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Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress (FSS) and the potential molecular mechanism. Methods Osteoblast MC3T3-E1 was treated with 1. 2 Pa FSS with time gradients of 0, 15, 30, 45, 60, 75 and 90 min, respectively. MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor. MC3T3-E1 cells were transfected with miR-199a-3p mimic and itsnegative control and then treated with 1. 2 Pa FSS for 45 min. The pc DNA NC, pc DNA-CABLES -1, si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells. The pc DNA-CABLES-1 and mir-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected, respectively. Cell activity was detected by CCK-8 assay. Real-time quantitative PCR (RT-qPCR) was used to detect expression levels of CABLES-1, miR-199a-3p, CDK 6, Cyclin D1 and PCNA. Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p. Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1, CDK 6, PCNA and Cyclin D1. Results Mir- 199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS. Over-expressed miR-199a-3p inhibitedosteoblast proliferation, and down-regulated miR-199a-3p expression promoted osteoblast proliferation. miR-199a- 3p could reverse the FSS-induced proliferation in osteoblasts. Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein. CABLES-1 could promote proliferation of osteoblasts. miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1. Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1. The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation, as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.
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Objective@# To investigate the relationship between serum miR-199a-5p and miR-378a-3p levels and the recurrence of infants with proliferative facial hemangioma relapsed after propranolol withdrawal in infants.@*Methods@#Ninety-three infants with proliferative facial hemangioma were selected, all of whom received propranolol treatment. The recurrence of the hemangioma after treatment was followed up, and the children were divided into a recurrence group (23 patients) and a nonrecurrence group (70 patients). Venous blood was collected before and after treatment, and the serum levels of miR-199a-5p and miR-378a-3p were detected by qRT-PCR, and the relationship between the serum levels of miR-199a-5p and miR-378a-3p and the recurrence of proliferative facial hemangioma relapsed after propranolol withdrawal in infants was analyzed.@*Results @# The serum expressions levels of miR-199a-5p and miR-378a-3p in non-relapsed group were increased after treatment compared with before treatment (P < 0.05), and there was no significant difference in the levels of miR-199a-5p and miR-378a-3p in the serum of the recurrence group after treatment compared with before treatment (P > 0.05). After treatment, the levels of miR-199a-5p and miR-378a-3p in the serum of the recurrence group were lower than those of the nonrecurrence group (P < 0.05). After treatment, the serum levels of miR-199a-5p and miR-378a-3p in patients with Ⅲ ~Ⅳ Norm grade hemangioma were higher than those in patients with Ⅰ~Ⅱ Norm grade hemangioma (P < 0.05). Logistic regression analysis showed that the low expression of miR-199a-5p, low expression of miR-378a-3p and tumor grade Ⅰ~Ⅱ after treatment were risk factors for the recurrence of proliferative facial hemangioma after propranolol withdrawal in infants (P < 0.05).@*Conclusion@# Low serum levels of miR-199a-5p and miR-378a-3p are associated with the recurrence of proliferative facial hemangioma after propranolol withdrawal in infants.
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Objective:To investigate the effect and potential mechanism of miR-199a-5p on the radiosensitivity of cervical cancer CaSki cells.Methods:Cervical cancer CaSki cells were cultured in vitro. MiR-199a-5p mimics (miR-199a-5p mimics) were transfected into cervical cancer CaSki cells (miR-199a-5p group) with liposome by Lipofectamine 2000. CaSki cells transfected with mimics control were used as negative control (NC group) and non transfected CaSki cells were used as blank control (control group). After X-ray irradiation, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-199a-5p in cells of each group. The effects of miR-199a-5p on radiosensitivity and apoptosis of CaSki cells were detected by clone formation assay and flow cytometry apoptosis assay. Bioinformatics software was used to predict the target gene of miR-199a-5p. Double luciferase reporter gene assay and Western blot were used to verify the targeting relationship between miR-199a-5p and thyroid hormone receptor interaction factor 4 (TRIP4). Results:The expression of miR-199a-5p in miR-199a-5p group was significantly higher than that in control group ( P<0.05). After X-ray irradiation, the expression of miR-199a-5p was more obvious ( P<0.05); Overexpression of miR-199a-5p could reduce the clonogenic ability and promote the apoptosis of CaSki cells ( P<0.05); Overexpression of miR-199a-5p could further reduce the clonal formation and promote the apoptosis of irradiated cells ( P<0.05); Double luciferase reporter gene experiment and Western blot confirmed that miR-199a-5p could target and negatively regulate TRIP4. Conclusions:miR-199a-5p can increase the radiosensitivity of cervical cancer CaSki cells by negatively regulating the expression of TRIP4.
