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Objective To investigate the role of PPARγ/GluT4 axis in insulin resistance(IR),cell proliferation and apoptosis of granulosa cells.Methods A total of 45 married women with PCOS who received routine IVF-ET assisted pregnancy treatment in Center of Reproductive Medicine,Qinghai Provincial People's Hospital were enrolled in this study from August 2018 to August 2020.All the patients were divided into IR group(PCOS-IR group,HOMA-IR≥2.57,n=23)and non-IR group(PCOS-NIR group,HOMA-IR<2.57,n=22)according to HOMA-IR.Meanwhile,21 married patients with infertility due to male or fallopian tube factors were enrolled as control group(Con).miR-27a mimics,miR-27a inhibitors(miR-27a inhibitor)and corresponding controls(mimics NC and inhibitor NC)transfected PCOS-IR granulosa cells,which were then divided into miR-27a mimics group,miR-27a inhibitor group,mimics-NC group and inhibitor-NC group.Double luciferase report test confirmed that miR-27a binded to PPARγ.Cell proliferation and apoptosis were detected by CCK-8 method and Annexinv-FITC/PI method.The expression of miR-27a,GluT4,PPARγ,Bax related to B lymphomas-2,Cleaved caspase-3 and B lymphomas-2(Bcl-2)were detected by RT-qPCR and Western blot respectively.Results Compared with Con group,the expression of miR-27a increased(P<0.01),while the expression of PPARγ mRNA and protein decreased in PCOS-NIR and PCOS-IR groups(P<0.01).Compared with PCOS-NIR group,the expression of miR-27a increased(P<0.05),while the expression of PPARγ mRNA and protein decreased in PCOS-IR group(P<0.01).The double luciferase report showed that there was a targeted binding site between PPARγ and miR-27a.Compared with inhibitor-NC group,the cell activity increased at 24 h,48 h,72 h and 96 h in miR-27a inhibitor group(P<0.05 or P<0.01),while the apoptosis rate decreased inmiR-27a inhibitor and mimics-NC group(P<0.05 or P<0.01).Compared with miR-27a inhibitor group,the apoptosis rate increased,and the cell activity decreased at 24,48,72 and 96 h in mimics-NC and miR-27a mimics groups(P<0.05 or P<0.01).Compared with the inhibitor-NC group,the expression of miR-27a,Bax and Cleaved caspase-3 increased(P<0.05 or P<0.01),while the expression of GluT4,PPARγ and Bcl-2 decreased in miR-27a mimics group(P<0.01).In miR-27a inhibitor group,the protein expressions of GluT4,PPARγ and Bcl-2 increased(P<0.05 or P<0.01),while miR-27a,Bax and Cleaved caspase-3 decreased(P<0.01).Compared with miR-27a inhibitor group,the expressions of miR-27a,Bax and Cleaved caspase-3 increased(P<0.01),while the expressions of GluT4,PPARγ and Bcl-2 decreased in mimics-NC and miR-27a mimics groups(P<0.05 or P<0.01).Conclusion The expression level of miRNA-27a is related to IR,cell proliferation,and apoptosis of granulosa cells,which may be related to PPARγ signal path.
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Objective @#To investigate the expression of miR-27a in colorectal cancer cell , and to analyze the effect of its targeted regulation of ( Secreted Frizzled Related Protein , SFRP1) on the biological behavior of colorectal cancer cells .@*Methods @#Real time fluorescent quantitative PCR(qRT-PCR) was employed to examine the expres sion of miR-27a and SFRP1 mRNA in colorectal cancer tissues and adjacent normal tissues . Western blot was used to detect the expression of SFRP1 protein in colorectal cancer tissues and adjacent normal tissues . TargetScan soft ware and dual luciferase reporter gene test were used to detect the targeted regulation of miR-27a on SFRP1 . HCT116 cells were transfected with miR-27a mimic , miR-27a inhibitor and negtive control (NC) . The expression of miR-27a and SFRP1 mRNA in each group was determined by qRT-PCR . MTT colorimetry was performed to eval uate the proliferation of each group cells . Transwell assay was used to evaluate the cell invasion and migration ability . Meanwhile , the protein expression levels of SFRP1 , key factors Wnt4 and β-catenin in the Wnt/β catenin signaling pathway were determined by Western blot.@*Results @#Compared with adjacent normal tissues , miR-27a was highly expressed in colorectal cancer tissues , while SFRP1 was low expressed in colorectal cancer tissues ( P < 0.05) . Target Scan software and dual luciferase reporter gene test showed that miR-27a targeted SFRP1 . Compared with NC group , the expression of miR-27a of miR-27a mimic group increased , the proliferation , invasion and migration ability enhanced , the expression of SFRP1 protein decreased , while Wnt4 and β-catenin protein expression increased ( P < 0.05 ) . Compared with miR-27a mimic group , the expression of miR-27a of miR-27a inhibitor group decreased , the proliferation , invasion and migration ability reduced , the expression of SFRP1 protein in creased , while Wnt4 and β-catenin protein expression decreased( P < 0.05) .@*Conclusion @#miR-27a can target SFRP1 , inhibit the proliferation , invasion and migration of colorectal cancer cells , mainly by up regulating SFRP1 and blocking the downstream Wnt/β-catenin signaling pathway , which provides a new direction for clinical treatment.
