence microscope indicated similar transfection efficiencies,and miRNA - 30c knockdown re-leased the activity of target gene Gli2. As a result,miRNA - 30c knockdown vector was constructed successfully(P ﹤0. 001). During differentiation of mouse P19 cells into myocardial cells,the beating cell clusters in miRNA - 30c knockdown cells were much lower than those in the control cells,and cTnT,NKX2. 5,GATA4 in miRNA - 30c knock-down cells showed significantly lower expression than those in the control cells( all P ﹤ 0. 05). Conclusions miRNA - 30c inhibits the P19 cell proliferation and differentiation. This study gives us a new insight of heart develop-ment and we need more efforts on exploring the deep function of heart diseases.

Effect of shRNA silenced aromatic hydrocarbon receptor on WNT signaling pathway during the differentiation of cardiac myocytes / 临床儿科杂志

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.

Clinical analysis of HAA induction regimen for newly diagnosed and refractory relapsed acute myeloid leukemia / 白血病·淋巴瘤

Objective To analyze the efficacy and safety of HAA induction regimen consisted of homoharringtonine (HHT),cytarabine (Ara-C) and aclacinomycin (ACM) in naive and refractory relapsed acute myeloid leukemia.Methods Data from 66 acute myeloid leukemia (AML) cases hospitalized and treated with HAA induction regimen was analyzed retrospectively.Results 45 of the 66 cases suffered from naive AML,and 21 were refractory relapsed.HAA efficacy in naive AML was evaluated in 41 cases with 36 in complete remission (CR) and 1 in partial remission (PR).The efficiency of HAA induction regimen was 90.2 ％ (37/41)in naive AML group and 42.9 ％ (9/21) in refractory relapsed group,respectively.There were no differences (P ＞ 0.05) when considering patient' s gender,age,disease subtype and white blood cell count at onset.14 patients in CR with naive AML were followed-up for a median time of 9 months (2-17 months),and 5 cases relapsed (35.7 ％) in a range of 2-8 months.The median myelosuppression period was 14 days (3-23 days).Nausea and vomiting [20 ％ (13/66)] were the major side effects of HAA regimen,and the other side effects were abdominal pain and diarrhea [9 ％ (6/66).After chemotherapy,53 ％ (35/66) of the cases experienced infection/fever due to neutropenia.Other severe non-hematological side effects did not occur.Conclusion HAA regimen may be an ideal choice for the induction chemotherapy of naive and relapsed refractory AML.