Objective To construct human recombinant lentiviral expression vector of microRNA-210 (miR-210)and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 wasconstructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfectedas control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescencedetection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. Theconcentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells werecultured in 96-well culture plate coated with Matrigel to assess the abil ity of capillary formation. Results The recombinantplasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescencedetection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-timefluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than thatin LV-GFP group (t= —11.10，P=0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group (12.52% ± 0.67%) was significantly lower than that in LV-GFP group (73.22% ± 1.45%) (t= —66.12，P=0.00).The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group[(305.29 ± 16.52) pg/mL vs. (42.52 ± 3.11) pg/mL, t= —27.06，P=0.00]. In vitro capillary-l ike formation assay showed that thenumber of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 ± 6.33 vs. 6.33 ± 2.33,t= —2.83，P=0.04). Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully andHUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facil itates further study on themolecular functions of miR-210 in angiogenesis.
Objective To review the advance in the experimental studies of microRNA(miRNA) and the relationship between miRNA and stem cells. Methods The related literature was reviewed, and the research findings of miRNA and stem cell were summarized. Results miRNA was noncoding small RNA (20-25 nt) involved in posttranscriptional change, that have been shown to regulate gene expressions. Ithas been reported that some kinds of miRNAs were likely important regulators forstem cells maintaining their state of selfrenewal，and play key roles in theirdifferentiation. Conclusion miRNA as regulation of gene expressions, can be served as a new way for stem cells research.
Hematopoietic stem cells (HSCs) are tissue specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Recently, people have learned a lot about the embryonic HSCs on their development and homing. During their differentiation, HSCs are regulated by the transcription factors, such as Runx1 and Notch signaling pathway, etc. MicroRNAs also regulate the self-renewal and differentiation of hematopoietic stem/progenitor cells on the post-transcriptional levels. Since the onset of circulation, the formation of HSCs and their differentiation into blood cells, especially red blood cells, are regulated by the hemodynamic forces. It would be of great significance if we could treat hematologic diseases with induced HSCs in vitro on the basis of fully understanding of hemotopoietic stem cell development. This review is focused on the advances in the research of HSCs' development and regulation.
microRNA (miRNA) is a kind of RNAs which involves in the regulation of proliferation, invasion and metastasis of tumors. It also closely connects to the colorectal cancer. This article reviews the relationship between miRNA and proliferation, invasion, metastasis, diagnosis and treatment of colorectal cancer.
ObjectiveTo summarize the regulating mechanism of microRNA in tumor microenvironment. MethodThe literatures about the studies on the mechanism regulated by microRNA for tumor microenvironment were reviewed according to the results searched from PubMed in recent years. ResultsmicroRNA might be participated in regulation of tumor microenvironment factors such as hypoxia-inducible factor, tumor associated fibroblasts, extracellular matrix, which leaded to a change in biological behavior of tumor cells by reforming the microenviroment. ConclusionsmicroRNA has been participated in regulating many factors of tumor microenvironment. The change of neoplastic microenvironment has been recognized to play a critical role in the development of cancer. Therefore revealing microRNA mechanism for tumor microenvironment could not only help exploring the biological behavior of tumor cells, but also come an important insight for new means of diagnosis and treatment of cancer.
ObjectiveTo investigate the function and possible mechanism of microRNA-199b-5p (miR-199b-5p) in Ewing's sarcoma cell lines so as to provide theoretical basis for biological treatment in the future. MethodsA673 cells and TC252 cells were adopted as Ewing's sarcoma cell lines in vitro. miR-199b-5p oligonucleotide fragments (mimic) and scramble control (mimic control) were used to transfect TC252 cells and A673 cells, respectively. The expression of miR-199b-5p was measured in 2 cell lines by real-time quantitative-PCR and compared with that in mesenchymal stem cells (MSCs). The cell proliferation was examined by cell counting kit 8. The cell cycle and apoptosis were detected by flow cytometry. The possible targets of miR-199b-5p were determined by luciferase assays. The protein expressions of possible targets were measured by Western blot. ResultsThe expression of miR-199b-5p in control group was significantly down-regulated in A673 cells and TC252 cells when compared with that in MSCs (P<0.05); and the expression of miR-199b-5p in experimental group was significantly up-regulated when compared with that in control group (P<0.05). G1 phase cells increased obviously and S phase cells decreased significantly compared with corresponding cells in control group (P<0.05); but no significant difference was found in G2/M phase cells between 2 groups (P>0.05). The apoptosis rate increased significantly in experimental group when compared with that in control group (P<0.05). The possible targets of miR-199b-5p were cyclin-L1 (CCNL1) and c-Kit by luciferase assays. Western blot results showed that the CCNL1 and c-Kit protein expression levels in experimental group were significantly lower than those in control group (P<0.05). ConclusionmiR-199b-5p can suppress the cell proliferation, block the cell cycle, and promote the cell apoptosis, so miR-199b-5p inhibits Ewing's sarcoma cell lines by targeting CCNL1 and c-Kit.