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Objective:To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1(LncRNA NEAT1)and miR-27a-3p in serum and cerebro-spinal fluid of patients with Alzheimer disease(AD).Methods:Sixty-six AD patients received by the department of neurology of our hospital from October 2019 to September 2021 were gathered,according to the clinical dementia rating scale score,they were grouped into mild group(≤ 1 point,n=41)and moderate-to-severe group(＞1 point,n=25).Another 66 cases of serum and cerebrospinal fluid sam-ples from outpatient physical examination personnel were regarded as the control group.The general infor-mation on all subjects was recorded and cognition was assessed;real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid;enzyme-linked immunosorbent assay was performed to measure the protein levels of β-amyloid precursor protein cleaving enzyme 1(BACE1),β-amyloid(Aβ)40 and Aβ42 in cerebrospinal fluid;Spearman's method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with mini-mental state examination(MMSE)and montreal cognitive assessment(MoCA)scores;Pearson method was per-formed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aβ deposition standard uptake value ratio(SUVR)and cerebrospinal fluid miR-27a-3p,NEAT1,BACE1,Aβ42 and Aβ40 levels.Results:The MMSE score[21(17,25),9(7,11)vs.27(21,34)],MoCA score[17(12,21),10(7,13)vs.27(21,31)],serum miR-27a-3p level(0.55±0.13,0.46±0.06 vs.0.97± 0.22),cerebrospinal fluid miR-27a-3p(0.48±0.10,0.35±0.10 vs.1.03±0.31),Aβ42 levels[(303.55±36.77)ng/L,(231.45±34.14)ng/L vs.(499.99±53.63)ng/L]and Aβ42/Aβ40 ra-tio(0.030±0.008,0.022±0.007 vs.0.048±0.010)of AD patients in mild group and moderate-to-severe group were all lower than those in the control group,and the moderate-to-severe group were lower than the mild group(all P＜0.05);the serum NEAT1 level(2.31±0.64,3.13±0.76 vs.1.05± 0.20),SUVR(1.50±0.29,1.76±0.52 vs.0.74±0.15),and cerebrospinal fluid NEAT1(3.51± 1.24,4.30±1.65 vs.1.01±0.23)and B ACE 1 levels[(55.78±5.98)μg/L,(72.32±16.08)μg/L vs.(21.39±3.73)μg/L]were higher than those in the control group,and the moderate-to-se-vere group were higher than the mild group(all P＜0.05).Serum NEAT1 level in AD patients was posi-tively correlated with SUVR,cerebrospinal fluid NEAT1 and BACE1(r=0.350,0.606,0.341,P＜0.05),and negatively correlated with MMSE score and MoCA score(r=-0.473,-0.482,all P＜0.05);serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level,MMSE score and MoCA score(r=0.695,0.424,0.412,all P＜0.05),and negatively correlated with SUVR and cerebrospinal fluid BACE1 level(r=-0.521,-0.447,all P＜0.05).Conclusion:The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are con-sistent,the level of NEAT1 is increased,and the level of miR-27a-3p is decreased.The levels of the two are negatively correlated,which is related to the degree of Aβ deposition in the brain of AD patients and is involved in the progression of AD.
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Objective To investigate the effect of GHET1 on the biological behavior of gallbladder cancer cells and the regulatory mechanism of GHET1 on miR-27b. Methods The expression of GHET1 and miR-27b in 50 samples of gallbladder cancer was detected by real-time quantitative PCR. The si-NC vector, si-GHET1 vector, miR-27b inhibitor, and si-GHET1 vector+miR-27b inhibitor were transfected into SGC-996 cells and set as the control group, GHET1 interference group, miR-27b interference group, and GHET1+miR-27b interference group. Cell proliferation, apoptosis, and metastasis in each group were detected by MTT, flow cytometry, and Transwell assays. The regulatory effect of GHET1 on miR-27b was validated by luciferase reporter gene assay. Results GHET1 expression was higher in cancer tissues than that in paracancerous ones. miR-27b expression was lower in cancer tissues than that in paracancerous tissues. GHET1 was negatively correlated with miR-27b expression (P<0.05), and GHET1 expression was associated with TNM staging and lymph node metastasis (P<0.05). High GHET1 expression was associated with poor prognosis of patients with gallbladder cancer (P<0.05). Compared with the control group, the GHET1 interference group showed decreased cell-proliferation ability, increased apoptosis rate, and reduced number of cell metastasis. The miR-27b interference group showed increased cell-proliferation ability, decreased apoptosis rate, and increased number of cell metastasis (P<0.05). Compared with the GHET1 interference group, the GHET1+miR-27b interference group showed increased cell-proliferation ability, decreased apoptosis rate, and increased number of cell metastasis (P<0.05). GHET1 inhibited miR-27b expression by acting as a sponge of miR-27b. Conclusion GHET1 promotes the proliferation and metastasis and inhibits the apoptosis of gallbladder cancer cells by targeting miR-27b, suggesting that GHET1/miR-27b axis plays a role in gallbladder cancer progression.
