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Objective:To explore the regulatory role of miR-210 in hypoxia-induced epithelial-mesenchymal transition (EMT) of pancreatic cancer PANC1 cells.Methods:PANC1 cells cultured in normoxia and hypoxia were established in normoxia group and hypoxia group. Recombinant plasmid carrrying miR-210 mimics and miR-210 antagomirs were constructed. The recombinant plasmids were transfected with PANC1 cells cultured in normoxia and hypoxia by liposome method to establish cell lines of miR-210 overexpression (miR-210 mimics normoxia group) and miR-210 expression inhibition (miR-210 antagomirs hypoxia group). The blank plasmids were transfected to establish blank plasmid normoxia group and blank plasmid hypoxia group. Relative expression levels of miR-210 for PANC1 cells were determined by qRT-PCR in each group. Western blot was used to measure the expressions of HIF-1α, NF-κB and EMT related protein such as E-cadherin, β-catenin, vimentin and N-cadherin. Cell relative viability under gemcitabine and in vitro cell invasion ability were detected by CCK8 and Transwell chamber experiments, respectively. Results:The relative expressions of miR-210 in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group. The expression levels of HIF-1α, NF-κB and mesenchymal cell markers such as vimentin and N-cadherin in hypoxia group were significantly higher than those in normoxia group (0.74±0.06 vs 0.40±0.05, 1.58±0.16 vs 1.09±0.13, 0.46±0.04 vs 0.17±0.02, 1.27±0.07 vs 0.40±0.03) and the epithelial cell markers such as E-cadherin and β-catenin were significantly lower (0.40±0.07 vs 0.77±0.10, 0.35±0.02 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 mimics normoxia group were significantly higher than those in blank plasmid normoxia group (0.91±0.08 vs 0.40±0.06, 1.52±0.17 vs 1.05±0.14, 0.82±0.06 vs 0.66±0.07, 0.76±0.04 vs 0.46±0.03) and E-cadherin and β-catenin were significantly lower (0.38±0.07 vs 0.65±0.09, 0.50±0.03 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 antagomirs hypoxia group were significantly lower than those in blank plasmid hypoxia group (0.31±0.05 vs 0.55±0.06, 0.68±0.05 vs 1.11±0.13, 0.41±0.03 vs 0.74±0.07, 0.69±0.06 vs 0.78±0.05), while E-cadherin and β-catenin were significantly higher (0.72±0.13 vs 0.50±0.07, 0.71±0.04 vs 0.54±0.05). All the differences among the groups were statistically significant (all P value <0.05). Under gemcitabine, the relative viability of PANC1 cells in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group at 48 h (1.10±0.10 vs 0.76±0.05, 1.46±0.11 vs 1.12±0.09) and 72 h (1.12±0.13 vs 0.76±0.05, 1.54±0.13 vs 1.12±0.09) accordingly. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group at 48 and 72 h (0.75±0.09 vs 1.10±0.10, 1.19±0.12 vs 1.46±0.11). All the differences among the groups were statistically significant (all P value <0.05). The number of transmembrane cells in hypoxia group and miR-210 mimics normoxia group was significantly higher than those in normoxia group and blank plasmid normoxia group, respectively (417.50±81.22 vs 228.30±47.71, 371.30±72.81 vs 245.00±33.62 per high field), while those in miR-210 antagomirs hypoxia group was significantly lower than those in blank plasmid hypoxia group (228.30±54.01 vs 433.30±65.63 per high field). All the differences among the groups were statistically significant (all P value <0.05). Conclusions:miR-210 could weaken the sensitivity to gemcitabine and promote the invasion of PANC1 cells by regulating the occurrence of the hypoxia-induced epithelial-mesenchymal transition.
