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BACKGROUND:Mesenchymal stem cell-derived exosomes may play a crucial role in tissue damage repair,and miRNA is an important component of exosomes for therapeutic effects.Among them,miR-29b-3p has the effect of reducing cell apoptosis,promoting axonal regeneration,and angiogenesis. OBJECTIVE:To study the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on a neural cell injury model simulated by H2O2-treated PC12 cells,and explore the relevant mechanisms. METHODS:(1)First,the collagenase digestion method was used to extract rat adipose-derived mesenchymal stem cells.Adipose-derived mesenchymal stem cells were transfected with miR-29b-3p mimics and inhibitors.Exosomes were extracted from the culture supernatant by ultracentrifugation and identified so as to construct adipose-derived mesenchymal stem cell-derived exosomes with high expression and knockdown miR-29b-3p.(2)By constructing a neural cell injury model simulated by PC12 cells treated with H2O2,the relevant mechanisms of the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on the simulated neuronal cell injury model were studied. RESULTS AND CONCLUSION:(1)Adipose-derived mesenchymal stem cell-derived exosome had a typical cup-shaped shape and a diameter distribution in the range of 50-140 nm,expressed membrane proteins Alix,CD63,and TSG101,which were specific markers on the surface of exosomes,and could be successfully ingested by PC12 cells.(2)Adipose-derived mesenchymal stem cell-derived exosome pretreatment could reduce cell apoptosis induced by H2O2 treatment in PC12 cells,and this protective effect was enhanced with the increase of miR-29b-3p expression in the exosomes and weakened with the decrease of miR-29b-3p expression in the exosomes.The mechanism of its effect was related to adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p promoting the expression of anti-apoptotic protein Bcl-2 and inhibiting the expression of apoptotic protein Bax.
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ObjectiveTo explore the mechanism and pathway of Gandou Fumu decoction （GDFMD） in the development of liver fibrosis in Wilson's disease （WD）. MethodFirst， 30 TX-j mice were randomly divided into the model group， high-dose， medium-dose， and low-dose GDFMD groups， and penicillamine group， with six mice in each group， and another six wild-type mice were used as the normal group. The high-dose， medium-dose， and low-dose GDFMD groups were intragastrically administered drugs of 13.92， 6.96， 3.48 g·kg-1. In the penicillamine group， 0.1 g·kg-1 of penicillamine was given by intragastric administration. The model group and the normal group were given equal volume of normal saline， once a day， for four consecutive weeks. Samples were collected four weeks after gavage， and enzyme-linked immunosorbent assay （ELISA） was used to detect type Ⅲ procollagen peptide （PCⅢ）， collagen type Ⅳ （Col Ⅳ）， hyaluronic acid （HA）， and laminin （LN）. Hematoxylin-eosin （HE）， Masson， and picric acid-Sirus red collagen （Sirus Red） staining were used to observe the histopathological changes of liver fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， immunohistochemistry， and Western blot were used to observe the expressions of α-smooth muscle actin （α-SMA） and collagen type Ⅰ （Col Ⅰ）， which were related to the activation of hepatic stellate cells （HSCs）. The expression of miR-29b-3p was observed by Real-time PCR. The expression of Unc-51-like kinase 1 （ULK1） and its downstream-related factors were observed by Western blot. The downstream genes of miR-29b-3p were verified by the dual luciferase reporter gene detection method. ResultCompared with the normal group， the four items of liver fibrosis （PCⅢ， Col Ⅳ， HA， and LN） in the model group were significantly abnormal （P<0.01）， and the pathology was significantly abnormal. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was up-regulated （P<0.05， P<0.01）， and miR-29b-3p expression was down-regulated （P<0.01）. ULK1， p-ULK1， autophagy-related gene 13 （Atg13）， p-Atg13， Beclin-1， FAK family kinase-interacting protein of 200 kDa （FIP200）， activating molecule in BECN1-regulated autophagy protein 1 （AMBKA1）， and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ） were up-regulated （P<0.05， P<0.01）. p62 protein expression was down-regulated （P<0.01）. Compared with the model group， the four items of liver fibrosis in the high-dose， medium-dose， and low-dose GDFMD groups and the penicillamine group were significantly improve （P<0.01）， and the pathological conditions were improved. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was down-regulated （P<0.05， P<0.01）， and the expression of miR-29b-3p was up-regulated （P<0.01）. ULK1， p-ULK1， Atg13， p-Atg13， Beclin-1， FIP200， AMBKA1， and LC3Ⅱ/Ⅰ were down-regulated （P<0.05， P<0.01）， and p62 protein expression was up-regulated （P<0.01）. The prediction software predicted that there was a binding site between miR-29b-3p and ULK1. The dual-luciferase reporter gene detection method indicated that the luciferase activity of the ULK1-WT plasmid-transfected cell group was reduced when miR-29b-3p mimics were co-cultured （P<0.01）. ConclusionGDFMD can regulate ULK1-mediated autophagy by up-regulating miR-29b-3p and further exert its anti-hepatic fibrosis effect in Wilson's disease.