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Objective To investigate the effect and mechanism of miR-27a-3p on nerve cell apoptosis induced by oxygen glucose deprivation/reoxygenation(OGD/R)through regulation of Rho family GTPase 3(Rnd3)expression.Methods PC12 neurons were cultured in vitro and reoxygenated for 3,6,9 h and 12 h after 2 h oxygen glucose deprivation.Cell viability,miR-27a-3p expression and Rnd3 mRNA expression were assessed at each time point and the optimal reoxygenation time point was screened.After transfection of miR-27a-3p Mimic,miR-27a-3p Inhibitor and their negative control,transfection of shRnd3 and its negative control,or co-transfection of shRnd3 and miR-27a-3p Inhibitor through lentivirus,CCK-8 assay was used to detect cell activity.The apoptosis rate of the cells was detected using flow cytometry.Expression of miR-27a-3p and Rnd3 mRNA was detected by RT-qPCR.Expression of apoptosis-related protein and Rnd3 protein was detected by Western blot.The dual luciferase reporter assay confirmed the targeting relationship between miR-27a-3p and Rnd3.Results Upregulation of miR-27a-3p increased cell viability,decreased total cell apoptosis rate,suppressed pro-apoptotic proteins Cleaved Caspase-3(C-caspase-3)and Bax,and promoted expression of anti-apoptotic protein Bcl-2(P<0.05);The opposite result was found when down-regulating miR-27a-3p.The double luciferase reporter gene assay showed that Rnd3 was the target gene of miR-27a-3p.Down-regulation of Rnd3 increased cell viability,decreased the total rate of apoptosis,suppressed the pro-apoptotic protein C-caspase-3,Bax,and promoted expression of the anti-apoptotic protein Bcl-2(P<0.05).However,miR-27a-3p Inhibitor reversed the protective effect of shRnd3.Conclusion miR-27a-3p alleviates OGD/R-induced damage to PC12 neurons by targeting Rnd3 to inhibit cell apoptosis.
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Objective:To investigate the effect of miR-27b-3p on radiation resistance of breast cancer cells.Methods:The relative expression levels of miR-27b-3p in normal tissues and breast cancer tissues were analyzed through GEO database and verified by the qRT-PCR assay. Cloning formation, immunofluorescence, and EDU assay were used to assess the functions of miR-27b-3p on radioresistance of breast cancer cells. The luciferase reporter assay was used to verify whether miR-27b-3p directly targeted PLK2 mRNA. A rescue experiment was performed to identify the influence of PLK2 overexpression on miR-27b-3p regulated radioresistance.Results:The expression level of miR-27b-3p in both breast cancer tissues ( t=2.99, P<0.01) and breast cancer cells ( t=21.21, 32.88, P<0.05) was significantly higher than those in normal breast tissues and cells, especially, it was elevated in radioresistant MCF-7R cells ( t=25.63, P<0.05). Overexpression of miR-27b-3p enhanced the cloning efficiency of MCF-7 cells ( t=10.32, P<0.05), and had a protective effect on the proliferation of irradiated MCF-7 cells ( t=8.77, 8.26, 8.03, P<0.05). But interference of miR-27b-3p reduced the cloning efficiency and proliferation of MCF-7R cells ( t=40.00, P<0.05) after irradiation with different doses( t=8.54, 8.32, 8.23, P<0.05). Moreover, PLK2 was verified to be a direct target of miR-27b-3p, and overexpression of PLK2 inhibited miR-27b-3p-mediated radioresistance of breast cancer cells (MCF-7: t=9.66, P<0.05; MCF-7R: t=6.42, P<0.05). Conclusions:miR-27b-3p contributes to the radioresistance of breast cancer cells by targeting PLK2.
