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@#Objective: To construct a hsa-microRNA-224(miR-224) lentiviral expression vector and to establish pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression. Methods: Pri-miR-224 gene fragment was designed and amplified by quantitative real-time polymerase chain reaction (qRT-PCR), and then loaded into GV369 lentiviral vectors (GV369-miR224) by gene recombination technology. GV369-miR-224 lentivrial expression vectors were then identified by PCR and DNA sequencing. The GV369-miR-224 vector fluid was then used to infect pancreatic mucinous cystadenocarcinoma MCC1 cell line to establish the MCC1 cell line stably over-expressing miR-224. The transfection efficiency of GV369-NC and GV369-miR-224 was observed under fluorescence microscopy; and the expression levels of miR-224 in MCC1, GV369-miR-224-MCC1 and GV369-NC-MCC1 cell lines were detected by RT-PCR. Results: The GV369-miR-224 lentiviral vectors were successfully constructed. GV369-miR-224-MCC1 and GV369-NC-MCC1 cells all emit green fluorescence under the fluorescence microscope. The expression level of miR-224 in GV369miR-224-MCC1 cell group was significantly higher than that in negative control GV369-miR-224-MCC1 group and blank control MCC1 cell group (23.45±1.94， 1.46±0.1 and 2.11±0.38, P<0.01), however, there was no significant difference between the two control groups (P>0.05). Conclusion: A pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression was successfully established, and this will provide a new cell model for exploring the function and pathogenesis of miR-224 in pancreatic mucinous cystadenocarcinoma.
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Objective To investigate the expression of miR-141 and miR-224 in non-small cell lung cancer (NSCLC) and its clinical significance.Methods The levels of serum miR-141,miR-224 and squamous cell carcinoma associated antigen (SCC-Ag) were detected by RT-PCR in 128 patients with NSCLC,60 patients with benign lesions (benign group) and 60 healthy subjects (control group).To analyze the relationship between the expression levels of miR-141 and miR-224 and the clinicopathological features of NSCLC.The sensitivity and specificity of miR-141,miR-224 and SCC-Ag in the diagnosis of NSCLC were evaluated by ROC curve,and the correlation between serum miR-141 and miR-224,SCC-Ag were analyzed by Pearson correlation analysis in NSCLC patients.Results The levels of serum miR-141,miR-224 and SCC-Ag in NSCLC group were significantly higher than those in the benign group and the control group[miR-141(2-△△Ct):2.56±0.48 vs 1.08±0.24 and 1.02±0.21,miR-224 (2-△△Ct):3.94±0.82 vs 1.42±0.35 and 1.26±0.30,SCC-Ag (ng/ml):2.75±1.36 vs 0.64±0.47 and 0.52±0.24,all P＜0.01].In patients with NSCLC,the levels of serum miR-141 and miR-224 were correlated with pathological stage,pathological grade and lymph node metastasis (P＜0.05).ROC curve analysis showed that the optimal cut-off values of serum miR-141,miR 224 and SCC Ag (ng/ml) for diagnosis of NSCLC were 1.84,2.85 and 2.03,respectively.The AUC (0.913) of the three combined diagnosis of NSCLC was the largest,and the sensitivity and specificity were better,with 92.5 ％ and 82.7 ％,respectively.Pearson correlation analysis showed that serum miR-141 were positively corre lated with miR-224 and SCC-Ag (r=0.782,0.594,all P＜0.01),and serum miR 224 was positively correlated with SCC-Ag (r=0.594,P＜0.01).Conclusion Serum miR-141 and miR-224 are up-regulated in patients with NSCLC and are associated with clinicopathological features,and may be a new biomarker for the diagnosis of NSCLC.
