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International Journal of Ophthalmology(English edition) 2020年第5期13卷 693-700页

作者： Qu-Zhen Deji Feng Yan Wang-Dui Zhaba Ya-Jun Liu Jie Yin Zhen-Ping Huang Department of Ophthalmology Affiliated Jinling HospitalMedical School of Nanjing UniversityNanjing 210002Jiangsu ProvinceChina Department of Neurosurgery Affiliated Jinling HospitalMedical School of Nanjing UniversityNanjing 210002Jiangsu ProvinceChina Department of Ophthalmology Affiliated Drum Tower HospitalMedical School of Nanjing UniversityNanjing 210008Jiangsu ProvinceChina

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P2-induced EMT(P2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

关键词： microRNA-let7c transforming growth factor-β2 epithelial-to-mesenchymal transition human retinal pigment epithelial cells nuclear factor-kappa B pathway

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

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International Journal of Ophthalmology(English edition) 2016年第1期9卷 29-32页

作者： Chao Liu Xao-Li Wu Xin-Yi Wu Zhen-Hua Zhang Xiao-Hua Liu Department of Ophthalmology Qilu Hospital Shandong University Jinan 250012 Shandong Province China Department of Ophthalmology Central Hospital of QingdaoQingdao 266042 Shandong Province China Department of Ophthalmology Rongjun Hospital of Shandong Jinan 250013 Shandong Province China Department of Stomatology Central Hospital of QingdaoQingdao 266042 Shandong Province China

AIM：To study the inhibition of nuclear factor kappa-B p65（NF-κB p65）antisense oligodeoxynucleotide（ASODN）on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2（TGF-β2）.·M ETHODS：NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide（MSODN）were designed and *** lens epithelial cell line（HLE B-3）cells were prepared for study and divided into 7 *** group was HLE B-3 cells cultured in dulbecco’s modified eagle medium（DMEM）.T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for *** negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent（Hi Per Fect）for *** morphology was observed at different time points using an inverted *** expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction（RT-PCR）,and the expression ofα-smooth muscle actin（α-SMA）protein was assayed with ELISA.·RESULTS：With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant（〈0.05）.NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β***-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β*** difference between control group and A＋T group was not statistically significant（〉0.05）,but the difference among A＋T group and other groups was statistically significant（〈0.05）.·CONCLUSION：NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiati

关键词： nuclear factor kappa-B p65 antisenseoligodeoxynucleotide transforming growth factor-β2 α-smooth muscle actin lens epithelial cells

All-trans retinoic acid regulates the expression of MMP-2 and TGF-β2 via RDH5 in retinal pigment epithelium cells

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International Journal of Ophthalmology(English edition) 2023年第6期16卷 849-854页

作者： Yu-Mei Mao Chang-Jun Lan Qing-Qing Tan Gui-Mei Zhou Xiao-Ling Xiang Jia Lin Xuan Liao Department of Ophthalmology Affiliated Hospital of North Sichuan Medical CollegeNanchong 637000Sichuan ProvinceChina Medical School of Ophthalmology and Optometry North Sichuan Medical CollegeNanchong 637000Sichuan ProvinceChina

·AIM: To investigate the effect of all-trans retinoic acid(ATRA) on retinol dehydrogenase 5(RDH5), matrix metalloproteinase-2(MMP-2) and transforming growth factor-β2(TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium(RPE) cells.·METHODS: After adult RPE cell line-19(ARPE-19 cells) intervened with gradient concentrations of ATRA(0-20 μmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative realtime polymerase chain reaction(q RT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 m RNA expression. Then, after ARPE-19 cells transfected with three different si RNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 m RNA within them was detected by q RT-PCR. ·RESULTS: Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 μmol/L and compared with the normal control group(P=0.027 and P=0.031, respectively). q RT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 m RNA(P2 and TGF-β2 m RNA(P=0.03 and P2). When RDH5 was knocked down for 48h, the results of q RT-PCR showed that the expressions of MMP-2 and TGF-β2 m RNA were significantly up-regulated(P2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesen

关键词： KEYWORDS:retinol dehydrogenase 5 matrix metalloproteinase-2 transforming growth factor-β2 all-trans retinoic acid ARPE-19

In vitro protective effect of recombinant prominin-1 combined with microRNA-29b on N-methyl-D-aspartateinduced excitotoxicity in retinal ganglion cells

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International Journal of Ophthalmology(English edition) 2023年第11期16卷 1746-1755页

作者： Jun-Hua Li Guan-Shun Yu Yu-Da Wang Tian-Kun Li Eye Hospital and School of Ophthalmology and Optometry Wenzhou Medical UniversityWenzhou 325027Zhejiang ProvinceChina Eye Hospital of Wenzhou Medical University Hangzhou Branch Hangzhou 310020Zhejiang ProvinceChina Tianjin Eye Hospital Tianjin 300020China