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Objective:To explore the expression significance of serum MicroRNA-27 (miR-27), MicroRNA-93 (miR-93) and bone morphogenetic protein 15 (BMP-15) in patients with polycystic ovary syndrome (PCOS) .Methods:63 PCOS patients admitted to Yantai Yantaishan Hospital from Aug. 2015 to Jan. 2019 were selected and divided into the obese group (obese PCOS, BMI≥25 kg/m 2, n=22) and the non-obese group (non-obese PCOS, BMI<25 kg/m 2, n=41) according to body mass index (BMI). 30 healthy women with normal physical examination during the same period were selected as the healthy group. Serum miR-27, miR-93 expression levels and bone morphogenetic protein 15 (BMP-15) concentration of the three groups were analyzed, and endocrine metabolism indicators of the three groups were compared. Receiver operating characteristic curve (ROS) was used to evaluate the predictive value of serum miR-27, miR-93 and BMP-15 for PCOS and obese PCOS patients. Results:(1) Endocrine indicators: Compared with the healthy group, FSH was lower in the obese group and the non-obese group, and T, LH/FSH and IR were higher ( P<0.05) ; Compared with the non-obese group, T and IR were higher in the obese group ( P<0.05) ; (2) Serum miR-27, miR-93 and BMP-15: Compared with the healthy group, serum BMP-15 concentration was lower in the obese group and the non-obese group, and serum miR-27, miR-93 expression levels were higher ( P<0.05) ; Compared with the non-obese group, the serum miR-27 and miR-93 expression levels in the obese group were higher ( P<0.05) ; (3) miR-27, miR-93 and BMP-15 had predictive value for PCOS, and the area under the curve of BMP-15 was the highest ( P<0.05) ; (4) miR-27 and miR-93 had predictive value for obese PCOS, and the area under the curve of miR-93 was the highest ( P <0.05) . Conclusions:In addition to significant endocrine index disorders in obese PCOS patients, serum miR-27 and miR-93 are highly expressed and the level of BMP-15 is relatively low. BMP-15 can be used as an effective parameter to assist in the diagnosis of PCOS, and miR-93 can be used as an effective parameter to assist in the diagnosis of obese PCOS.
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@#AIM: To investigate the clinical value of microRNA(miRNA, miR)-27 expression in patients with diabetic retinopathy(DR).<p>METHODS: A total of DR 80 patients(DR group)treated between January 2019 and January 2020 were retrospectively reviewed. Meanwhile, 40 patients with simple type 2 diabetes mellitus(T2DM)(T2DM group)and 40 normal healthy persons(control group)were enrolled, and plasma RNA was extracted. Real time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)was adopted to determine plasma miR-27 expression, and enzyme-linked immunosorbent assay was performed to determine the vascular endothelial growth factor(VEGF)level. Plasma miR-27 and serum VEGF expression in different groups and in patients with different severities of DR was comparatively analyzed. Multivariate Logistic regression analysis was performed to screen factors influencing the expression of miR-27 in patients with DR, and Pearson correlation analysis of miR-27, serum VEGF and blood glucose indexes was conducted. Meanwhile, significance of miR-27 in pathogenesis of DR was summarized.<p>RESULTS: DR group had the highest plasma miR-27 and serum VEGF levels, followed by T2DM group, and then the control group(<i>P</i><0.05). Proliferative diabetic retinopathy(PDR)patients had higher levels of plasma miR-27, serum VEGF, fasting blood glucose and glycated hemoglobin than those with non-proliferative diabetic retinopathy(NPDR)(<i>P</i><0.05). It was found that course of disease(<i>OR</i>=3.206), fasting blood glucose(<i>OR</i>=2.570), glycated hemoglobin(<i>OR</i>=2.787), VEGF(<i>OR</i>=3.442)and severity of DR(<i>OR</i>=5.842)were influencing factors of plasma miR-27 expression in DR patients(<i>P</i><0.05). In DR patients, relative expression of plasma miR-27 was positively correlated with serum VEGF, fasting blood glucose and glycated hemoglobin(<i>r</i>=0.548, 0.398, 0.522, all <i>P</i><0.05).<p>CONCLUSION: DR patients have higher plasma miR-27 expression level than those with simple T2DM and normal healthy people. The duration of diabetes, fasting blood glucose, glycated hemoglobin and severity of DR all affect the expression of miR-27. Besides, miR-27 is positively correlated with serum VEGF, glycated hemoglobin and fasting blood glucose. It is speculated that miR-27 may mediate the pathogenesis and progression of DR by regulating glucose metabolism and promoting angiogenesis.