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Objective: To investigate the role of MicroRNA-210 (miR-210) in the growth process of ovarian cancer cells and its relationship with radiosensitivity, and to elucidate the effect of miR-210 on the development and treatment of ovarian cancer the possible molecular mechanism. Methods: The human ovarian cancer OVCAR3 and SKOV3 cells were cultured in vitro, miR-210 mimic and anti miR-210 inhibitors were transfected into the human ovarian cancer cell lines OVCAR3 and SKOV3, respectively, by cell transfection, and identified by Real-time PCR method; miR-210 overexpression and low expression of ovarian cancer cell models were obtained The two kinds of cells were divided into control group, miR-210 overexpression model group and miR-210 low expression model group, and the cell proliferation activity was detected by MTT The cells in control group and miR-210 overexpression model group were irradiated with different doses (30, 50, and 100 Gy) of ionizing radiation, and the cell proliferation activity in each group was detected by MTT method Western blotting method was used to detect the expression levels of apoptosis-related proteins in the ovarian cancer cells exposed to 50 Gy radiation dose. All the experimental cells were cultured with three double holes and repeated three times. Results: Compared with control group, the proliferation activity of the cells in miR-210 overexpression ovarian cancer cells group was enhanced, and the proliferation activity of the cells in miR-210 low expression cells group was reduced (P<0.05). After ionizing radiation, the proliferation activity of ovarian cancer cells in miRNA-210 overexpression group was enhanced compared with control group (P<0.05); the expression levels of apoptosis-related protein BAX in OVCAR3 and SKOV3 cells were significantly decreased, while the expression level of BCL-2 were significantly increased. Conclusion: miR-210 can promote the growth of ovarian cancer cells, and reduce the sensitivity of ovarian cancer cells to radiation therapy by inhibiting the apoptosis.
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Objective To investigate the expression and clinical significance of microRNA-210 (miR-210)in breast cancer tissues,and to investigate its effect on the proliferation and metastasis of human triple negative breast cancer cell line MDA-MB-231 in vitro. Methods The breast cancer tissues and paracancerous tissues in 82 patients were collected in the Department of Pathology of Hunan Cancer Hospital from December 2013 to September 2015. Quantitative real-time polymerase chain reaction (qRT-PCR)technique was used to detect the expression level of miR-210 in tissues and cells. The relationship between the expression of miR-210 and clinical data and prognosis of patients were analyzed. The triple negative breast cancer cell line MDA-MB-231 transfected with full-length miR-210 plasmid was regarded as test group,and the cell transfected with blank vector was regarded as control group. CCK-8 assay was used to detect the proliferation ability of cells in both groups. Transwell invasion and migration assays were used to detect the metastasis and invasion ability of cells. Results The results of qRT-PCR showed that the expression level of miR-210 was 0. 198 ± 0. 014 in breast cancer tissues,which was significantly higher than that in paracancerous tissues (0. 084 ± 0. 009),and the difference was statistically significant (t = 8. 141,P < 0. 001). The expression level of miR-210 in triple nega-tive breast cancer tissues was 0. 254 ± 0. 026,which was significantly higher than that in non-triple negative breast cancer tissues (0. 167 ± 0. 015),and the difference was statistically significant (t = 3. 175,P =0. 003). There were significant differences in TNM staging and molecular typing between the patients with high and low expression of miR-210 (χ2 = 7. 859,P = 0. 005;χ2 = 7. 053,P = 0. 008). The 4-year survival rate of patients with high expression of miR-210 was significantly lower than that of patients with low expression of miR-210 (49. 37％ vs. 76. 80％),and the difference was statistically significant (χ2 = 4. 743,P = 0. 024). The results of qRT-PCR showed that the expression of miR-210 in cells in test group was 0. 517 ± 0. 038,which was significantly higher than that in control group (0. 284 ± 0. 022),and the difference was statistically significant (t = 9. 280,P < 0. 001). The results of CCK-8 assay showed that the proliferation abilities of the test group were significantly higher than those of the control group in 48,72 and 96 h (3. 771 ± 0. 452 vs. 3. 206 ± 0. 314;7. 662 ± 0. 619 vs. 6. 736 ± 0. 552;15. 477 ± 1. 425 vs. 11. 592 ± 1. 243),and the differences were statistically significant (t = 2. 296,P = 0. 025;t = 2. 496,P = 0. 019;t = 4. 594,P = 0. 001). The results of Transwell invasion assay showed that the cell number of test group in inferior surface was 107. 8 ± 13. 0,which was significantly higher than that of control group (74. 4 ± 10. 9),and the difference was statistically significant (t = 3. 732,P = 0. 001). The results of Transwell migration assay showed that the cell number of test group in inferior surface was 136. 5 ± 18. 5,which was significantly higher than that of control group (87. 4 ± 15. 7), and the difference was statistically significant (t = 4. 256,P < 0. 001). Conclusion The expression of miR-210 in breast cancer tissues is high,and its expression is closely related to progression,malignancy and progno-sis of patients. In vitro,miR-210 can promote the malignant behavior of triple negative breast cancer cell line MDA-MB-231. It is a potential molecular marker and targeted treatment site.