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Abstract Objectives: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. Methods: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of cir-cUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. Results: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. Conclusion: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.
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Two new ursane triterpenoids along with twelve known compounds were isolated from 80% ethanol extract of Agastache rugosa (Fisch. et. Mey.) O. Kuntze by using silica gel column, MCI column, ODS column and HPLC. The structures of the new compounds were identified as 2α,3α-dihydroxy-24-nor-urs-4(23),12(13)-dien-28-oic acid (1) and 2α,3α-dihydroxy-24-nor-urs-4(23),12(13),20(30) -trien-28-oic acid (2) by HR-ESI-MS, NMR and ECD spectral data, named agasursacid A and agasursacid B. In addition, compounds 3, 4, 6, 8 showed anti-coxsackievirus B3 (CVB3) activities with a IC50 as 4.77, 1.59, 11.11 and 25.87 μmol·L-1, resepectively.
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ObjectiveTo investigate the effect of myosin heavy chain 7 gene-derived miRNA-208b-3p on the fibrotic phenotype of cardiac fibroblasts. MethodsmiRNA chip array was performed to detect the dysregulated miRNAs in the myocardium of diabetic db/db mice and db/m control mice. Neonatal mouse ventricular cardiomyocytes （NMVCs） and cardiac fibroblasts （CFs） were isolated from C57BL/6 mice and cultured. Real-time quantitative PCR （RT-qPCR） was conducted to determine the expression of miR-208b-3p in mouse CFs and NMVCs subjected to angiotensinⅡ（AngⅡ） and high glucose plus glucose oxidase （G/Go） treatment， respectively. Cell counting kit 8（CCk8） assay， flow cytometry and determination of fibrosis-related protein， including COL1A1， COL3A1and α-SMA， were performed in mCFs transfected with miR-208b-3p. Dual luciferase reporter assay was performed to confirm the interaction between miR-208b-3p and the 3'-UTR of metal response element binding transcription factor 2 （Mtf2） and progesterone receptor membrane component 1（Pgrmc1）， respectively. The expressions of Mtf2 and Pgrmc1 at the mRNA and protein levels in mCFs after miR-208b-3p mimic transfection were determined using RT-qPCR and Western blot assay， respectively. The small interfering RNA （siRNA） was used to inhibit Mtf2 and Pgrmc1 expression in mCFs， and the effects of Mtf2 siRNA， Pgrmc1 siRNA and miR-208b-3p on fibrosis-related protein expression in mCFs were investigated. ResultsResults of miRNA chip array and RT-qPCR assay showed that miR-208b-3p was up-regulated in the myocardium of the diabetic db/db mice. miR-208b precursor and the host gene of Myh7 were consistently increased in db/db mice. miR-208b-3p and Myh7 mRNA were expressed in mCFs and NMVCs， but the levels of miR-208b-3p and Myh7 mRNA in NMVCs were much higher than those in mCFs. miR-208b-3p was up-regulated in mCFs and NMVCs subjected to Ang Ⅱ and G/Go treatment， respectively. miR-208b-3p could significantly enhance fibrosis-related protein， including COL1A1， COL3A1 and α-SMA， in mCFs， without affecting the proliferation activity and cell cycle distribution of mCFs. Dual luciferase reporter assay revealed the interactions of miR-208b-3p with the 3'-UTR of Mtf2 and Pgrmc1. The results of RT-qPCR and Western blotting confirmed that miR-208b-3p inhibited Mtf2 and Pgrmc1 expression at the post- transcriptional level. Transfection with miR-208b-3p mimic， Mtf2 siRNA and Pgrmc1 siRNA could consistently enhance the fibrosis-related protein expression in the cardiac fibroblasts. ConclusionsmiR-208b-3p enhances fibrosis-related gene expression by targeting Mtf2 and Pgrmc1in mCFs.