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Aim: To explore the effects of microRNA- 27a-3p(miR-27a-3p) on collagen type I (Col I) and collagen type III (Col III) synthesis in pulmonary fibroblasts and the underlying mechanisms. Methods: Human pulmonary fibroblasts MRC-5 were cultured and then transfected with miR-27a-3p mimic or its inhibitor. qPCR and Western blot were used to detect miR- 27a-3p levels and nuclear β-catenin content, respectively. The expression of Col I, Col III and Wnt3a was measured using qPCR and Western blot. Bioinformatics predicted the potential of miR-27a-3p bound to Wnt3a 3'-untranslated region (3'-UTR). Dual luciferase reporter gene analyzed the effects of miR-27a-3p mimic transfection on luciferase activity of wild type and mutant Wnt3a 3'-UTR. MRC-5 cells were treated with the Wnt3a/β-catenin signaling pathway inhibitor Dkkl, followed by transfection with miR-27a-3p mimic or its inhibitor. Col I and Col III expression was detected by qPCR and Western blot. Results: miR-27a- 3p mimic markedly increased miR-27a-3p levels and decreased Col I, Col III, Wnt3a and β-catenin expression (P 0. 05), revealing Wnt3a as a target of miR-27a-3p. In addition, Dkkl pretreatment almost fully reversed the inductive effect of miR-27a-3p inhibitor on Col I and Col III expression in MRC-5 cells (P < 0. 05). Conclusions: miR-27a-3p inhibits the biosynthesis of Col I and Col III in pulmonary fibroblasts, which is attributed to inactivated Wnt3a/β-catenin signaling pathway.
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Objective@#To investigate the effects of miR-27b targeting ten-eleven translocation methylcytosine dioxygenase 2 (TET2) on oxidized low-density lipoprotein (ox-LDL) induced inflammatory responses and apoptosis in endothelial cells.@*Methods@#Double luciferase reporter gene analysis verified the targeting effect of miR-27b on TET2. Human umbilical vein endothelial cells (HUVECs) were induced by ox-LDL in vitro. Eight groups were set up including control group, ox-LDL group, ox-LDL+ anti-miR-con group, ox-LDL+ anti-miR-27b group, ox-LDL+ pcDNA group, ox-LDL+ pcDNA-TET2 group, anti-miR-27b+ si-con group and anti-miR-27b+ si-TET2 group. qRT-PCR was used to detect the expression of miR-27b and TET2 at mRNA level. Cell viability was detected by MTT assay. Cell apoptosis rate was detected by flow cytometry. Western blot was used to detect the expression of TET2, Cyclin D1 and caspase-3 at protein level.@*Results@#TET2 was the target gene of miR-27b. TET2 expression could be negatively regulated by miR-27b. ox-LDL increased the expression of miR-27b and reduced the expression of TET2 in HUVECs. The secretion of inflammatory factors and apoptosis rates of HUVECs in the control, ox-LDL+ anti-miR-27b, ox-LDL+ pcDNA-TET2 and anti-miR-27b+ si-con groups were significantly lower than those in the ox-LDL, ox-LDL+ anti-miR-con, ox-LDL+ pcDNA and anti-miR-27b+ si-TET2 groups, respectively (P<0.05).@*Conclusions@#miR-27b promoted ox-LDL-induced inflammatory responses and apoptosis in endothelial cells through down-regulating the expression of TET2.
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PURPOSE: The aim of this study was to explore the function of microRNA-27b (miR-27b) in fibroblast-like synoviocytes (FLSs) stimulated by tumor necrosis factor α (TNF-α). MATERIALS AND METHODS: mRNA expression of miR-27b in FLS cells (MH7A) treated with or without TNF-α was determined by q-PCR. MiR-27b mimics was transfected into MH7A cells to upregulate miR-27b expression. MTT assay and flow cytometry analysis were performed to investigate the effect of miR-27b on MH7A cell viability and apoptosis. The targets of miR-27b were predicted by TargetScan. The direct regulation of miR-27b on IL-1β expression was verified by luciferase assay. The protein expression levels of apoptosis-related proteins, IL-1β, and NF-κB signaling-related proteins were detected by Western blot. RESULTS: We discovered that miR-27b expression was decreased in MH7A cells stimulated by TNF-α. Upregulation of miR-27b by miR-27b mimics significantly inhibited the proliferation and promoted the apoptosis of TNF-α-stimulated MH7A cells. Consistently, upregulation of miR-27 decreased the level of Bcl-2 and increased Bax and caspase-3 expression in MH7A cells stimulated by TNF-α. Luciferase assay revealed that IL-1β was indeed a target of miR-27b. By quantitative real-time PCR and Western blot, we found that the expression of IL-1β is negatively regulated by miR-27b. Moreover, the NF-κB signaling pathway was significantly inhibited by miR-27b. CONCLUSION: Taken together, our results illustrated that enhanced miR-27b expression results in the suppression of proliferation and the promotion of apoptosis in FLSs stimulated by TNF-α, partially by regulating IL-1β expression and NF-κB signaling.