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Objective:To investigate the role of MicroRNA-210(miR-210)in the growth process of ovarian cancer cells and its relationship with radiosensitivity,and to elucidate the effect of miR-210 on the development and treatment of ovarian cancer the possible molecular mechanism.Methods:The human ovarian cancer OVCAR3 and SKOV3 cells were cultured in vitro,miR-210 mimic and anti miR-210 inhibitors were transfected into the human ovarian cancer cell lines OVCAR3 and SKOV3,respectively,by cell transfection,and identified by Real-time PCR method;miR-210 overexpression and low expression of ovarian cancer cell models were obtained.The two kinds of cells were divided into control group,miR-210 overexpression model group and miR-210 low expression model group,and the cell proliferation activity was detected by MTT.The cells in control group and miR-210 overexpression model group were irradiated with different doses(30,50,and 100 Gy)of ionizing radiation,and the cell proliferation activity in each group was detected by MTT method.Western blotting method was used to detect the expression levels of apoptosis-related proteins in the ovarian cancer cells exposed to 50 Gy radiation dose.All the experimental cells were cultured with three double holes and repeated three times.Results:Compared with control group,the proliferation activity of the cells in miR-210 overexpression ovarian cancer cells group was enhanced,and the proliferation activity of the cells in miR-210 low expression cells group was reduced(P<0.05).After ionizing radiation,the proliferation activity of ovarian cancer cells in miRNA-210 overexpression group was enhanced compared with control group(P<0.05);the expression levels of apoptosis-related protein BAX in OVCAR3 and SKOV3 cells were significantly decreased,while the expression level of BCL-2 were significantly increased. Conclusion:miR-210 can promote the growth of ovarian cancer cells,and reduce the sensitivity of ovarian cancer cells to radiation therapy by inhibiting the apoptosis.
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Background and purpose: miR-210 was closely related to the occurrence and development of gastric cancer, but its mechanism and clinical significance were still not clear. The purpose of this study was to explore the expression of miR-210 in gastric cancer tissues and its clinical significance. Methods: The expression of miR-210 was detected in gastric cancer tissues and the corresponding adjacent tissues. The relationship between the expression of miR-210 and clinical pathological factors and prognosis was analyzed. Results: Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) showed that the expression of miR-210 in gastric cancer was higher than that in adjacent tissues, and there was a significant difference between the two groups (P<0.05). The expression of miR-210 was closely related to tumor size, lymph node metastasis and TNM stage, but was not related to age, gender, tumor dif-ferentiation and depth of invasion. The 5-year survival rate of patients with low miR-210 expression was 48.2%, where-as the 5-year survival rate of patients with high miR-210 expression was 30.4% (χ2=4.216, P=0.040). Conclusion: The expression of miR-210 in gastric cancer was higher than that in adjacent tissues, and maybe related to the development and prognosis of gastric cancer. miR-210 is expected to be a new diagnostic marker and therapeutic target for gastric cancer.