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Objective:To study the effects of Zuogui Pills on the expressions of miR-133b-3p and RhoA in osteoclasts of postmenopausal osteoporosis rats; To discuss its potential mechanism.Methods:SD female rats were randomly divided into normal group, model group, sham-operation group, and Zuogui Pills group using a random number table method, with 6 rats in each group. The model group and Zuogui Pills group were treated with oophorectomy to construct a rat model of osteoporosis. Zuogui Pills group was orally administered with Zuogui Pills decoction at a concentration of 10 g/kg for 12 consecutive weeks. Colorimetric method was used to measure the serum calcium and phosphorus levels of rats, and ELISA method was used to detect ALP levels. Bone density meter was used to measure the bone density of the femurs of rats in each group. The osteoclast of each group were cultured, and the expressions of RANKL and RUNX2 protein were detected by Western blot. MiRNA sequencing and differential expression analysis were performed on bone tissues of rats. Osteoclasts were treated with miR-133b-3p mimic and its negative control. The cell proliferation activity of osteoclasts was detected by cell counting kit-8 (CCK-8). The osteoclast differentiation activity was detected by the tartrate-resistant acid phosphatase staining. The dual-luciferase reporter assay was used to detect the relationship between miR-133b-3p and RhoA. The "rescue" experiment of miR-133b-3p mimic and RhoA co-expression were used to study the molecular regulatory mechanism of Zuogui Pills on osteoclast activity.Results:Compared with the model group, the bone mineral density of Zuogui Pills group significantly increased ( P<0.05, P<0.01), the levels of calcium and phosphorus in serum increased, the level of alkaline phosphatase ALP decreased ( P<0.05), the expression of RANKL protein decreased, and the expression of RUNX2 protein increased. Sequencing results showed that rno-miR-133b-3p was down-regulated in osteoclasts of postmenopausa osteoporosis rats treated with Zuogui Pills with the maximum difference ( P<0.01). Q-PCR results showed that the expression of miR-133b-3p in osteoclasts of Zuogui Pills group was significantly lower than that of the model group. The upregulation of miR-133b-3p could significantly promote the cell proliferation and differentiation of osteoclasts. RhoA overexpression could reverse the excessive proliferation and differentiation of osteoclasts caused by miR-133b-3p overexpression. Conclusions:RhoA is the target gene regulated by miR-133b-3p. Zuogui Pills can inhibit the activity of osteoclasts by regulating miR-133b-3p/RhoA axis, relieving the symptoms of osteoporosis.
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Objective:To detect the expression of acetaldehyde dehydrogenase (ALDH) 2 and microRNA (miR)-135b-3p in endometrial cancer (EC) tissues, and to explore their correlation with clinical characteristics.Methods:94 endometrial cancer tissue specimens (EC group) and 60 normal endometrial specimens (control group) were selected from Northwest Women′s and Children′s Hospital from February 2019 to February 2022. Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression changes of ALDH2 mRNA and miR-135b-3p in endometrial tissue of two groups, and immunohistochemistry was used to detect the positive expression rate of ALDH2 protein. The relationship between the expression levels of ALDH2 and miR-135b-3p and the clinicopathological characteristics was analyzed.Results:The expression of ALDH2 mRNA in the EC group (0.67±0.15) was lower than that in the control group (1.05±0.12), and the expression level of miR-135b-3p (1.52±0.26) was higher than that in the control group (1.01±0.06). The positive expression rate of ALDH2 in cancer tissue of EC group was 30.85%(29/94), which was lower than 51.67%(31/60) in normal endometrial tissue of the control group ( P<0.05). The expression levels of ALDH2 mRNA and miR-135b-3p in EC patients were related to International Federation of Obstetrics and Gynecology (FIGO) stage, lymph node metastasis, differentiation and myometrium invasion (all P<0.05), but not to age, pathological type, menopause, HPV infection, menarche age, parity, tumor length and BMI (all P>0.05). Conclusions:In EC tissues, the expression of ALDH2 is down-regulated and the expression of miR-135b-3p is up-regulated, which may be involved in the occurrence and development of EC.