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Apoptosis , Arthritis, Rheumatoid , Blotting, Western , Caspase 3 , Cell Survival , Flow Cytometry , Luciferases , Real-Time Polymerase Chain Reaction , RNA, Messenger , Tumor Necrosis Factor-alpha , Up-RegulationABSTRACT
Aim To study the effect of circulating mi-crovesicles containing miR-27 a on blood brain barrier tight junction injury of ischemia stroke mice and its mechanism. Methods The middle cerebral artery occlusion mouse model was established, and the mi-crovesicles in the supernatant of 2 h of ischemic stroke brain tissues were separated by centrifugal ultrafiltra-tion method. The transmission electron microscopy was used to observe microvesicle morphology, and the di-ameter of microvesicles was detected. Based on the 2 h ischemia stroke mouse model, mice were injected via femoral vein with microvesicles at 5 mg · kg-1 . TTC staining was applied to detect infarction volume of is-chemia brain, while HE staining was applied to detect the expression change of tight junction protein occludin and claudin-5 . Western blot was subjected to detect occludin, claudin-5, TLR4, NF-κB and p38 protein expression. ELISA method was used to measure the contents of cytokines IL-1β and TNF-α. Results The microvesicle shape was approximately circular bi-lateral membrane structure, with an average diameter of 160 nm, which conformed to the morphological char-acteristics of microvesicles. Compared with the ische-mic stroke group, injection of microvesicles could ag-gravate the damage of brain tissues in ischemic mice, further increase the infarct volume and reduce the posi-tive staining areas of occludin and claudin-5 in ische-mia brain tissues. Meanwhile, the protein expressions of occludin and claudin-5 further decreased, and fur-ther up-regulated TLR4 and phosphorylation of NF-κB and p38 compared with the ischemia stroke group. Al-so, more content of IL-1βand TNF-αwere detected in ischemia stroke group injected with microvesicles com-pared with those in ischemia stroke group with signifi-cant difference, while the injection of antagomir-27a could alleviate brain damage and reduce the activation of TLR4, NF-κB and p38 in ischemia stroke mice. Conclusions Microvesicles containing miR-27 a could significantly attenuate brain injury in ischemia stroke mice, while aggravate the tight junction damage of ischemia brain. The mechanism might be correlated with the up-regulation of the expression of TLR4 , the phosphorylation of NF-κB, p38, and the release of cy-tokines IL-1β and TNF-α.
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Objective@#To detect the expression level of miR-27a during the osteogenic differentiation of beagle maxillary sinus membrane stem cells (MSMSCs) and explore the role of miR-27a in the osteogenic differentiation of MSMSCs.@*Methods@#Beagle MSMSCs were cultured in vitro. The expression level of miR-27a was detected via RT-PCR after an osteogenic inductive culture was prepared. The mRNA expression levels of Runx2 and OPN were examined via RT-PCR, and the protein expression levels of Runx2 and OPN were examined via Western blot after the cells were transfected with pre-miR-27a or anti-miR-27a. Finally, osteoprogenitor cells transfected with pre-miR-27a were composited with Bio-Oss particles and subcutaneously implanted into nude mice to form ectopic bone formation models, and then the inhibition of bone formation from miR-27a was observed in vivo. @*Results@#The expression level of miR-27a in the beagle MSMSCs decreased after osteogenic inductive culturing. The relative miR-27a levels were significantly decreased at day 1 (t=3.795, P=0.023), day 3 (t=4.493, P=0.011), day 7 (t=11.591, P < 0.001), day 14 (t=12.542, P < 0.001), and day 21 (t=5.621, P=0.008) compared with day 0. In addition, the expression levels of Runx2 mRNA (t=4.923, P=0.007) and protein (t=4.425, P=0.008) were reduced after the cells were transfected with pre-miR-27a. The expression levels OPN mRNA (t=5.253, P=0.006) and protein (t=5.132, P=0.006) were also reduced. In contrast, the mRNA expression levels of Runx2 (t=3.925, P=0.013) and OPN (t=3.712, P=0.019) were increased after the cells were transfected with anti-miR-27a, and bone formation was observed after the subcutaneous implantation of beagle MSMSCs composited with Bio-Oss in nude mice. Nevertheless, ectopic bone formation was inhibited by pre-miR-27a-transfected beagle MSMSCs composited with Bio-Oss (t=7.219, P=0.0020). @* Conclusion @# MiR-27a negatively regulates the osteogenic differentiation of MSMSCs.