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Objective The aim of this study was to evaluate the value of serum exosomal miRNAs,originating from tumor tissue cells,could be used as noninvasive biomarker for distinguishing clear cell renal cell carcinoma (ccRCC).Methods 30 pairs of tissue samples and the corresponding serum samples were collected from 20 ccRCC male patients and 10 female ccRCC patients,operated in our department from June 2015 to June 2016.Their age ranged from 45 to 70 years old,mean 57 years old.Based on the miRNA microarray analysis of ccRCCs miRNA expression profiles,we picked up four miRNAs,including miR-210,miR-224,miR-452,and miR-34a,to confirm our hypothesis.Then the expression quantity of these four miRNAs in tissues,serums and serum exosomes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).Sensitivity,specificity and area under curve (AUC) for serum miRNA levels were determined using receiver operator characteristic (ROC) analysis.Results Expression of miR-210,miR-224,and miR-452 were higher in tumor tissues compared to those in adjacent noncancerous tissues (P＜0.05),increasing by 20.51-fold,54.08-fold and 2.48-fold respectively.But only miR-210 was significantly higher in ccRCC patients compared to healthy controls (HCs) in serum and serum exosome (P ＜0.05),increasing by 2.45-fold and 2.32-fold respectively.ROC curve analysis indicated that the serum exosomal miR-210 level might serve as a useful biomarker for differentiating patients with ccRCC from those with HCs;the AUC was 0.8789 (95％ CI 0.7803-0.9775) and the sensitivity and specificity was 92.1％ and 80.0％,respectively.Conclusion The detection of miR-210 in the serum exosome is useful for early diagnosis of clear cell renal cell carcinoma.
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Objective The aim of this study was to evaluate the value of serum exosomal miRNAs,originating from tumor tissue cells,could be used as noninvasive biomarker for distinguishing clear cell renal cell carcinoma (ccRCC).Methods 30 pairs of tissue samples and the corresponding serum samples were collected from 20 ccRCC male patients and 10 female ccRCC patients,operated in our department from June 2015 to June 2016.Their age ranged from 45 to 70 years old,mean 57 years old.Based on the miRNA microarray analysis of ccRCCs miRNA expression profiles,we picked up four miRNAs,including miR-210,miR-224,miR-452,and miR-34a,to confirm our hypothesis.Then the expression quantity of these four miRNAs in tissues,serums and serum exosomes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).Sensitivity,specificity and area under curve (AUC) for serum miRNA levels were determined using receiver operator characteristic (ROC) analysis.Results Expression of miR-210,miR-224,and miR-452 were higher in tumor tissues compared to those in adjacent noncancerous tissues (P＜0.05),increasing by 20.51-fold,54.08-fold and 2.48-fold respectively.But only miR-210 was significantly higher in ccRCC patients compared to healthy controls (HCs) in serum and serum exosome (P ＜0.05),increasing by 2.45-fold and 2.32-fold respectively.ROC curve analysis indicated that the serum exosomal miR-210 level might serve as a useful biomarker for differentiating patients with ccRCC from those with HCs;the AUC was 0.8789 (95％ CI 0.7803-0.9775) and the sensitivity and specificity was 92.1％ and 80.0％,respectively.Conclusion The detection of miR-210 in the serum exosome is useful for early diagnosis of clear cell renal cell carcinoma.
