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 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.
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.
ObjectiveTo quantitate expression of microRNA-21 (miRNA-21) in gastric cancer of different tumor stages and discuss its clinical value. Method The relative expressions of miRNA-21 were quantitated in the cancer tissues, corresponding normal gastric tissues adjacent to gastric cancer, and serums of 50 gastric cancer patients received opera-tion and confirmed gastric cancer by pathology and the serums of nongastric cancer patients in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine and its Chongming Branch from January 2015 to January 2016 by real time quantitative PCR. ResultsThe relative expression level of miRNA-21 in the gastric cancer tissues was significantly higher than that in the normal gastric tissues adjacent to gastric cancer. Among the TNM stageⅠ, Ⅱ, Ⅲ of gastric cancer patients, the relative expression levels of miRNA-21 in the cancer tissues were 2.17 (1.48-2.90), 4.08 (2.30-4.86), 8.64 (5.82-18.20), respectively and the differences among these three stages were statistically significant (P<0.05). The relative expression level of the serum miRNA-21 in the gastric cancer patients was significantly higher than that in the nongastric cancer patients, which in the serums for stageⅠ, Ⅱ, and Ⅲpatients were 31.00 (24.60-37.15), 39.10 (28.90-39.80), 44.15 (38.95-56.68), respectively and the differences among three stages were statistically significant (P<0.05). The relative expression level of miRNA-21 in the serums and cancer tissues had a positive correlation (r=0.86, P<0.05). ConclusionMiRNA-21 appears to have a potential association with TNM stage of gastric cancer, which cautiously suggests that it might be a potential indicator for prediction of preoperative TNM stage of gastric cancer.
ObjectiveTo investigate the expression level of exosome microRNA-21 (miRNA-21) in the bile and its clinical diagnostic value for the patients with cholangiocarcinoma. MethodsIn this study, 45 cases of cholangiocarcinoma admitted to Dongfeng General Hospital from August 2019 to December 2021 and met the inclusion criteria were selected and 35 patients with benign diseases of bile duct (choledocholithiasis or benign stricture of bile duct) during the same period were selected as control. The exosome in the bile was extracted by hypervelocity centrifugation method and identified. The exosome miRNA was extracted from the bile using a kit, then the expression level of miRNA-21 was detected by real-time fluorescent quantitative PCR. The diagnostic value of exosome miRNA-21 in the bile for cholangiocarcinoma was analyzed by receiver operating characteristic curve (ROC). ResultsThe isolated exosome in the bile conformed to the characteristics of recognized exosome and the concentration was higher. The average expression level of exosome miRNA-21 in the bile of the patients with cholangiocarcinoma was statistically higher than that in the patients with benign diseases of bile duct (59.45 verses 25.41, t=3.445, P<0.001). The area under the ROC curve was 0.715 [95%CI (0.602, 0.827), P=0.001]. The sensitivity and specificity of miRNA-21 in the diagnosis of cholangiocarcinoma were 75.6% and 62.9%, respectively. ConclusionFrom the results of this study, exosome miRNA-21 expression in bile is higher and it may be a potential early diagnostic marker for patients with cholangiocarcinoma.
Objective To explore the effect of long non-coding RNA H19 (lncRNA H19) on chronic heart failure (CHF) rats and its possible mechanism. Methods CHF (SD male rats, with a weight of 300±10 g, 10 weeks old) rat model was established by abdominal aortic coarctation. The 84 rats successfully modeled were randomly divided into a model group, a si-NC group [transfected lncRNA H19 small interfering RNA (siRNA) negative control], a si-H19 group (transfected lncRNA H19 siRNA), a si-miR-NC group [transfected microRNA-214 (miR-214) siRNA negative control], a si-miR-214 group (transfected miR-214 siRNA), a si-H19+si-miR-NC group (co-transfected lncRNA H19 siRNA and miR-214 siRNA negative control), and a si-H19+si-miR-214 group (co-transfected lncRNA H19 siRNA and miR-214 siRNA), 12 rats in each group. Another 12 rats were set up in a sham operation group (rats were only threaded without ligation, and the other operations were the same as the model group). After 4 weeks of intervention, the cardiac function, serum myocardial injury markers, heart failure markers, inflammatory related factors, apoptosis related factors and myocardial histopathological changes were compared. The expressions of lncRNA H19 and miR-214 in myocardial tissue were detected by real-time fluorescence quantitative PCR, and the targeting relationship between lncRNA H19 and miR-214 was detected by double luciferase reporter gene. Results Compared with those in the sham operation group, the myocardium of rats in the model group was severely damaged and a large number of inflammatory cells infiltrated; the lncRNA H19, cardiac function indexes (left ventricular end systolic diameter, left ventricular end diastolic diameter), serum myocardial injury markers (creatine kinase MB, cardiac troponin I), heart failure markers (brain natriuretic peptide, N-terminal pro brain natriuretic peptide), inflammatory related factors (interleukin-1β, interleukin-18, tumor necrosis factor-α, interleukin-6), cardiomyocyte apoptosis rate, apoptosis related proteins [B lymphocytoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cysteinyl aspartate specific proteinase-1 (Caspase-1)] in the myocardial tissue of the model group were significantly increased (P<0.05); miR-214 of myocardial tissue, cardiac function indexes (left ventricular ejection fraction, left ventricular fractional shortening) and Bcl-2/Bax ratio were significantly decreased (P<0.05). Compared with the model group, silencing lncRNA H19 could significantly improve the cardiac function and the changes of the above indexes in CHF rats, and reduce myocardial injury (P<0.05); down-regulation of miR-214 could significantly reverse the protective effect of si-H19 on myocardial injury in CHF rats (P<0.05). Conclusion Silencing lncRNA H19 can up-regulate the expression of miR-214, inhibit the expression of Caspase-1, inhibit the apoptosis and inflammatory reaction of cardiomyocytes, and alleviate myocardial injury in rats with CHF.