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The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.
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Animals , Rats , Cardiomegaly/genetics , Isoproterenol , Myocardium , Myocytes, Cardiac , Oleanolic Acid/analogs & derivatives , Saponins/pharmacologyABSTRACT
Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.
Subject(s)
Animals , Mice , Autophagy , Autophagy-Related Protein 5 , MicroRNAs/genetics , Non-alcoholic Fatty Liver Disease/genetics , Panax , Saponins/pharmacologyABSTRACT
OBJECTIVE@#To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats.@*METHODS@#Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting.@*RESULTS@#BYHWD treatment significantly promoted the recovery of neurological function (@*CONCLUSIONS@#Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.
Subject(s)
Animals , Male , Rats , Brain Ischemia/drug therapy , Drugs, Chinese Herbal/therapeutic use , Ischemic Stroke/drug therapy , MicroRNAs/genetics , Neurogenesis/drug effects , Rats, Sprague-Dawley , Up-Regulation/drug effects , Vascular Endothelial Growth Factor A/geneticsABSTRACT
@# Objective: To investigate the effect of long non-coding RNA (lncRNA) lung cancer associated transcript 1 (LUCAT1) on proliferation and migration of clear cell renal cell carcinoma (ccRCC) 786-O cells and the underlying mechanism. Methods: A total of 40 pairs of pathologically confirmed tumor tissues and corresponding adjacent normal tissues from ccRCC patients, who underwent surgical resection in the Department of Urology, the First People's Hospital of Yichang during June 2013 and June 2017, were selected for this study. ccRCC cell lines (786-O, ACHN, UM-RC-2) and normal renal epithelial KiMA cells were also used in this study. qPCR was used to detect the mRNA expressions of LUCAT1, miR-199a-5p and hypoxia inducible fator 1α (HIF-1α) in above mentioned tissues and cell lines; CCK-8 assay was used to evaluate the proliferation of 786-O cells; Transwell assay was used to evaluate the migration of 786-O cells; Dual luciferase reporter gene assay was performed to validate the relationship between LUCAT1 and miR-199a-5p; and Western blotting was conducted to detect the effect of LUCAT1 and miR-199a-5p on the protein expression of HIF-1α. Results: LUCAT1 was significantly up-regulated in ccRCC tissues and cell lines (all P<0.01), and its knockdown significantly inhibited the proliferation and migration of 786-O cells (all P<0.01). miR-199a-5p was low-expressed in ccRCC tissues and cell lines (all P<0.01), StarBase analysis showed that LUCAT1 contained a conserved target site for miR-199a-5p. miR-199a-5p exerted significant suppression on the luciferase activity of LUCAT1-Wt (P<0.01), and LUCAT1 knockdown significantly reduced miR-199a-5p expression (P< 0.01). LUCAT1 was low-expressed in 786-O cells transfected with miR-199a-5p mimics, however, it was attenuated after co-transfection with LUCAT1. The mRNA and protein expressions of HIF-1α in 786-O cells transfected with miR-199a-5p mimics were up-regulated, which was then reversed by LUCAT1 over-expression (P<0.05 or P<0.01). miR-199a-5p over-expression suppressed the proliferation and migration of 786-O cells, which was partially attenuated by LUCAT1 transfection (P<0.05 or P<0.01). Conclusion: LUCAT1 exerts oncogenic function in ccRCC via regulating miR-199a-5p/HIF-1α axis.·
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Objective@#To investigate the role of miR-199a-3p in children with severe mycoplasmal pneumonia (MP) and to study its effect on Th17 cells.@*Methods@#Sixty children with severe MP (severe group), 50 children with recovery MP (recovery group) and 40 healthy children (control group) were enrolled. Venous blood samples were collected from all subjects, and the expression of miR-199a-3p in the peripheral blood leukocytes was detected by RT-PCR. The levels of IL-17a, IL-13, GM-CSF and IL-6 in serum were determined by enzyme-linked immunosorbent assay (ELISA). The effect of miR-199a-3p on the differentiation of Th17 cells was investigated by inducing differentiation of Th17 cells and transfecting miR-199a-3p mimics or inhibitors in vitro.@*Results@#Compared with the control group, the expression of miR-199a-3p in peripheral blood leukocytes of children with severe MP was significantly decreased (P<0.05). The levels of IL-17a, IL-13, GM-CSF and IL-6 in serum were significantly higher than those in the control group (all P<0.05). High expression of miR-199a-3p inhibited the differentiation of Th17 cells in vitro.@*Conclusions@#The expression of miR-199a-3p is decreased in the pathogenesis of MP. Th17 cells are the target cells of miR-199a-3p, and their differentiation is regulated by miR-199a-3p. The high expression of miR-199a-3p significantly inhibites the differentiation of Th17 cells.
