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Abstract Objectives Some previous studies indicated that the excessive proliferation and migration of Pulmonary Artery Smooth Muscle Cells (PASMCs) could be observed in pulmonary artery intima after Pulmonary Embolism (PE) occurred. In addition, recent studies identified some miRNAs that are differentially expressed in the blood of PE patients, which might be used as a diagnostic biomarker for PE, including let-7a-5p, let-7b-5p, and miR-150-5p. Hence, the authors sought to explore the effects of let-7b-5p in PASMC proliferation and migration and the corresponding regulatory mechanism. Methods Platelet-Derived Growth Factor (PDGF) was utilized to induce the hyper-proliferation model in PASMCs. The mRNA and protein expression levels were detected by RT-qPCR and western blot, respectively. The proliferation of PASMCs was evaluated by the detection of PCNA expression, as well as CCK-8 and Edu assays. Wound healing and Transwell assays were exploited to assess the migration ability of PASMCs. The targets of let-7b-5p were predicted based on two bioinformatics online tools. Dual-luciferase and Ago2 pull-down assays were applied to confirm the interaction between let-7b-5p and IGF1. Results 40 ng/mL PDGF was selected as the optimal concentration to induce PASMCs. let-7b-5p mimics suppressed the proliferation and migration of PDGF-induced PASMCs, while let-7b-5p inhibitor led to the opposite result. In further mechanism exploration, IGF1 was predicted and confirmed as the direct target gene of let-7b-5p. The promotion role of IGF1 overexpression on the proliferation and migration of PDGF-induced PASMCs was dramatically countered by let-7b-5p mimics. Conclusion let-7b-5p prohibits the proliferation and migration of PDGF-induced PASMCs by modulating IGF1.
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Objective To investigate the significance of serum Let-7b in prostate cancer (PCa). Methods From July 2015 to September 2016, 72 patients with PCa (PCa group) and 34 healthy people (control group) were selected. The Let- 7b was detected by Western blot and fluorescence quantitative polymerase chain reaction (PCR), and the Let-7b was compared between 2 groups. Results The Let-7b in PCa group was significantly lower than that of control group (0.81 ±0.38 vs.1.31 ±0.34), and there was statistical difference (P<0.01). In PCa group, the Let- 7b in patients with TNM T3 and T4 stage was significantly lower than that in patients with TNM T1 and T2 stage (0.74 ± 0.39 vs. 0.88 ± 0.38), and there was statistical difference (P<0.05); the Let-7b in patients with hormone dependence was significantly higher than that in patients with hormone resistance (1.03 ± 0.40 vs. 0.64 ± 0.27), and there was statistical difference (P<0.05). Receiver operating characteristics curve analysis result showed that Let-7b sensitivity and specificity were 72.2％ and 88.2％ in the diagnosis of PCa. Conclusions Let-7b can indicate the degree of malignancy of the tumor and the hormone resistance. For patients with the high risk of early diagnosis of refractory PCa, the patients′ individualized treatment plan could be adjusted.
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Objective To explore the role of fibroblast growth factor receptor ( FGFR ) 1 in endothelial homeostasis via an induction of microRNA let-7s, with effects on AcSDKP(N-acetyl-seryl-aspartyl-lysyl-proline) and associated mitochondrial biogenesis. Methods Blocking FGFR1 signaling pathway, Western blot and immunofluorescence staining were used to measure mitochondrial fusion ( mitofusin-2, MFN2;optic atrophy protein 1, OPA1 ) and fission ( dynamin-related protein-1, DRP1 ) proteins and mitochondrial biogenesis by MitoTraker Green. Also real-time quantitative PCR(qPCR) was performed to test microRNA let-7' expression. Results FGFR1 signaling pathway was critical for AcSDKP maintaining mitochondrial biogenesis through induction of microRNA let-7b. In endothelial cells, the AcSDKP restored the triple[TGF-β2, interleukin (IL)-1β, tumor necrosis factor (TNF)-α]-suppressed microRNA let-7b-5p expression and associated with mitochondrial biogenesis. Such effect of AcSDKP was lost in either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1 treated-cells. Similarly, AcSDKP lost its effect on mitochondrial biogenesis in microRNA let-7b-5p inhibitor-treated-cells. In addition, microRNA let-7b-5p mimic reversed the FRS2 siRNA-suppressed mitochondrial biogenesis in endothelial cells. Conclusion These findings demonstrated that FGFR1 is critical for maintaining mitochondrial biogenesis through control of microRNA let-7b-5p in endothelial cells.
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Objective To explore the change of microRNA let-7b in heat acclimatization/heat stroke rat models and its relation with HSP70,IL-6,TNF-α,TGF-3 and IL-12,and analyze its clinical significance.Methods 60 male rats with almost the same anal temperature,weight,and weeks' age were selected from Laboratory Animal Center in the Second Military Medical University.They were randomly divided into control,heat acclimatization,heat stroke groups averagely.Heat acclimatization/heat stroke rat models were built in hot climate simulated animal tank,blood was collected from cordis apex and serum was separated.Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to test microRNA let-7b in serum,enzyme-linked immunosorbent reaction (ELISA) was to measure the protein level of HSP70,IL-6,TNF-α,TGF-β and IL-12 in serum.Kruskal-Wallis H test was used to analyze quantitative data in the three groups,Spearman rank correlation coefficient was used to reveal relation between two variables in heat stroke group.Results M values of miRNA let-7b in control,heat acclimatization,heat stroke groups were 0.99,1.04 and 1.93 separately,Q values were 0.30,0.25 and 0.44 (x2 =38.95,P＜0.001),separately,with statistical significance.The results of pairwise comparison showed no statistical difference in control and heat acclimatization (P＞0.05),but there were differences in control and heat stroke,heat acclimatization and heat stroke groups statistically (P＜0.05).Spearman correlation analysis showed that in heat stroke group,let-7b was positively related with HSP70,TNF-α and IL-6 (rs =0.579,0.498 and 0.609,P＜0.05) with statistical significance.Conclusion miR let-7b might involve in the pathology of heat stroke,it provided a potential biomarker for monitoring patients in high temperature and humidity clinically.