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Objective To explore the regulatory mechanism of microRNA-224 (miR-224) in the apoptosis and proliferation of human cholangiocarcinoma (CC) cells and examine whether the pathway of miR-224-HOXD10-PAK4 exist in cc cells or not.Methods The expression of miR-224 in CC and adjacent normal tissue were measured by RT-PCR.QBC939 cells were chosen and divided into two groups:cell groups transducted with negative control viruses (NC group) and cell groups transducted with target gene miRNA-down virus (DOMN group).The rate of apoptosis and the number of colonies formed in QBC939 cell lines of DOWN group and NC group were counted.The expression of miR-224,API5,PAK4 and HOXD10 in QBC939 cell lines of DOWN group and NC group were measured by RT-PCR and western blotting.Results (1) MiR-224 expression was higher in CC tissues than that in normal bile duct tissues (P ＜ 0.05).(2) In QBC939 cells lines,miR-224 expression was lower in DOMN group than that in NC group.The clone formation of QBC939 cells in DOWN group was significantly lower than that of NC group.The apoptosis rate of QBC939 cells of DOWN group was significantly higher than that of NC group.(3) The expression of API5 and HOXD10 in QBC939 cells of DOWN group was significantly higher than that of NC group in the RT-PCR and western blotting experiments.(4) The expression of PAK4 in QBC939 cells of DOWN group was significantly lower than that of NC group in the RT-PCR and western blotting.Conclusions MiR-224 has anti-apoptotic effects and can promote the proliferation of QBC939 cells and may serve as an important regulator of QBC939 cell proliferation in vitro.There are miR-224-HOXD10-PAK4 signaling pathways in cholangiocarcinoma.
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Colorectal cancer is the third most common cancer in humans and poses a serious threat to human health.MicroRNAs (miRNAs) affected the mechanism of occurrence and development of colorectal cancer.As an important member of miRNA,miR-224 modulates the migration,proliferation,metastases and drug resistance of colorectal cancer by acting on its downstream targets.The expression of miR-224 affects the prognosis of colorectal cancer.Therefore,this review summarizes the role of miR-224 and its downstream targets in colorectal cancer,it may provide a new method for diagnosis and treatment to colorectal cancer at the cellular and molecular level.
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OBJECTIVE@#To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells.@*METHODS@#Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively.@*RESULTS@#The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = -4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05).@*CONCLUSIONS@#A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.
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Objective To explore the effect of microRNAs 224 and 21 on human glioma stem cells survival and the possible molecular mechanisms.Methods qPCR was used to detect the dysregulated expression of microRNAs in malignant glioma samples, human GBM stem cells, artificially established GBM stem cell lines and human tissues.Caspase 3/7 assay, Annexin V apoptosis/fluorescence assay were performed to determine the effect of miR-21 or miR-224 mimics and inhibitor on cell apoptosis.Living cells count was used to assess miR-21 or miR-224 mimics and inhibitor on cell growth.TargetScan was used to explore potential targets of miR-21 and miR-224, and dual luciferase reporter assay was used to identify whether the 3’UTR of Caspase 3, Caspase 9 and Bim mRNA was a binding target of miR-21 or miR-224.Western blot was used to detect the expression of Caspase 3, Caspase 9 and Bim protein after transfection of miR-21 or miR-224 mimics or inhibitors.Results miR-21 and miR-224 are strongly upregulated in GSC samples, multiple GBM human tumor specimens, and GBM neurosphere stem cell lines ( P<0.05 ) .Caspase 3/7 assay and Annexin V apoptosis/fluorescence assay results showed that miR-224 and miR-21 regulated GSC apoptosis.Living cells count results demonstrated that miR-224 and miR-21 regulated GSC growth.miR-224 and miR-21 regulate pro-apoptotic gene expression by directly targeting Caspase 3, Caspase 9, and Bim 3’-UTRs. Conclusion These results indicate that miR-224 and miR-21 are important physiologic drivers of GSC resistance to apoptosis, providing new points of therapeutic leverage against these treatment-resistant cells.
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Objective To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells. Methods Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. Results The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = −4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05). Conclusions A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and miRNA-122a plasmids possessed good transfection efficiency. The cell growth, invasion and colony-forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.
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miR-224 is a small noncoding RNA that usually binds to response element of target mRNA through the seed sequences,leading to the suppression of translation or mRNA degradation. Recent researches indicate that miR-224 not on?ly aberrantly expresses in multiple tumors(hepatocellular carcinoma,gastric carcinoma, diffuse large B-cell lymphoma, prostatecancer,colorectal cancer, etc),playing an important role in tumor suppressor or oncognen,but also has an effect on the chemosensitivity of tumor.In this article,we review recent studies about the mechanism of miR-224 in the tumor develop?ment ,progression and chemosensitivity.