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@#【Objective】To investigate the role and the potential target of miR-199a-3p in mouse cardiac hypertrophy.【Methods】Neonatal mouse ventricular cardiomyocytes（NMVC）were isolated from the hearts of 0- 3- day- old newborn C57BL/6 mice. MiR-199a-3p mimic and retinoblastoma transcriptional corepressor 1（Rb-1）siRNA were transfected in? to NMVC to elevate the level of miR-199a-3p and inhibit Rb-1 expression，respectively. NMVC were stained with FITC- phalloidin solution to determine the size of NMVC. Dual luciferase reporter assay was performed to identify the interaction between miR- 199a- 3p and the 3’UTR of Rb- 1. mRNA and protein expression of cardiac hypertrophy associated genes were determined by RT-qPCR and Western blotting assay，respectively.【Results】（1）Over-expression of miR-199a-3pcould significantly enhance the expression of cardiac hypertrophy-related genes in NMVC ；（2）Dual-luciferase reporter assay results verified that miR- 199a-3p can interact with the 3’UTR of Rb-1. MiR-199a-3p could suppress Rb-1 ex? pression at the post-transcriptional level；（3）Functionally，miR-199a-3p mimic，consistent with Rb-1 siRNA，could increase cell size and the expression of Nppa，Acta1 and Myh7 in NMVC，and promote the nuclear translocation of E2f2 in NMVC.【Conclusions】MiR-199a-3p promotes the entry of E2f2 into the nucleus through inhibiting the expression of Rb-1，contributing to cardiomyocyte hypertrophy.
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Objective To investigate the role of miR-199a-3p in children with severe mycoplasmal pneumonia (MP) and to study its effect on Th17 cells. Methods Sixty children with severe MP (severe group), 50 children with recovery MP (recovery group) and 40 healthy children (control group) were enrolled. Venous blood samples were collected from all subjects, and the expression of miR-199a-3p in the peripheral blood leukocytes was detected by RT-PCR. The levels of IL-17a, IL-13, GM-CSF and IL-6 in serum were determined by enzyme-linked immunosorbent assay (ELISA). The effect of miR-199a-3p on the differentiation of Th17 cells was investigated by inducing differentiation of Th17 cells and transfecting miR-199a-3p mimics or inhibitors in vitro. Results Compared with the control group, the expression of miR-199a-3p in peripheral blood leukocytes of children with severe MP was significantly decreased (P<0.05). The levels of IL-17a, IL-13, GM-CSF and IL-6 in serum were significantly higher than those in the control group (all P<0.05). High expression of miR-199a-3p inhibited the differentiation of Th17 cells in vitro. Conclusions The expression of miR-199a-3p is decreased in the pathogenesis of MP. Th17 cells are the target cells of miR-199a-3p, and their differentiation is regulated by miR-199a-3p. The high expression of miR-199a-3p significantly inhibites the differentiation of Th17 cells.
