目的探讨食管鳞癌组织中microRNA let-7a-3甲基化状态与血浆中类胰岛素样生长因子2(Insulin like growth factor 2,IGF-Ⅱ)表达的相关性。方法采用甲基化特异性PCR法(Methylation specific PCR,qMSP)检测83例食管癌及相对应的癌旁正常...目的探讨食管鳞癌组织中microRNA let-7a-3甲基化状态与血浆中类胰岛素样生长因子2(Insulin like growth factor 2,IGF-Ⅱ)表达的相关性。方法采用甲基化特异性PCR法(Methylation specific PCR,qMSP)检测83例食管癌及相对应的癌旁正常组织中let-7a-3甲基化状态,采用酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测血浆中IGF-Ⅱ的表达水平。结果83例食管鳞癌患者癌组织中的microRNA let-7a-3甲基化程度显著高于癌旁正常组织(P<0.001)。83例食管鳞癌患者血浆中IGF-Ⅱ的表达水平与let-7a-3基因的甲基化程度总体上呈正相关,具有统计学意义(r=0.600,P<0.001)。结论microRNA let-7a-3可能通过对下游分子的甲基化调控参与食管鳞癌的发生发展,这对了解食管鳞癌形成的机制具有重要意义,可为食管鳞癌的诊断和预后提供依据。展开更多
AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinfor...AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.展开更多
This study aimed to investigate the role of insulin-like growth factor-binding protein-3 （IGFBP-3） in erectUe dysfunction （ED） in two-kidney one-clip （2K-1C） hypertensive rats treated with the β-blocking agent ...This study aimed to investigate the role of insulin-like growth factor-binding protein-3 （IGFBP-3） in erectUe dysfunction （ED） in two-kidney one-clip （2K-1C） hypertensive rats treated with the β-blocking agent propranolol. Adult male Wistar rats were randomly divided into three groups： a normal control group, a hypertensive control group and a propranolol treatment group （n=9）. After 4 weeks of propranolol treatment, intracavemous pressure （ICP） responses to electrical stimulation of the cavernous nerves were evaluated. The expression of IGFBP-3 and insulin-like growth factor-1 （IGF-1） mRNA and protein in the rat cavernous tissue were detected by quantitative real-time PCR and Western blot, respectively. The concentration of cyclic guanosine monophosphate （cGMP） in the cavernous tissue was determined by enzyme-linked immunosorbent assay （ELISA）. Cavernosal pressure in response to cavernous nerve stimulation was decreased 4 weeks after propranolol treatment （P〈0.01, compared to the hypertensive control group）. IGFBP-3 mRNA and protein expression was increased in the propranolol treatment group compared to the hypertensive control group （P〈O.01）, whereas IGF-1 expression was decreased in the propranolol treatment group compared to the hypertensive control group （P〈0.01）. In addition, cavernous cGMP concentration was decreased in the prepranolol treatment group compared to the hypertensive control group （P〈0.01）. Taken together, these results suggest that the upregulation of IGFBP-3 may play a role in the development of ED in hypertensive rats.展开更多
OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like g...OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like growth factor-binding protein-3(IGFBP-3)suppresses HCC cell proliferation in both IGF-dependent and independent manners.The present study is to investigate whether treatment with exogenous IGFBP-3 inhibits bF GF and PDGF production and the cell proliferation of HCC cells.METHODS Cell Counting Kit 8 assay were designed to detect HCC cell proliferation,transcription factor early growth response-1(EGR1)involving in IGFBP-3 regulation of b FGF and PDGF were detected by RT-PCR and Western blot assays.Western blot assay was adopted to detect the IGFBP-3 regulating insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS The present study demonstrates that IGFBP-3 suppressed IGF-1-induced b FGF and PDGF expression while it does not affect their expression in the absence of IGF-1.To delineate the underlying mechanism,Western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1(EGR1)is involved in IGFBP-3 regulation of b FGF and PDGF.IGFBP-3 inhibition of type 1 insulin-like growth factor receptor(IGF1R),ERK and AKT activation is IGF-1-dependent.Furthermore,transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1,b FGF and PDGF expression.CONCLUSION In conclusion,these findings suggest that IGFBP-3suppresses transcription of EGR1 and its target genes b FGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation,suggesting that IGFBP-3 could be a target for the treatment of HCC.展开更多
文摘AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.
基金ACKNOWLEDGMENTS We thank Prof. B. W. Peng and Dr S. R, Guo for their excellent technical support. This study was supported by the National Natural Science Foundation of China （no. 30872572）.
文摘This study aimed to investigate the role of insulin-like growth factor-binding protein-3 （IGFBP-3） in erectUe dysfunction （ED） in two-kidney one-clip （2K-1C） hypertensive rats treated with the β-blocking agent propranolol. Adult male Wistar rats were randomly divided into three groups： a normal control group, a hypertensive control group and a propranolol treatment group （n=9）. After 4 weeks of propranolol treatment, intracavemous pressure （ICP） responses to electrical stimulation of the cavernous nerves were evaluated. The expression of IGFBP-3 and insulin-like growth factor-1 （IGF-1） mRNA and protein in the rat cavernous tissue were detected by quantitative real-time PCR and Western blot, respectively. The concentration of cyclic guanosine monophosphate （cGMP） in the cavernous tissue was determined by enzyme-linked immunosorbent assay （ELISA）. Cavernosal pressure in response to cavernous nerve stimulation was decreased 4 weeks after propranolol treatment （P〈0.01, compared to the hypertensive control group）. IGFBP-3 mRNA and protein expression was increased in the propranolol treatment group compared to the hypertensive control group （P〈O.01）, whereas IGF-1 expression was decreased in the propranolol treatment group compared to the hypertensive control group （P〈0.01）. In addition, cavernous cGMP concentration was decreased in the prepranolol treatment group compared to the hypertensive control group （P〈0.01）. Taken together, these results suggest that the upregulation of IGFBP-3 may play a role in the development of ED in hypertensive rats.
基金supported by National Natural Science Foundation of China(81502123 and81330081)Natural Science Foundation of Anhui Province(1308085QH130)Anhui Province Nature Science Foundation in University(KJ2014A119)
文摘OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like growth factor-binding protein-3(IGFBP-3)suppresses HCC cell proliferation in both IGF-dependent and independent manners.The present study is to investigate whether treatment with exogenous IGFBP-3 inhibits bF GF and PDGF production and the cell proliferation of HCC cells.METHODS Cell Counting Kit 8 assay were designed to detect HCC cell proliferation,transcription factor early growth response-1(EGR1)involving in IGFBP-3 regulation of b FGF and PDGF were detected by RT-PCR and Western blot assays.Western blot assay was adopted to detect the IGFBP-3 regulating insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS The present study demonstrates that IGFBP-3 suppressed IGF-1-induced b FGF and PDGF expression while it does not affect their expression in the absence of IGF-1.To delineate the underlying mechanism,Western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1(EGR1)is involved in IGFBP-3 regulation of b FGF and PDGF.IGFBP-3 inhibition of type 1 insulin-like growth factor receptor(IGF1R),ERK and AKT activation is IGF-1-dependent.Furthermore,transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1,b FGF and PDGF expression.CONCLUSION In conclusion,these findings suggest that IGFBP-3suppresses transcription of EGR1 and its target genes b FGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation,suggesting that IGFBP-3 could be a target for the treatment of HCC.