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Objective To investigate diagnostic value of miRNAs for esophageal cancer .Methods Blood samples were obtained from patients with esophageal cancer .MiRNAs including miR-30a ,miR-126a ,let-7b were detected and compared with healthy con-trols .After adjusting smoking and insobriety ,Logistic regression was used to judging which was best for clinical guidance .ROC curve was used to analyzed the clinic value of these miRNAs .Results Age of the two groups had no significant difference(P>0 .05) .The levels of smoking ,alcoholism and family history of cancer in the two groups had significant difference (P<0 .05) .Two groups′miR-30a levels in plasma had no significant differences (P>0 .05) .Let-7b and miR-126a levels in plasma in patients with e-sophageal were significantly higher in healthy volunteers (P<0 .05) .After adjustment the factors of smoking and alcohol ,let-7b lev-els were associated with the risk of esophageal cancer (P<0 .05) .Area under the ROC curve was 0 .712 ,which had a certain esoph-ageal cancer diagnostic performance .Conclusion Plasma let-7b could be treated as a biomarker for the diagnosis of esophageal canc-er.
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Objective To investigate diagnostic value of miRNAs for esophageal cancer .Methods Blood samples were obtained from patients with esophageal cancer .MiRNAs including miR-30a ,miR-126a ,let-7b were detected and compared with healthy con-trols .After adjusting smoking and insobriety ,Logistic regression was used to judging which was best for clinical guidance .ROC curve was used to analyzed the clinic value of these miRNAs .Results Age of the two groups had no significant difference(P>0 .05) .The levels of smoking ,alcoholism and family history of cancer in the two groups had significant difference (P<0 .05) .Two groups′miR-30a levels in plasma had no significant differences (P>0 .05) .Let-7b and miR-126a levels in plasma in patients with e-sophageal were significantly higher in healthy volunteers (P<0 .05) .After adjustment the factors of smoking and alcohol ,let-7b lev-els were associated with the risk of esophageal cancer (P<0 .05) .Area under the ROC curve was 0 .712 ,which had a certain esoph-ageal cancer diagnostic performance .Conclusion Plasma let-7b could be treated as a biomarker for the diagnosis of esophageal canc-er.
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Objective Vaccination is a most effective method for the prevention of severe diseases caused by pandemic influenza and microRNA ( miRNA) mediated gene silencing has offered a novel approach to the construction of new vaccines.Our study aimed to construct a recombinant influenza A ( H1 N1 ) virus with the PB1 gene that carries the target fragment of miRNA Let-7b. Methods After comparing the sequence of the A/Nanjing/108/2009 H1N1 viral fragments with that of Let-7b, we selected PB1 as the optimal gene sequence, inserted the Let-7b binding target gene into PB1, ligated the modified fragments with pDP 2000, and named the recombinant plasmids pDP-mu-PB1 and pDP-sclb-PB1, respectively.We co-transfected the MDCK and 293T cells with the recombinant and other seven plasmids and injected the supernatant into the allantoic cavity of the chickenembryo for virus propagation, followed by detection of the virus by hemagglutination ( HA) assay and measurement of the viral titer by TCID50 .We amplified the viral cRNA by RT-PCR and identified the viruses by agarose gel electrophoresis and nucleotide sequence analysis. Results PB1 was the optimal sequence ( 83 bp -107bp) for the attenuation of viruses.The HA-titers of miRT-H1N1 and scbl-H1N1 were 1∶32 and 1∶64, and their viral loads were 4.68 ×105 and 7.94 ×104 TCID50/mL, respectively.Nucleotide sequence analysis showed the expected fragment in the rescued virus. Conclusion A recombinant strain vaccine was successfully constructed, which has laid the foundation for fur-ther assessment of virulence.
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BACKGROUND: MicroRNA (miRNA) is a class of noncoding protein RNA as a promising biomarker for various diseases. In this study, the expression of let-7b, miR-30c, and miR-200c was studied in breast cancer tissues to evaluate the potential relationship with known clinicopathological parameters. METHODS: Quantitative real-time polymerase chain reaction was performed to determine the expression level of three miRNAs in 37 pairs of noncancerous normal and cancer tissues and an additional 38 cancer tissues from patients with invasive ductal carcinoma. RESULTS: miR-200c expression was higher in cancer tissues compared to noncancerous normal tissues, and its ratio was correlated with patient age at surgery, type of surgery, and Ki-67 expression. The expression level of let-7b in cancer tissues was inversely correlated with lymph node metastasis, histological grade, and Ki-67 expression but positively correlated with estrogen and progesterone receptor expression. miR-200c expression level was positively correlated with Her-2 expression. The miR-30c expression level in breast cancer was not correlated with any parameters. CONCLUSIONS: miR-200c and let-7b could be used as biomarkers in patients with breast cancer, but its pathological mechanism should be determined.