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Aim To investigate the effect of sevoflurane preconditioning on spinal cord ischemia-reperfusion in-jury ( SCIRI ) of miR-199a-5p in rats. Methods Twenty-four SD rats were randomly divided into: group of sham ( Sham group) , group of ischemia-reperfusion ( I/R group ) , and group of sevoflurane and control ( SEVO+I/R group) . Sham group received the same operation, but did not inhale sevoflurane or close the aortic arch; SCIRI model was established after 14 min of the aortic arch of the non-invasive arterial clip in I/R group. SEVO+I/R group, before the establishment of SCIRI model, was inhaled 2.4% sevoflurane for 3 h. Basso Beattie Bresnahan scoring method was used to evaluate neuromotor function and TUNEL staining to observe apoptosis. Evans blue ( EB) was used to de-termine blood-spinal cord barrier ( BSCB) permeabili-ty; real-time qPCR to detect miR-199a-5p content;Western blot to measure the levels of caspase-9 and Bcl-2 in spinal cord tissues. Results Compared with sham group, the score of neuromotor function in I/R group decreased, cell apoptosis rate increased, BSCB permeability increased, miR-199a-5p expression de-creased, caspase-9 expression increased, and Bcl-2 expression decreased; compared with I/R group, the neurological motor function score of SEVO+I/R group increased, the apoptotic rate decreased, the BSCB per-meability decreased, the expression of miR-199a-5p increased, the expression of caspase-9 decreased, and the expression of Bcl-2 increased. Conclusion Sevoflurane pretreatment may reduce BSCB permeabili-ty and neuronal apoptosis by up-regulation of miR-199a-5p and protection of SCIRI in rats.
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Objective To investigate the effect of miRNA-199a-3p overexpression on the expression of MAP3K4 protein in gastric cancer. Methods 35 gastric cancers and the matched adjacent tissue specimens were collected. Expression of miRNA-199a-3p and MAP3K4 were detected by stem-loop real-time reverse transcription polymerase chain reaction and western blot. Cell transfection was employed to explore the regulation of miRNA-199a-3p on MAP3K4 gene. Luciferase reporter assay was performed to identify target MAP3K4 gene. Results Com-pared with the adjacent tissue specimens ,miRNA-199a-3p was upregulated in the gastric cancers ,and MAP3K4 protein was down-regulated in the gastric cancers. Cells transfected with miR-199a-3p mimics showed lower MAP3K4 protein. MAP3K4 was identified as target gene of miR-199a-3p. Conclusions miRNA-199a-3p acts as an oncogene in gastric cancer and functions by targeting MAP3K4.
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Objective To study the effect of quercetin on proliferation of hepatocarcinoma cells and the expression of miR-199a. Methods Hepatocellular carcinoma cell line HepG2 cells were transfected with 20 μm/L quercetin. MTT proliferation assay were conducted to compare the proliferation, and Annexin V/PI apoptosis assay were adopted to compare the apoptosis of cells in quercetin group and NC group at 12 h, 24 h, 36 h and 48 h. The expression of miR-199a were determined by RT-qPCR and western blotting assays were used to detect the Western blotting was used to detect the expression of VEGFA, Bax and Caspase3. Results MTT showed that quercetin inhibited the proliferation of HepG2 cells (P<0.01). The apoptosis of hepatocellular carcinoma was markedly increased after treated with quercetin (P<0.01). Moreover, quercetin can up-regulate the expression of miR-199a in HepG2 cells. The quercetin decreased the expression of VEGFA, increased the expression of Bax and Caspase 3 on both mRNA and protein level (P<0.01). Conclusion Quercetin can inhibit the proliferation and promote apoptosis of hepatocarcinoma cells by up-regulating the expression of miR-199a.
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Objective To investigate the effect of miRNA-199a-3p overexpression on the expression of MAP3K4 protein in gastric cancer. Methods 35 gastric cancers and the matched adjacent tissue specimens were collected. Expression of miRNA-199a-3p and MAP3K4 were detected by stem-loop real-time reverse transcription polymerase chain reaction and western blot. Cell transfection was employed to explore the regulation of miRNA-199a-3p on MAP3K4 gene. Luciferase reporter assay was performed to identify target MAP3K4 gene. Results Com-pared with the adjacent tissue specimens ,miRNA-199a-3p was upregulated in the gastric cancers ,and MAP3K4 protein was down-regulated in the gastric cancers. Cells transfected with miR-199a-3p mimics showed lower MAP3K4 protein. MAP3K4 was identified as target gene of miR-199a-3p. Conclusions miRNA-199a-3p acts as an oncogene in gastric cancer and functions by targeting MAP3K4.
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Objective To invstigate the effect of ultrasound microbubble mediated miRNA delivery on mi-gration,invasion and cloning ability of human hepatoma HepG2 cells. Methods The migration,invasion and col-ony forming ability of HepG2 cells were measured after transfection with antisense miRNA-21/221 and miRNA-199a mimic via the optimal ultrasound microbubble transfection method. Results Compared with the control group ,the migration ,invasion and cloning ability of cells were significantly inhibited after transfection with miRNA mimics(P < 0.05,respectively),especially for miR-199a(relative cell migration rate was 31.05%,the number of invasive cells were 38.67 ± 4.51 and the number of clones were 105.67 ± 5.86). Conclusion The pres-ent study may provide new ideas and clues for gene therapy and prognosis of hepatocell ular carcinoma through ana-lyzing the effect of miRNAs on the biological characteristics of human hepatoma HepG2 cells.