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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 effect of miR-23b-3p regulation on osteogenic differentiation of renal intersti-tial fibroblasts(hRIFs)on the formation of Randall plaque and its possible mechanism.Methods qRT-PCR was used to detect the expression levels of miR-23b-3p and osteogenic marker:myocyte enhancer factor 2C(MEF2C),osteocalcin(OCN),osteopontin(OPN),runt-related transcription factor 2(Runx2)mRNA in Randall plaque tis-sue of CaOx stone patients(RP)and normal papillary tissue of kidney tumor patients undergoing nephrectomy(nRP).Isolation and culture of human normal hRIFs were isolated and cultured in vitro.The miR-23b-3p overex-pression plasmid pSi-miR-23b-3p and its negative no-load plasmid pSi-NC,the MEF2C lentivirus overexpression plasmid Lv-MEF2C and the no-load plasmid Lv-NC were transfected into hRIFs cells,and the cells were induced to osteogenic differentiation for 14 days.The activity of alkaline phosphatase(ALP)was determined by ELISA.Aliz-arin red staining was used to observe the formation of mineralized nodules.The expression levels of miR-23b-3p and MEF2C,OCN,OPN,Runx2 mRNA were detected by qRT-PCR.The expression level of MEF2C protein was de-tected by Western blot.Dual luciferase reporter gene assay verified the targeting relationship between miR-23b-3p and MEF2C.Results ① Compared with the nRP group,miR-23b-3p was low expressed and MEF2C,OCN,OPN,and Runx2 were highly expressed in the RP group.② 14 days after osteogenic induction of hRIFs cells,the activity of ALP in cells significantly increased,the ability of cells to form mineralized nodules was enhanced,the expression level of miR-23b-3p significantly decreased,the mRNA expression levels of MEF2C,OCN,OPN,and Runx2 significantly increased,and the expression level of MEF2C protein significantly increased.③ Overexpres-sion of miR-23b-3p decreased the activity of ALP in hRIFs cells after osteogenic induction,inhibited the formation of mineralized nodules in cells,and down-regulated the mRNA expression levels of OCN,OPN,and Runx2 in cells.④ Overexpression of MEF2C reversed the inhibitory effect of miR-23b-3p overexpression on osteoblast differ-entiation of hRIFs cells.⑤ MEF2C was the downstream target gene of miR-23b-3p.Conclusion miR-23b-3p is underexpressed in RP tissues and during osteoblastic differentiation of hRIFs cells.Up-regulation of miR-23b-3p in-hibits osteogenic differentiation of hRIFs cells,and its mechanism may be related to targeted silencing MEF2C.
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Objective:To investigate whether miR-548b-3p can regulate the malignant biological traits of hepatocellular carcinoma cells by down-regulating the expression of decoy receptor 3( DcR3). Methods:The miR-548b-3p levels in 5 common hepatoma cell lines Hep3b, HepG2, Huh7.5.1, PLC and 97H were detected by real-time fluorescence quantitative PCR. The experimental hepatoma cell lines were purchased from Shanghai Cancer Institute. The subcutaneous tumor formation experiment in nude mice included two parts: miR-548b-3p over expression group and down expression group. miR-548b-3p over expression experiment included Huh7.5.1- miR-548b-3p cell group and Huh7.5.1-NC cell group. The experimental group with low expression of miR-548b-3p included PLC- miR-548b-3p-sponge group and PLC-NC cell group, with 5 nude mice in each group. The nude mice used in the experiment were male Balb/c strain, body weight 20-25 g, 4-6 weeks old. Starbase software was used to predict whether there were binding sites between miR-548b-3p and DcR3. Dual luciferase reporting assay to verify whether DcR3 is the target gene of miR-548b-3p. Rescue experiments were conducted to verify whether the effects of miR-548b-3p on the proliferation, invasion and migration of liver cancer cells were realized through DcR3. Statistical analysis was performed using Graphpad Prism 9. Measurement data with normal distribution were expressed as mean±standard deviation( ± s), and t-test was adopted for comparison between general measurement data groups. Results:The relative expression level of 2 -ΔΔCt was used as the parameter for real-time fluorescence quantitative PCR comparison. Hep3b expression level was 1, HepG2 expression level was 0.902, Huh7.5.1 expression level was 0.712, PLC expression level was 1.293, and 97H expression level was 0.818. The final results showed that, The highest expression of miR-548b-3p was in PLC cells, and the lowest expression was in Huh7.5.1 cells. The subcutaneous tumor formation experiment of nude mice showed that after 4 weeks, the tumor volume of Huh7.5.1- miR-548b-3p was(444.77±142.34) mm 3, and that of Huh7.5.1-NC was(918.80±139.21) mm 3. The tumor volume of PLC- miR-548b-3p-sponge was(407.49±58.50) mm 3, and that of PLC-NC was(218.62±47.55) mm 3. The binding sites of miR-548b-3p and DcR3 were predicted by Starbase software. Dual luciferase assay showed that DcR3 was the target gene of miR-548b-3p. Rescue experiments verified that the effects of miR-548b-3p on the proliferation, invasion and migration of liver cancer cells were realized through DcR3. Conclusion:miR-548b-3p can regulate the proliferation, invasion, migration and other malignant biological traits of liver cancer cells, and it is achieved by down-regulating the expression level of DcR3.