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Background and purpose:MiR-224 is overexpressed in hepatocellular carcinoma, and participate in invasion and metastasis of cancer. The aim of this study was to investigate the effects of miR-224 antisense oligonucleotide (ASO) on the proliferation and apoptosis of Hep3B cells.Methods:After transfection with miR-224 ASO, and detecting the miR-224 mRNA expression of Hep3B cells by real-time quantitative PCR; the miR-224 expression in Hep3B cells was measured and cell proliferation was analyzed by MTT assay and the colony formation experimentin vitro andin vivo. The cell apoptosis was analyzed by flow cytometry.Results:Compared with the control group, miR-224 ASO significantly reduced the miR-224 mRNA expression in the Hep3B cell(P<0.05), MTT assay results showed that Hep3B cells survived rate decreased greatly after transfection with miR-224 ASO. Clone formation assay revealed that the colony formation rate in miR-224 ASO group was significantly lower than that in the control group.In vivo study further confirmed that miR-224 ASO could inhibit the proliferation of Hep3B cells,and miR-224 ASO group grew substantially slow compared with the negative control. Flow cytometry indicated that miR-224 ASO group promoted apoptosis significantly.Conclusion:miR-224 was overexpressed in Hep3B cells. Reducing the expression of miR-224 can effectively inhibit the growth of Hep3B cells and promote apoptosis. miR-224 may become a new target for the regulation of gene expression in hepatocellular carcinoma.
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Objective To investigate the expression level of miR-21, miR-146a and miR-224 and their clinical significances in cervical cancer .Methods miR-21, miR-146a and miR-224 were extracted from the normal cervical tissues and cervical cancer tissues , and their expression levels were quantified by real-time quan-titative polymerase chain reaction analysis .t test was used to analyze the expression difference of the two tissues and examine the associations between miR expression and the clinicopathological characters .The overall survival was analyzed by log-rank test, and survival curves were plotted according to Kaplan-Meier.Results miR-21 ex-pression levels of cervical cancer tissues and normal cervical tissues were 11.02 ±4.83 and 10.36 ±4.79, and miR-146a expression levels were 24.68 ±7.92 and 22.43 ±7.25, and there was no significant difference for both miR-21 and miR-146a expression between groups (P>0.05).miR-224 expression levels were 5.78 ±1.03 and 3.35 ±0.93 in cervical cancer tissues and normal cervical tissues , and the difference had statistical significance (P<0.001).For cervical cancer tissues, the miR-224 levels were significantly higher in tumors less differentia-ted, in advanced FIGO stage and with lymph nodes metastasis (P<0.05).miR-224 levels showed obvious corre-lation with survival time and the median survival time was shorter in patients with higher expression level of miR -224(P <0.05).COX multivariate analysis showed miR-224 expression level was an independent factor in patients'survival and prognosis ( P<0.05 ) .Conclusion miR-224 is independent predictor for aggressive pro-gression and poor prognosis in cervical cancer , and it might serve as a biomarker for predicting clinical outcome of cervical cancer patients .
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Objective:To investigate the serum level of miR-224 in hepatocellular carcinoma (HCC) patients and its clinical diag-nostic significance. Methods:The serum level of miR-224 was detected by real-time quantitative PCR. This study included 42 cases of patients with HCC, 36 patients with liver cirrhosis (LC), 55 patients with chronic hepatitis B (CHB), and 40 healthy persons (NC). The relative expressions of miR-224 were calculated. The receiver operating characteristic (ROC) curves were analyzed to determine the sensitivity and specificity of miR-224 expression levels in HCC diagnosis. Results:Result shows that the relative miR-224 expression was higher in the serum of HCC patients than that in the CHB, LC, and NC groups. The difference was statistically significant (P0.05). ROC analysis shows that the best critical value of the relative expression levels of miR-224 was 3.47, with sensi-tivity of 82.2%, specificity of 92.8%, and area under the curve of 0.935. Conclusion:The serum level of miR-224 in HCC patients has high specificity, and miR-224 has great potential to become a new serological marker for the diagnosis of HCC.
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Objective To investigate the expression and clinical significance of microRNA-224 and microRNA-378e in colorectal cancer tissues and normal mucosa adjacent to tumor lesions. Methods The gene chip technology was used to detect the different expression of miRNA in colorectal carcinoma tissues and adjacent normal tissues, which was then confirmed by real-time PCR. The relationship between the pathology and clinical data was analyzed. Results The expres-sion level of miR-224 was significantly up-regulated in tumor tissue, while miR-378e was down-regulated in tumor tissue, which was confirmed by real-time PCR. The expression of miR-224 was strongly associated with histological types, while miR-378e was strongly associated with the infiltration depth of colorectal cancer. Conclusion miR-224 is a potent tumor promoter, while miR-378e is a potent tumor suppressor. Both miR-224 and miR-378e can be used as potential colorectal cancer molecular markers.