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Objective:To investigate the effect and the related mechanism of c-Myc on the proliferation,invasion and migration ability of malignant melanoma B16-F10 cells. Methods:We detected the expression of PIK3R3 in malignant melanoma and normal tis-sues. Efficiency of gene silencing of c-Myc and PIK3R3 was determined by qPCR and Western blot. We detected the proliferate ability of B16-F10 cells after silencing c-Myc and PIK3R3 using EdU assay. We detected the migration and invasion ability of B16-F10 cells after silencing c-Myc and PIK3R3 using wound healing assays and Transwell matrigel invasion assays. The expression of miR-199a after silencing c-Myc and PIK3R3 using qPCR. The expression of c-Myc and PIK3R3 was detected by qPCR after transfecting miR-199a mimics or miR-199a inhibitor. Dual-luciferase reporter assay system was used to detect miR-199a regulating c-Myc and PIK3R3. Results:Compared normal skin tissue,expression of PIK3R3 was significantly increased in malignant melanoma tissue;after silencing c-Myc and PIK3R3 gene,the proliferation,invasion and metastasis of melanoma cell line B16-F10 were significantly reduced;expression of miR-199a upregulated after silencing c-Myc and PIK3R3 genes, expression of c-Myc and PIK3R3 decreased after transfecting miR-199a mimics, expression of c-Myc and PIK3R3 upregulated after transfecting miR-199a inhibitor, dual luciferase reporter system test results revealed miR-199a can directly regulate c-Myc and PIK3R3 transcription activity. Conclusion: miR199a regulated the expression of c-Myc,then promote proliferation,migration and invasion in malignant melanoma cells.
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Objective To explore the mechanism of vessel endothelial dysfunction in rats under intermittent hypoxia (IH). Methods The respiratory simulation system was used to simulate IH. Sixty C57BL/6J rats (male) were randomized into control group and IH group. The rats of IH group were exposed to IH 8 hours per day for 6 weeks. The serum levels of hypoxia inducible factor (HIF)-1a and stromal cell derived factor (SDF)-1a were assessed by ELISA. The serum levels of reactive oxygen species (ROS) were detected in two groups. The serum expression of miR-199a-5p was detected by real-time fluorescent quantitative PCR in two groups. The dual luciferase report system and point mutation test were used to verify target gene for HIF-1a. Results The serum levels of HIF-1a and SDF-1a were significantly higher in IH group than those of control group (μg/L:1.60±0.02 vs. 1.19±0.02, 1 823.00±8.97 vs. 1 444.00±17.90, P<0.01). The serum level of ROS was significantly higher in IH group than that of control group (U/mL:487.66±35.73 vs. 211.57±23.82, P<0.01). The serum level of miR-199a-5p expression was significantly lower in IH group compared to that of control group (1.31±0.07 vs. 3.47± 0.17, P<0.01). The result of dual luciferase reporter gene detection confirmed that target gene of miR-199a-5p was HIF-1a. Conclusion The serum level of miR-199a-5p is decreased first due to IH, and then its target gene (HIF-1a) is increased. HIF-1a can induce the increased level of SDF-1a, and its receptor (CXCR-4 ) is also increased. Finally, HIF-1a can increase the serum level of ROS, resulting in the endothelial dysfunction.
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Objective To study the effect of quercetin on proliferation of hepatocarcinoma cells and the expression of miR-199a. Methods Hepatocellular carcinoma cell line HepG2 cells were transfected with 20 μm/L quercetin. MTT proliferation assay were conducted to compare the proliferation, and Annexin V/PI apoptosis assay were adopted to compare the apoptosis of cells in quercetin group and NC group at 12 h, 24 h, 36 h and 48 h. The expression of miR-199a were determined by RT-qPCR and western blotting assays were used to detect the Western blotting was used to detect the expression of VEGFA, Bax and Caspase3. Results MTT showed that quercetin inhibited the proliferation of HepG2 cells (P<0.01). The apoptosis of hepatocellular carcinoma was markedly increased after treated with quercetin (P<0.01). Moreover, quercetin can up-regulate the expression of miR-199a in HepG2 cells. The quercetin decreased the expression of VEGFA, increased the expression of Bax and Caspase 3 on both mRNA and protein level (P<0.01). Conclusion Quercetin can inhibit the proliferation and promote apoptosis of hepatocarcinoma cells by up-regulating the expression of miR-199a.