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Objective:To investigate the effect of microRNA-27b-3p (miR-27b-3p)/nuclear factor-E2-related factor 2 (Nrf2) on metabolic memory impairment of human retinal pigment epithelial (RPE) cells and to explore its regulatory mechanism.Methods:ARPE-19 cells were divided into normal control group, metabolic memory group, miR-27b-3p control group, miR-27b-3p inhibitor group, and liraglutide group.Cells in normal control group were cultured in 5.5 mmol/L normal glucose medium for 6 days.Cells in metabolic memory group were cultured in 30 mmol/L glucose for 3 days and changed to 5.5 mmol/L for 3 days.Cells in miR-27b-3p inhibitor group were added with puromycin after lentiviral transfection to select the successfully transfected cells, and were cultured in 30 mmol/L glucose for 3 days then 5.5 mmol/L glucose for 3 days.Cells in liraglutide group were cultured in 30 mmol/L glucose with liraglutide for 3 days then 5.5 mmol/L glucose for 3 days.The regulatory relationship between miR-27b-3p and Nrf2 was verified by lentiviral transfection.Expressions of miR-27b-3p, Nrf2, NAD(P)H dehydrogenase[quinone]1 (NQO1), heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed by real-time quantitative PCR.Total and nuclear Nrf2 protein expressions were detected by Western blot.The cell proliferation rates of various groups were determined by cell counting kit-8 (CCK-8).The reactive oxygen species (ROS) level was detected by the DHE kit.Results:The miR-27b-3p mRNA relative expression of normal control group, metabolic memory group, miR-27b-3p control group, miR-27b-3p inhibitor group was 1.000±0.000, 1.881±0.034, 1.683±0.088 and 0.111±0.008, respectively, with a statistically significant difference ( F=850.815, P<0.001).The miR-27b-3p mRNA relative expression level was lower in normal control group than in metabolic memory group, lower in miR-27b-3p inhibitor group than in normal control group, and the differences were statistically significant (both at P<0.01).The expression levels of Nrf2 mRNA, total protein, and nuclear protein were decreased in metabolic memory group in comparison with normal control group and were significantly increased in miR-27b-3p inhibitor group in comparison with miR-27b-3p control group, showing statistically significant differences (all at P<0.01).The NQO1 and HO-1 mRNA expressions were decreased in metabolic memory group in comparison with normal control group, and were significantly higher in miR-27b-3p inhibitor group compared with miR-27b-3p control group, showing statistically significant differences (all at P<0.01).The fluorescence intensity of Nrf2, NQO1, and HO-1 was lower in metabolic memory group than in normal control group, and was higher in miR-27b-3p inhibitor group than in miR-27b-3p control group, showing statistically significant differences (all at P<0.01).Compared with metabolic memory group, the relative expression of miR-27b-3p mRNA declined in liraglutide group, with a statistically significant difference ( P<0.05).The relative expression levels of Nrf2 mRNA, NQO1 mRNA, HO-1 mRNA, total and nuclear Nrf2 protein of liraglutide group were enhanced in comparison with metabolic memory group, with statistically significant differences (all at P<0.05).The fluorescence intensity of Nrf2, NQO1, and HO-1 was enhanced in liraglutide group in comparison with metabolic memory group, and the differences were statistically significant (all at P<0.05).Compared with normal control group and liraglutide group, the cell proliferation viability was decreased in metabolic memory group, and the differences were statistically significant (both at P<0.01).The relative content of ROS was higher in metabolic memory group than in normal control group and liraglutide group, and the difference was significant (all at P<0.01). Conclusions:Liraglutide reverses the inhibition of metabolic memory on Nrf2, NQO1, and HO-1 by downregulating miR-27b-3p.
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@#Abstract: Objective To genotype and analyze whole genomic features of Coxsackievirus B3 (CVB3) isolated in Tianjin, to improve evolution information of CVB3 virus in Tianjin, and to provide basis for surveillance and early warning of related diseases. Methods Viral RNA was extracted from five CVB3 strains isolated in Tianjin, whole genome sequence of the virus was amplified by RT-PCR and sequenced by next-generation sequencing method, and phylogenetic and recombinant analysis were carried out. Results The open reading frame 1(ORF) of the five CVB3 strains contained 6 555 nucleotides and encoded 2 185 amino acids, and ORF2 was composed of sequences encoding 68 amino acids. The nucleotide sequence similarity ranged from 78.3%-100%, and the amino acid sequence similarity ranged from 95.7%-100%. Compared with the CVB3 prototype strain, the nucleotide sequence similarity of the five viruses was between 78.2%-79.1%, and the similarity of amino acid sequences was 94.9%-95.3%. All five viruses exhibited a T151A mutation on the VP2 protein. Additionally, the encephalitis isolate showed a K158E mutation on the VP2 protein, while one of the sewage isolates had a C234T mutation in 5' noncoding region. The five strains belonged to two different genotypes, among which the encephalitis isolate in 2016 belonged to the D genotype, while the sewage isolates in 2021 belonged to the E genotype. This is also the first report of E genotype CVB3 in northern China. The CVB3 strain may have recombinant events in non-structural protein regions, in which encephalitis isolate may recombine with a Coxsackievirus B5 (CVB5) strain, while sewage isolates may have recombinant events with a strain of ECHO virus 18 (E18). Conclusions The CVB3 isolates in Tianjin belong to D and E genotypes, and recombination events may exist in non-structural protein region of the viral genome. The results of CVB3 virus genome analysis in sewage suggests presence of CVB3 infection in the population of Tianjin, and its epidemic dominant genotype may have changed.