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MicroRNAs (miRNAs) have been reported to be associated with heart valve disease, which can be caused by inflammation. This study aimed to investigate the functional impacts of miR-27a on TNF-α-induced inflammatory injury in human mitral valve interstitial cells (hMVICs). hMVICs were subjected to 40 ng/mL TNF-α for 48 h, before which the expressions of miR-27a and NELL-1 in hMVICs were altered by stable transfection. Trypan blue staining, BrdU incorporation assay, flow cytometry detection, ELISA, and western blot assay were performed to detect cell proliferation, apoptosis, and the release of proinflammatory cytokines. We found that miR-27a was lowly expressed in response to TNF-α exposure in hMVICs. Overexpression of miR-27a rescued hMVICs from TNF-α-induced inflammatory injury, as cell viability and BrdU incorporation were increased, apoptotic cell rate was decreased, Bcl-2 was up-regulated, Bax and cleaved caspase-3/9 were down-regulated, and the release of IL-1β, IL-6, and MMP-9 were reduced. NELL-1 was positively regulated by miR-27a, and NELL-1 up-regulation exhibited protective functions during TNF-α-induced cell damage. Furthermore, miR-27a blocked JNK and Wnt/β-catenin signaling pathways, and the blockage was abolished when NELL-1 was silenced. This study demonstrated that miR-27a overexpression protected hMVICs from TNF-α-induced cell damage, which might be via up-regulation of NELL-1 and thus modulation of JNK and Wnt/β-catenin signaling pathways.
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Humans , Male , Female , Adult , Middle Aged , Inflammation/chemically induced , MicroRNAs/metabolism , Mitral Valve/drug effects , Nerve Tissue Proteins/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Apoptosis , Cell Proliferation , Cell Survival , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Heart Valve Diseases/prevention & control , Inflammation/pathology , Mitral Valve/cytology , Mitral Valve/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Up-RegulationABSTRACT
Objective To investigate the miRNA expression profiles in rectal cancer tissues and their associations with clinical pathological stage, depth of tumor invasion, and lymph node metastasis, and to evaluate the potential of miRNA as diagnostic and prognostic markers of rectal cancer. Methods Human miRNA microarray was used to profile miRNA expression in rectal cancer tissues and matched adjacent normal tissues (n=71). The up-regulated miR-93-5p and down-regulated miR-27a-3p were screened out, and the top differentially expressed miRNA were validated by quantitative real-time polymerase chain reaction ( qRT-PCR) . The relationship between the expression of miRNA and clinical parameters was analyzed by ANOVA and Spearman correlation. Results The expression of miR-27a-3p was down-regulated in miRNA microarray, but was up-regulated in qRT-PCR analysis;the data were relatively discrete. The expression of miR-93-5p was up-regulated in both miRNA microarray and qRT-PCR analysis;the expression level of miR-93-5p in rectal cancer tissues was 3165 times that in adjacent normal tissues ( P=00058);the expression level was correlated with tumor volume ( P= 0004 ) , and was positively correlated with the level of carcinoembryonic antigen ( CEA) before treatment ( P=0001) and the number of lymph nodes metastases (rs=0534, P=0005). Conclusions There is a differential miRNA expression pattern between rectal cancer tissues and matched adjacent normal tissues. The miR-93-5p is highly up-regulated in rectal cancer tissues and may serve as a diagnostic and prognostic marker of rectal cancer.
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Objective To investigate the miRNA expression profiles in rectal cancer tissues and their associations with clinical pathological stage, depth of tumor invasion, and lymph node metastasis, and to evaluate the potential of miRNA as diagnostic and prognostic markers of rectal cancer. Methods Human miRNA microarray was used to profile miRNA expression in rectal cancer tissues and matched adjacent normal tissues (n=71). The up-regulated miR-93-5p and down-regulated miR-27a-3p were screened out, and the top differentially expressed miRNA were validated by quantitative real-time polymerase chain reaction ( qRT-PCR) . The relationship between the expression of miRNA and clinical parameters was analyzed by ANOVA and Spearman correlation. Results The expression of miR-27a-3p was down-regulated in miRNA microarray, but was up-regulated in qRT-PCR analysis;the data were relatively discrete. The expression of miR-93-5p was up-regulated in both miRNA microarray and qRT-PCR analysis;the expression level of miR-93-5p in rectal cancer tissues was 3165 times that in adjacent normal tissues ( P=00058);the expression level was correlated with tumor volume ( P= 0004 ) , and was positively correlated with the level of carcinoembryonic antigen ( CEA) before treatment ( P=0001) and the number of lymph nodes metastases (rs=0534, P=0005). Conclusions There is a differential miRNA expression pattern between rectal cancer tissues and matched adjacent normal tissues. The miR-93-5p is highly up-regulated in rectal cancer tissues and may serve as a diagnostic and prognostic marker of rectal cancer.