AIM:To determine the in vitro protective effect of recombinant prominin-1(Prominin-1)+microRNA-29b(P1M29)on N-methyl-D-aspartate(NMDA)-induced excitotoxicity in retinal ganglion cells(RGCs).METHODS:RGC-5 cells were cultured,and NMDAinduced excitotoxicity at the range of 100–800μmol/L was assessed using the MTT ***(800μmol/L)was selected as the appropriate concentration for preparing the cell *** evaluate the protective effect of P1M29 on the cell model,Prominin-1 was added at the concentration of 1–6 ng/mL for 48h,and the cell survival was investigated with/without *** obtaining the appropriate concentration and time of P1M29 at 48h,real-time polymerase chain reaction(PCR)was utilized to detect the relative mRNA expression of vascular endothelial growth factor(VEGF)and transforming growth factor(TGF)-β*** blot detection was applied to measure the phosphorylation levels of protein kinase B(AKT)and extracellular regulated protein kinases(ERK)in RGC-5 cells after treatment with *** study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of *** analysis was used to analyze the expression levels of VEGF and TGF-β***:MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group(P<0.05).Real-time PCR data indicated that the expression levels of VEGF were significantly increased in both Prominin-1 and P1M29 groups compared NMDA and microRNA-29b group(P<0.05),while TGF-β2 were significantly decreased in both microRNA-29b and P1M29 groups compared NMDA and Prominin-1 group(P<0.05).Western blot results showed that both Prominin-1 and P1M29 groups significantly increased the phosphorylation levels of AKT and ERK compared to NMDA and microRNA-29b groups(P<0.05).Flow cytometry analysis revealed that P1M29 could prevent RGC-5 cell apoptosis in the early stage of apoptosis,while immunofluorescence results sho

关键词： Prominin-1 microRNA-29b vascular endothelial growth factor transforming growth factor-β2

Inhibition of TGF-β2-induced migration and epithelial-mesenchymal transition in ARPE-19 by sulforaphane

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International Journal of Ophthalmology(English edition) 2021年第7期14卷 973-980页

作者： Yan-Bing Huang Ping-Ping Liu Hui Zheng Xiu-Xia Yang Cheng-Cheng Yang Ye Liu Yang Liu Department of Ophthalmology the Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000Guangdong ProvinceChina Department of Pathology the Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000Guangdong ProvinceChina

AIM: To investigate the effects of sulforaphane(SFN) on transforming growth factor(TGF)-β2 stimulated migration and epithelial-mesenchymal transition(EMT) in ARPE-19 ***: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay(LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt ***: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

关键词： transforming growth factor-β2 epithelial-mesenchymal transition sulforaphane ARPE-19

Modulation of TGFβ_2 and dopamine by PKC in retinal Müller cells of guinea pig myopic eye

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International Journal of Ophthalmology(English edition) 2011年第4期4卷 357-360页

作者： Jun-Feng Mao, Shuang-Zhen Liu, Wen-Juan Qin, Qian Xiang Department of Ophthalmology, Xiangya Hospital of Central South University, Changsha 410008, Hunan Province, China Department of Ophthalmology Xiangya Hospital of Central South University Changsha 410008 Hunan Province China.

AIM: To investigate the effect of protein kinase C (PKC) on transforming growth factor-β2 (TGFβ2) and dopamine in retinal Müller cells of guinea pig myopic eye. METHODS: Myopia was induced by translucent goggles in guinea pig, whose retinal Müller cells were cultured using the enzyme-digesting method. Retinal Müller cells were divided into 5 groups: normal control, myopia, myopia plus GF109203X, myopia plus PMA, myopia plus DMSO. PKC activities were detected by the non-radioactive methods. TGFβ2 and tyrosine hydroxylase (TH) proteins were analyzed by Western Blotting in retinal Müller cells. Dopamine was determined by the high-performance liquid chromatography- electrochemical detection in suspensions. RESULTS: After 14 days deprived, the occluded eyes became myopic with ocular axle elongating. Müller cells of guinea pigs were obtained using enzyme digestion. Compared with normal control group, the increase in PKC activity and the up-regulation in TGFβ2 expression were found in retinal Müller cells of myopic eyes, with the decrease of TH and dopamine content (P 2 and TH content were up-regulated with the increase of dopamine content (P 203X, proteins of TGFβ2 and TH were down-regulated in the myopic eyes, with the decrease of dopamine content (P 2 and dopamine are modulated by PKC in Müller cells of the myopic eyes in guinea pig.

关键词： protein kinase C retinal Müller cell transforming growth factor-β2 dopamine myopia

Effect of Endothelial Microparticles Induced by Hypoxia on Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells by Delivering MicroRNA-19b

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Chinese Medical Journal 2018年第22期131卷 2726-2733页

作者： Hui-Zhu Liang Su-Fang Li Feng Zhang Man-Yan Wu Chang-Long Li Jun-Xian Song Chongyou Lee Hong Chen Department of Cardiology Beijing Key Laboratory of Early Prediction and Intervention of Acute Myocardial InfarctionCenter for Cardiovascular Translational ResearchPeking University People's HospitalBeijing 100044China

Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial ***: Human umbilical vein endothelial cells (HUVECs) were culturedin vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student’st-test was used when two groups were ***: Compared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40,P 235.00 ± 16.52,P transforming growth factor-β2 (TGFβ2)

关键词： Endothelial Microparticle MicroRNA-19b Hypoxia Cell Migration Angiogenesis transforming growth factor-β2