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This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.
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Animais , MicroRNAs/metabolismo , Cabras/genética , PPAR gama/metabolismo , Adipogenia/genética , Diferenciação Celular/genética , Luciferases , RNA MensageiroRESUMO
OBJECTIVE@#To investigate the association between the expression level of platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3 ) gene in bone marrow CD138+ cells of patients with multiple myeloma (MM) treated with autologous hematopoietic stem cell transplantation (AHSCT) and the prognosis within 2 years.@*METHODS@#147 MM patients treated with AHSCT in The First and The Second Affiliated Hospital of Nantong University from May 2014 to May 2019 were included in the study. Expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells of the patients was detected. Patients with disease progression or death during 2 years of follow-up were included in progression group, and the rest were included in good prognosis group. After comparing the clinical data and PAFAH1B3 mRNA expression levels of the two groups, the patients were divided into high PAFAH1B3 expression group and low PAFAH1B3 expression group based on the median PAFAH1B3 mRNA expression level of the enrolled patients. Progression-free survival rate (PFSR) between the two groups was compared by the Kaplan-Meier method. The related factors of prognosis within 2 years were analyzed by univariate analysis and multivariate COX regression analysis.@*RESULTS@#At the end of follow-up, there were 13 patients lost to follow-up. Finally, 44 patients were included in the progression group and 90 patients were included in the good prognosis group. Age in the progression group was higher than that in the good prognosis group, the proportion of patients with CR+VGPR after transplantation in the progression group was lower than that in the good prognosis group, and there was a statistical difference between two groups in the cases distribution of ISS stage (all P<0.05). PAFAH1B3 mRNA expression level and the proportion of patients with LDH>250U/L in the progression group were higher than those in the good prognosis group, and platelet count in the progression group was lower than that in the good prognosis group (all P<0.05). Compared with the low PAFAH1B3 expression group, the 2-year PFSR of the high PAFAH1B3 expression group was significantly lower (log-rank χ2=8.167, P=0.004). LDH>250U/L (HR=3.389, P=0.010), PAFAH1B3 mRNA expression (HR=50.561, P=0.001) and ISS stage Ⅲ(HR=1.000, P=0.003) were independent risk factors for prognosis in MM patients, and ISS stage Ⅰ (HR=0.133, P=0.001) was independent protective factor.@*CONCLUSION@#The expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells is related to the prognosis of MM patients treated with AHSCT, and detecting PAFAH1B3 mRNA expression can bring some information for predicting PFSR and prognostic stratification of patients.
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Humanos , Progressão da Doença , Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo/tratamento farmacológico , Prognóstico , Estudos Retrospectivos , Transplante Autólogo , 1-Alquil-2-acetilglicerofosfocolina Esterase/genéticaRESUMO
OBJECTIVE@#To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis.@*METHODS@#The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells.@*RESULTS@#The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01).@*CONCLUSION@#GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.
Assuntos
Humanos , Proliferação de Células , MicroRNAs/metabolismo , Artrite Reumatoide/genética , Gentisatos/farmacologia , Movimento Celular/genéticaRESUMO
Objective @#The purpose of this study was to investigate the effects of plasma exosome⁃derived miR⁃29b⁃3p in myocardial ischemia⁃reperfusion injury ( MIRI) rats on hypoxia/reoxygenation ( H/R) cardiomyocyte after sevoflurane (SEV) postconditioning through targeting IGF1 . @*Methods @# The GEO database was used to screen differentially expressed miRNAs in MIRI , and cardiomyocytes were treated with H/R to construct a MIRI cell model. The expression of miR⁃29b⁃3p and IGF1 in the MIRI cell model post⁃treated with SEV was intervened , and then the survival rate of cardiomyocytes was detected by MTT , apoptosis was detected by flow cytometry , and inflammatory factors (IL⁃1β and TNF⁃α ) in cardiomyocytes in each group were detected by ELISA.@*Results @#Compared with Normal group , the expression of miR⁃29b⁃3p in plasma exosomes of MIRI rats was enhanced (P < 0. 05) , and the target⁃binding relationship between miR⁃29b⁃3p and IGF1 was confirmed ( P < 0. 05) . After SEV post⁃treatment , the expression of miR⁃29b⁃3p in H/R⁃stimulated cardiomyocytes decreased , while the expression of IGF1 increased (both P < 0. 05) . Overexpression of miR⁃29b⁃3p in plasma exosomes could significantly inhibit the survival rate of H/R cells after SEV treatment , aggravate apoptosis and inflammatory response , while knockdown of miR⁃29b⁃3p showed a opposite effects (all P < 0. 05) . The rescue experimental data showed that overexpression of IGF1 could partially reverse the effects of overexpression of miR⁃29b⁃3p on H/R cell injury after SEV treatment ( all P <0. 05) .@*Conclusion @#Plasma exosome⁃derived miR⁃29b⁃3p promotes H/R cardiomyocyte injury after SEV treatment by targeting IGF1 .