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Objective To investigate the changes of microRNA(miR)-27a and miR-23a in patients with premature ovarian failure(POF)after treatment,and to analyze the feasibility of these miRsfor assessing the efficacy.Methods 77 women with POF and 50 healthy women were assigned to a study group and a control group. miR-27a and miR-23a in peripheral blood were detected,andthe diagnostic value of these miRs for POF were ana-lyzed. miRs in the study group were detected again after treatment. The correlation between these miRs and modi-fied Kupperman score was analyzed.According to the cut-off values of miRs,the study group was subdivided into a high-efficacy group and a low-efficacy group,the differences among the two groups and the control group were com-pared. Results MiR-27a and miR-23a were reliable for diagnosis of POF,the best cut-off value was 2.29 and 1.95 respectively.After treatment,the miRs decreased in the study group(P<0.05).MiR-27a and miR-23a were positively correlated with the modified Kupperman score(P < 0.001). All indexes detected in high-efficacy group were close to those in the control group(P>0.05).Conclusions MiR-27a and miR-23a obviously decreases in women with POF after treatment,these miRs are helpful for assessing the efficacy.
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Objective To explore the mechanism of mTOR-mediated liver cancer cell invasion.Methods q-PCR was used to check the expression of miR-27a and GP73;miR-27a mimics were transfected into GP73-high expressing M97H cells and miR-27a inhibitors were transfected into GP73-low expressing HepG2 cells,q-PCR and Western blot were performed to observe the expression of GP73;Dual-luciferase assay was also performed to verify the binding sites of miR-27a in GP73 3'UTR;miR-27a mimics were transfected into M97H cells and miR-27a inhibitors were transfected into HepG2 cells,Transwell assay was used to measure cell invasion.Results mTOR downregulated miR-27a and upregulated GP73;GP73 was downregulated by miR-27a and upregulated by miR-27a suppression;GP73 was a target gene of miR-27a;miR-27a inhibited the invasion of M97H cells rather than HepG2 cells.Conclusions miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73.
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OBJECTIVE:To investigate the expression difference and its mechanism of miR-27a-3p and HOXB8 protein in cer-vical cancer tissues of HPV16-positive and HPV16-negative patients. METHODS:A total of 120 patients with cervical cancer in our hospital during Jan. 2012-Jan. 2016 were divided into HPV16-positive group(60 cases)and HPV16-negative group(60 cases) according to HPV16 infection situation. The expression of miR-27a-3p mRNA and HOXB8 mRNA in cervical cancer tissue were de-tected by RT-qPCR. The expression of HOXB8 protein was detected by Western blotting assay. DNA methylation level of miR-27a-3p promoter region was detected by nested-down methylation specific PCR (nMS-PCR). Histone methylation level of miR-27a-3p promoter region was detected by chromatin immunoprecipitation PCR (CHIP-PCR). The expression of miR-27a-3p mRNA and HOXB8 mRNA were detected after transfecting miR-27a-3p mimic and inhibitor into Human cervical cancer cell line SiHa,respectively. RESULTS:The expression of miR-27a-3p in HPV16-positive group was significantly lower than HPV16-nega-tive group,while HOXB8 mRNA and protein expression,DNA and histone methylation levels of miR-27a-3p promoter region were significantly higher than HPV16-negative group,with statistical significance (P<0.05 or P<0.01). After transfecting miR-27a-3p mimic into SiHa cells,the expression of miR-27a-3p was increased significantly,while that of HOXB8 mRNA was de-creased significantly;after miR-27a-3p inhibitor transfection,the expression of miR-27a-3p was decreased significantly,while that of HOXB8 mRNA was increased significantly,with statistical significance(P<0.01). CONCLUSIONS:HPV16 may down-regu-late the expression of miR-27a-3p through DNA methylation and histone methylation of promoter region,so as to influence the gen-eration and development of cervical cancer. HOXB8 may be the target protein of miR-27a-3p.
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Increasing attention is focused on the down-regulation of miRNAs in cancer process. Nuclear receptor subfamily 2 (NR2F2, also known as COUP-TFII) is involved in the development of many types of cancers, but its role in gastric cancer remains elusive. In this experiment, oncomine and Kaplan-meier database revealed that NR2F2 was up-regulated in gastric cancer and that the high NR2F2 expression contributed to poor survival. MicroRNA-27b was targeted and down-regulated by NR2F2 in human gastric cancer tissues and cells. The ectopic expression of miR-27b inhibited gastric cancer cell proliferation and tumor growth in vitro and in vivo. Assays suggested that the overexpression of miR-27b could promote MGC-803 cells' migration and invasion and retard their metastasis to the liver. In addition, down-regulation of miR-27b enhanced GES-1 cells' proliferation and metastasis in vitro. These findings reveal that miR-27b is a tumor suppressor in gastric cancer and a biomarker for improving patients' survival.