Objective To explore the diagnostic value of miRNAs for pancreatic cancer. Methods PubMed, Scopus, Web of Science, CBM, CNKI, WanFang Data and VIP databases were retrieved from inception to December 31st 2015, to collect diagnostic accuracy studies about miRNAs for pancreatic cancer. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies by using the QUADAS-2 tool. Then meta-analysis was performed using MetaDiSc 1.4 and Stata 12.0 softwares. Results A total of 40 articles involving 109 studies were included. The results of meta-analysis showed that the pooled Sen, Spe, +LR, –LR and DOR were 0.81 (95%CI 0.80 to 0.82), 0.77 (95%CI 0.75 to 0.78), 3.15 (95%CI 2.78 to 3.58), 0.27 (95%CI 0.24 to 0.31) and 13.58 (95%CI 10.89 to 16.94), respectively. The AUC of SROC was 0.86 (95%CI 0.84 to 0.88). Subgroups analysis showed that: as to diagnostic accuracy, Caucasian was superior to Asian (AUC=0.89vs. 0.84); multiple-miRNAs profiling-based assays was superior to single miRNA assays (AUC=0.91vs. 0.84). Conclusion Current evidence suggests that miRNA has potential diagnostic value for pancreatic cancer, particularly using multiple miRNAs. Due to limited quality of the included studies, more high quality studies are needed to verify the above conclusion.
Objective To determine the expression levels of micro RNA (miR)-196, miR-217, and transforming growth factor β receptor 1 (TGFβR1) protein in the pancreatic ductal adenocarcinoma tissues and its adjacent tissues, to reveal the relationship among them in the pathological process of pancreatic ductal adenocarcinoma. Methods A total of 30 cases’ pancreatic ductal adenocarcinoma tissues and its adjacent tissues were collected. The expression levels of miR-196b and miR-217 in the pancreatic ductal adenocarcinoma and adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction method, the level of TGFβR1 protein was detected by Western blotting method. Results In the pancreatic ductal adenocarcinoma tissues, the expression levels of miR-196b and TGFβR1 protein were significantly higher than those of adjacent tissues (P<0.001), while the level of miR-217 was significantly lower than that of adjacent tissues (P=0.001). For further detection, the level of miR-196b in pancreatic ductal adenocarcinoma tissues was significantly positively correlated with the expression level of TGFβR1 protein (r=0.803, P<0.001), while the expression level of miR-217 was negatively correlated with the expression level of TGFβR1 protein (r=–0.839, P<0.001). Conclusions Expression TGFβR1 protein in pancreatic ductal adenocarcinoma tissues may be bi-directionally regulated by miR-196b and miR-217. This two-way regulating mechanism may be one of the important mechanisms for restricting the development of pancreatic ductal adenocarcinoma, implying a potential target for treatment of pancreatic cancer.
Objective To summarize the mechanism of microRNA in the recurrence and metastasis of hepatocellular carcinoma (HCC) and its possible clinical application. Methods The relevant literatures at home and abroad were reviewed to summarize the results of various scholars. Results microRNA played an important role in cell proliferation and apoptosis of HCC. microRNA of different types promoted or inhibited the recurrence and metastasis of HCC through different action targets and molecular pathways. Conclusions microRNA has a regulation role in the recurrence and metastasis of HCC, and the depth mechanisms study of microRNA in the recurrence and metastasis of HCC provides great significance to clinical therapy. The miRNA is expected to be one of the new target on the prediction and treatment of recurrence and metastasis in HCC.
Objectives To evaluate the expression levels of serum microRNA-21 (miRNA-21) and microRNA-155 (miRNA-155) from patients and rats with pancreatic cancer, and to explore its value in the diagnosis of pancreatic cancer. Methods The clinical materials and the serum samples from 18 patients with pancreatic cancer (pancreatic cancer group) and 12 patients with benign pancreatic disease (benign pancreatic disease group) admitted to Fujian Medical University Union Hospital between January 2016 and December 2016 were collected prospectively. The real-time fluorescent quantitative PCR was performed to detect the levels of serum miRNA-21 and miRNA-155. 7, 12-dimethylbenz (a) anthracene (DMBA)-induced pancreatic cancer rat models (n=20) and the models of the blank control group (sham operation, n=10) were established and the serum samples from the pancreatic cancer group and the blank control group were measured by the real-time fluorescent quantitative PCR, to detect the levels of miRNA-21 and miRNA-155. Results The median expression levels of serum miRNA-21 and miRNA-155 were 1.99 (1.43–5.30) and 7.06 (4.98–21.48) in the pancreatic cancer group, as well as 1.28 (0.58–2.01) and 2.20 (1.76–3.02) in the benign pancreatic disease group. The expression levels of serum miRNA-21 and miRNA-155 were significantly higher in the pancreatic cancer group (Z=–2.621,P=0.009; Z=–3.430,P=0.001). In animal studies, the rat models of pancreatic cancer were successfully established and 11 rats with pancreatic cancer were acquired, as well as 9 rats in the blank control group were acquired. The median expression levels of serum miRNA-21 and miRNA-155 were 2.12 (1.33–2.72) and 16.45 (7.18–25.40) in the rat pancreatic cancer group, as well as 1.00 (0.45–1.60) and 1.49 (1.25–1.97) in the blank control group. The expression levels of serum miRNA-21 and miRNA-155 were significantly higher in the rat pancreatic cancer group (Z=–2.621,P=0.009; Z=–3.609,P<0.001). For distinguishing pancreatic cancer from benign diseases, the best cutoff value of serum miRNA-21 level was 4.21 and the sensitivity and specificity were 75.0% and 61.1% respectively; the best cutoff value of serum miRNA-155 level was 4.67 and the sensitivity and specificity both were 83.3%. Conclusions The serum miRNA-21and miRNA-155 levels are elevated both in patients and rats with pancreatic cancer. Detection of serum miRNA-155 will be helpful to some extent to distinguish pancreatic cancer from benign diseases.