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Objective @#To analyze the effect of miR⁃148b⁃3p on the proliferation of keloid derived fibroblasts. @*Methods @#The expression levels of miR⁃148b⁃3p and SPARC in human keloid derived fibroblasts (HKF) and normal human fibroblasts (NFS) were analyzed by real time PCR. The expression level of SPARC protein was detected by Western blot. The effects of miR⁃148b⁃3p and SPARC on HKF proliferation were analyzed by CCK⁃8 method the luciferase reporter plasmid was constructed. The targeted binding site of miR⁃148b⁃3p and the target gene was analyzed by luciferase reporter gene method.@*Results @#miR⁃148b⁃3p was low expressed in HKF and SPARC was high expressed in HKF. Transfection of miR⁃148b⁃3p in HKF cells could down regulate the expression of SPARC and inhibit cell proliferation. Online analysis software predicted that miR⁃148b⁃3p could target the 3 ′⁃ UTR binding SPARC ; The results of dual luciferase reporter gene further confirmed that miR⁃148b⁃3p could target the 3 ′⁃ UTR of SPARC. Transfection of SPARC eukaryotic expression plasmid into HKF transfected with miR⁃148b⁃3p could counteract the effect of miR⁃148b⁃3p and restore cell proliferation. @*Conclusion @# miR⁃148b⁃3p can inhibit the proliferation of HKF by targeting the 3 ′⁃ UTR of SPARC and inhibiting its expression.
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Objective:To explore the effect of myricitrin on the injury of human retinal microvascular endothelial cells (HRMECs) induced by high glucose and its regulation mechanism.Methods:HRMECs were divided into normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml myricitrin groups.HRMECs transfected with pcDNA and pcDNA-circZNF292, respectively and then cultured in high-glucose medium containing 25 mmol/L D-glucose for 24 hours were assigned as pcDNA group and pcDNA-circZNF292 group.HRMECs transfected with siR-NC and siR-circZNF292, respectively and then cultured in medium containing 50.0 μg/ml myricitrin and 25 mmol/L D-glucose for 24 hours were assigned as myricitrin+ siR-NC group and myricitrin+ siR-circZNF292 group.The cell apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in cells were detected by enzyme-linked immunosorbent assay (ELISA) kits.The expression levels of circZNF292 and miR-23b-3p were detected by real-time fluorescence quantitative PCR.The targeting relationship between circZNF292 and miR-23b-3p was detected by dual-luciferase reporter assay.The relative expression levels of B-cell lymphoma-2 (bcl-2) and bcl-2-related X protein (bax) were assayed by Western blot.Results:Significant differences were found in the relative expressions of bax and bcl-2 proteins, cell apoptosis rate, MDA concentration, SOD activity, circZNF292 and miR-23b-3p among normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml, 50.0 μg/ml myricitrin groups ( F=105.707, 111.835, 74.515, 109.651, 135.020, 219.919, 116.304; all at P<0.001).With the increase of myricitrin concentration, the relative expression levels of bax protein, cell apoptosis rate, MDA concentration and miR-23b-3p in cells gradually decreased, while the relative expression levels of bcl-2 protein, SOD activity and circZNF292 increased, with statistically significant differences among groups with different concentrations of myricitrin (all at P<0.05).In the co-transfected wild-type (WT)-circZNF292 cells, the relative luciferase activity in miR-23b-3p group was 0.35±0.03, which was lower than 0.96±0.09 in microRNA-negative control group, and the difference was statistically significant ( t=11.137, P<0.001).Compared with pcDNA group, the relative expression levels of bcl-2 protein, circZNF292 and MDA concentration in cells of pcDNA-circZNF292 group were significantly increased, and the relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and SOD activity were significantly decreased (all at P<0.05).The relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and MDA concentration were reduced and relative expression levels of bcl-2 protein, circZNF292 and SOD activity were enhanced in myricitrin group and myricitrin+ siR-NC group in comparison with high glucose group and myricitrin+ siR-circZNF292 group, showing statistically significant differences (all at P<0.05). Conclusions:Myricitrin can inhibit cell apoptosis and oxidative stress by regulating the expression of circZNF292/miR-23b-3p, thereby reducing the damage of HRMECs induced by high glucose.