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<p><b>OBJECTIVE</b>To investigate the microRNAs (miRNAs) expression profile of acute myocardial infarction (AMI) rats and the regulating effects of Huoxue Anxin Recipe (, HAR) on angiogenesis-related miRNAs and genes.</p><p><b>METHODS</b>Forty-five Wistar rats were randomly assigned to 3 groups according to a random number table: sham, AMI, and AMI+HAR groups (15 in each group). AMI rats were established by ligation of the left descending coronary artery. HAR was intragastrically administered to rats of the AMI+HAR group for successive 21 days since modeling, meanwhile the same volume of 0.9% normal saline was administered to rats of the sham and AMI groups. Doppler echocardiography was used for noninvasive cardiac function test. Hematoxylin and eosin staining was used to observe the histopathological change. miRNAs expression profile was detected by quantitative realtime polymerase chain reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor (VEGF), and a target gene of miR-210 was further detected by qRT-PCR and Western blot, respectively. The microvessels density of myocardium was evaluated by CD31 immunostaining.</p><p><b>RESULTS</b>Compared with the sham group, ejection fraction (EF) and fractional shortening (FS) values were decreased significantly in the AMI group (P<0.01), while the infarction area and the interstitial collagen deposition were increased obviously. As for the AMI+HAR group, EF and FS values were increased significantly (P<0.05 vs. AMI group), and the infarction area was reduced and the interstitial collagen deposition were alleviated significantly. Total of 23 miRNAs in the AMI group expressed differently by at least 1.5 folds compared with those in the sham group; 5 miRNAs in the AMI+HAR group expressed differently by at least 1.5 folds compared with those in the AMI group. Among them, miR-210 was low in the AMI group and high in the AMI+HAR group. The relative mRNA and protein expressions of VEGF were decreased significantly in the AMI group (P<0.05 vs. sham group), and increased significantly in the AMI+HAR group (P<0.01 vs. AMI group). CD31 expression area and optical intensity were decreased significantly in the AMI group (P<0.05 vs. sham group), and increased significantly in the AMI+HAR group (P<0.01 vs. AMI group).</p><p><b>CONCLUSIONS</b>HAR could reduce the infarction area, alleviate the interstitial fibrosis and improve the cardiac function of AMI rats. Those effects could be related to promoting myocardium angiogenesis of HAR by up-regulating miR-210 and VEGF.</p>
Subject(s)
Animals , Male , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Heart Function Tests , MicroRNAs , Genetics , Metabolism , Microvessels , Pathology , Myocardial Infarction , Drug Therapy , Genetics , Myocardium , Pathology , Neovascularization, Physiologic , Genetics , RNA, Messenger , Genetics , Metabolism , Rats, Wistar , Up-Regulation , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
Objective To investigate the expression of serum microRNA-210 (miR-210) in patients with acute cerebral infarction(ACI),and to evaluate its clinical significance. Methods A retrospective study was conducted. Eighty patients with ACI admitted to Tianjin Hospital from January 2011 to March 2014 within 48 hours of onset were enrolled,and 30 healthy volunteers served as controls. The peripheral blood was collected,and the expression of serum miR-210 was determined by reverse transcription-polymerase chain reaction(RT-PCR). The receiver operating characteristic curve(ROC)was drawn to analyze the role of miR-210 in the diagnosis of ACI. According to the pathological and physiological characteristics of patients receiving treatment,the relationship between miR-210 and clinical physiology index was analyzed. Results The expression of miR-210 in serum of patients with ACI was significantly lower than that of the healthy control group(2-ΔΔCt:1.349±0.043 vs. 1.923±0.107,t=6.567, P60 years:1.32±0.12 and 1.31±0.09,t=2.351,P=0.264;large infarction,small infarction,lacunar infarction:respectively 1.31±0.02, 1.33±0.11, 1.31±0.06, F=1.236, P=0.087), or with the severity of cerebral infarction,and there was a tendency in lowering of expression of miR-210(2-ΔΔCt,light,medium,severe:1.53±0.11, 1.33±0.11,1.08±0.04,F=5.394,P=0.014).Conclusion The serum level of miR-210 in ACI was significantly lower than that in normal healthy persons,and it may be an important new serological marker in screening and diagnosis of ACI.
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The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1alpha activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1alpha. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1alpha expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1alpha was important for MSC survival under hypoxic conditions.