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OBJECTIVE: To explore the expression of miR-199a in ulcerative colitis (UC) rats induced by 2, 4, 6-trinitrobenzene sulfonic (TNBS)/ethanol and the study on the effect of TWP on them. METHODS: Through injecting TNBS/ethyl alcohol acid liquid into the anus of the rats to establish the UC rat model. The colitics commom morphous damage and grade the histopathological score (CMDI) of colon mucosa injury were evaluated. Chip analysis and Real-time PCR were used to verify the expression of miR-199a in each colon mucosa tissue. Based on the expression profile, the downstream target genes mRNA in milwalk database was selected, then the expression of target genes mRNA by Real-time PCR in each group was veritied, at last the relevant signal pathway in the DAVID database was analyzed. Doing these to analyse the target gene mRNA regulated by the miR-199a in the inflammatory activity of UC. RESULTS: Compared with the model group, TWP high dose group was significantly lower on gross morphological damage score and histopathological injury score(P < 0.01). Chip analysis showed that in model group, the expression of miR-199a was significantly higher than the normal group(P < 0.01), and expression of the AZA group was significantly lower than the model group(P < 0.01, P < 0.05). The expression of miR-199a-3p in medium dose group and the expression of miR-199a-5p in high dose group were significantly lower than the model group(P < 0.05). The results of Real-time PCR showed that expression of miR-199a in the model group was significantly increased than that in the normal group(P < 0.01). The expression of miR-199a-3p in TWP medium dose group, high dose group and AZA group were decreased than that in model group(P < 0.05). Meanwhile, the expression of miR-199a-5p in TWP medium dose group was decreased than that in model group(P < 0.05). The gene expression profile showed that FASL was the target gene of miR-199a. In the model group, the expression of FASL was higher than that in the normal group. The expression of FASL in AZA group was significantly decreased than that in the model group(P < 0.01). The results by the Real-time PCR of the target gene FASL showed that in the model group, the expression of FASL was higher than that in the normal group (P < 0.01). The expression of FASL in medium dose group, high dose group and AZA group were significantly decreased than that in the model group (P < 0.01). CONCLUSION: miR-199a is up-regulated in TNBS/Ethanol UC rats, and FASL is the downstream target gene of miR-199a. TWP can reduce the UC's inflammatory effectively and decrease the up-regulated miR-199a in UC. FASL is up-regulated in UC's inflammatory activity. TWP can reduce downstream target gene FASL of miR-199a.
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Background and purpose:Multiple microRNAs (miRNAs) are abnormally expressed in breast cancer and play an important role in the regulation of breast cancer. miRNAs may be a new target for the treatment of breast cancer. This study aimed to investigate the expression of miR-199a-3p in breast cancer and the effect of miR-199a-3p on proliferation and apoptosis of breast cancer.Methods:Real-time PCR was used to test the expression of miR-199a-3p in breast cancer tissues, normal breast tissues, breast cancer cells and normal breast cells. Overexpression (or silencing the expression) of miR-199a-3p was conducted by transfecting MDA-MB-231 with miR-199a-3p mimics (or inhibitors). The proliferation of MDA-MB-231 was detected by MTT method. The apoptosis of MDA-MB-231 was investigated by Hoechst staining and caspase-3 activity assay kit.Results:Compared to corresponding non-tumor breast tissues (or normal breast cell HBL-100), lower levels of miR-199a-3p were expressed in breast cancer tissues or breast cancer cells. Overexpression of miR-199a-3p induced by miR-199a-3p mimic inhibited the proliferation and promoted the apoptosis of MDA-MB-231, while silencing the expression of miR-199a-3p induced by miR-199a-3p in-hibitor increased the proliferation and suppressed the apoptosis of MDA-MB-231.Conclusion:The expression of miR-199a-3p is lower in breast cancer, which shows its tumor suppression effect by regulating the proliferation and apoptosis of breast cancer cells.