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Objective:To investigate the regulatory effects of microRNA-23b-3p (miR-23b-3p) on the autophagy and apoptosis of human lens epithelial cells induced by high glucose.Methods:Thirty diabetic cataract (DC) patients as DC group and 30 patients with simple cataract as simple cataract group were enrolled in The First Affiliated Hospital of Xi'an Medical University from September 2019 to October 2020.Conventional phacoemulsification and intraocular lens transplantation were performed in both groups.The anterior capsular tissue was collected during the operation.The expression of miR-23b-3p in the anterior lens capsule was detected by real-time fluorescence quantitative PCR (RT-qPCR). Human lens epithelial cell line HLEB3 cells were cultured in vitro and divided into normal control group and high-glucose group, which were cultured in normal and high-glucose medium, respectively.The targeting relationship between proto-cadherin 17 (PCDH17) and miR-23b-3p was predicted according to the bioinformatics database, and was verified by the dual-luciferase reporter gene experiment.High glucose-cultured HLEB3 cells were divided into miR-23b-3p mimics group, negative control (NC) mimics group, NC-siRNA group, PCDH17-siRNA group, miR-23b-3p mimics+ Vector group, miR-23b-3p mimics+ pcDNA-PCDH17 group, and were transfected with corresponding reagents according to grouping.The expression of miR-23b-3p and PCDH17 mRNA was detected by RT-qPCR.The expressions of a mammalian homolog of yeast Atg6/Vps30 (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3B), c-Jun N-terminal kinases (JNK), phosphorylated (p-) JNK, c-Jun, p-c-Jun, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) proteins were assayed by Western blot.The apoptosis rate was detected by flow cytometry.The study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Medical College (No.LSL2019037). Written informed consent was obtained from each patient. Results:The relative expression of miR-23b-3p in the anterior lens capsule of DC group was 0.35±0.15, which was significantly lower than 1.00±0.09 of simple cataract group ( t=44.627, P<0.01). There were significant differences in the relative expression levels of miR-23b-3p, LC3B Ⅱ/Ⅰ, Beclin-1, Bcl-2 and Bax proteins among normal control group, high glucose group, high glucose+ NC mimics group and high glucose+ miR-23b-3p mimics group ( F=21.325, 28.318, 17.634, 15.482, 22.325, 26.537; all at P<0.01). Compared with normal control group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax protein expressions in high glucose group were significantly increased, and the Bcl-2 protein expression was significantly decreased (all at P<0.05). Compared with NC mimics group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1, and Bax protein expressions were significantly decreased and the Bcl-2 protein expression was significantly increased in miR-23b-3p mimics group (all at P<0.05). The results of bioinformatics and dual-luciferase reporter gene experiments showed that PCDH17 was a target gene of miR-23b-3p, and the relative expression of PCDH17 mRNA in miR-23b-3p mimics group was significantly lower than that in NC mimics group ( P<0.05). Compared with NC-siRNA group, the apoptosis rate, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax protein expressions in PCDH17-siRNA group were significantly decreased, and the Bcl-2 protein expression was significantly increased ( t=9.116, 12.413, 5.349, 3.273, 8.419; all at P<0.01). There were significant differences in the relative expression levels of p-JNK/JNK, p-c-Jun/c-Jun, LC3B Ⅱ/Ⅰ, Beclin-1, Bcl-2 and Bax proteins in NC mimics group, miR-23b-3p mimics group, miR-23b-3p mimics+ Vector group and miR-23b-3p mimics+ pcDNA-PCDH17 group ( F=24.724, 19.319, 23.418, 17.562, 20.263, 15.249; all at P<0.05). Compared with the miR-23b-3p mimics+ Vector group, the expressions of p-JNK/JNK, p-c-Jun/c-Jun, LC3B Ⅱ/Ⅰ, Beclin-1 and Bax were significantly increased, and the expression of Bcl-2 protein was decreased in miR-23b-3p mimics+ pcDNA-PCDH17 group (all at P<0.05). Conclusions:MiR-23b-3p have a protective effect on HLEB3 cells in a high-glucose environment, mainly by targeting PCDH17 to regulate the JNK signaling pathway to inhibit high glucose-induced autophagy and apoptosis.
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In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.