Objective:To investigate the mechanism of MiR-34a-mediated mTOR/4E-BP1 signaling pathway on apoptosis of esophageal cancer cells.Methods:Esophageal cancer cell lines were purchased from the ATCC cell bank and randomly...Objective:To investigate the mechanism of MiR-34a-mediated mTOR/4E-BP1 signaling pathway on apoptosis of esophageal cancer cells.Methods:Esophageal cancer cell lines were purchased from the ATCC cell bank and randomly divided into three groups. The first group, miR-34a group (miR-34a group): transfected miR-34a mimics into esophageal cancer cells by transfection;The second group, esophageal cancer group (EC group): simple esophageal cancer cell line without other treatment, normal culture;third group, idling group (ID group): transfected miR-34a negative control into esophageal cancer cell line . To explore MiR by qRT-PCR, TUNEL, cck-8 and Westernblot in the detection of miR-34a mRNA expression, apoptosis, proliferation, and protein content of mTOR/4E-BP1 in esophageal cancer cells. -34a mediates the mechanism of mTOR/4E-BP1 signaling pathway on apoptosis of esophageal cancer cells.Results:The results of qRT-PCR showed that the expression of miR-34a mRNA was the lowest in esophageal cancer cells in EC group, and the highest in miR-34a group in miR-34a group, miR-34a group and ID group, EC Compared with the group, the miR-34a mRNA content increased significantly, indicating successful transfection (allP<0.05). The results of TUNEL showed that the number of apoptosis of esophageal cancer cells in EC group was the least, and the apoptosis of esophageal carcinoma cells in miR-34a group was significantly increased. The apoptosis rate of miR-34a group was significantly higher than that of EC group and ID group (P<0.05). There was no significant difference in apoptosis between EC group and ID group. The OD value of cell proliferation in ID group and EC group was higher than that in miR-34a group, and there was significant difference between groups (P<0.05). The proliferation of esophageal cancer cells in EC group was the highest, and the number of esophageal cancer cells in miR-34a group was the least. There was no significant difference in the proliferation of esophageal cancer cells in the ID group compared with the EC group (P>0.05). The mTOR/4E-BP1 protein in esophageal cancer cells of EC group, ID group and miR-34a group was immunoblotted by Western blot. The gray scale showed that the mTOR/4E-BP1 protein content of EC group was the highest, EC group and Compared with the ID group, the protein content was similar, no significant (P>0.05), and the miR-34a group had the lowest protein content. Compared with the EC group and the ID group, the miR-34a group had lower protein content in esophageal cancer cells (allP< 0.05).Conclusions:MiR-34a may promote the apoptosis of esophageal cancer cells by inhibiting the expression of mTOR/4E-BP1 signaling pathway.展开更多
Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode...Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode components of the translational apparatus. This mode of regulation involves TSC and Rheb, as knockout of TSC1 or TSC2 or overexpression of Rheb rescued TOP mRNA translation in oxygen-deprived celts. Stress-induced translational repression of these mRNAs closely correlates with the hypophosphorylated state of 4E-BP, a translational repressor. However, a series of 4E-BP loss- and gain-of-function experiments disprove a cause-and- effect relationship between the phosphorylation status of 4E-BP and the translational repression of TOP mRNAs under oxygen or growth factor deprivation. Furthermore, the repressive effect of anoxia is similar to that attained by the very efficient inhibition of mTOR activity by Torin 1, but much more pronounced than roptor or rictor knockouL Likewise, deficiency of raptor or rictor, even though it mildly downregulated basal translation efficiency of TOP mRNAs, failed to suppress the oxygen-mediated translational activation of TOP mRNAs. Finally, co-knockdown of TIA-1 and TIAR, two RNA-binding proteins previously implicated in translational repression of TOP mRNAs in amino acid-starved cells, failed to relieve TOP mRNA translation under other stress conditions. Thus, the nature of the proximal translational regulator of TOP m RNAs remains elusive.展开更多
Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders,the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown.Here,we report that the expression of ...Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders,the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown.Here,we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem celis(hMSCs).Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration,increases mitochondrial reactive oxygen species(Ros)production,and accelerates cellular senescence.Mechanistically,the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes,especially several key subunits of complex III including UQCRC2.Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs.These findings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis,particularly for the mitochondrial respiration complex Il,thus providing a new potential target to counteract human stem cell senescence.展开更多
目的:筛选不同转移潜能人大肠癌细胞株的差异信号通路,探讨大肠癌转移的分子机制。方法:利用Full Moon抗体芯片(Phospho Explorer Array PEX100)对31条信号通路、432个信号蛋白的679个磷酸化位点进行高通量检测,分析来自同一病人的原位...目的:筛选不同转移潜能人大肠癌细胞株的差异信号通路,探讨大肠癌转移的分子机制。方法:利用Full Moon抗体芯片(Phospho Explorer Array PEX100)对31条信号通路、432个信号蛋白的679个磷酸化位点进行高通量检测,分析来自同一病人的原位和转移大肠癌细胞株SW480和SW620信号通路蛋白磷酸化水平的变化谱。通过生物信息学分析,了解大肠癌转移相关信号通路并进行Western-blot检测。结果:SW480和SW620两个细胞株存在169个差异磷酸化蛋白(31个上调,138个下调)。差异表达的蛋白质中,大部分与基因转录、信号转导、细胞凋亡等通路相关。Western-blot证实PI3K-AKT-mTOR信号通路分子在原位和转移性大肠癌中存在明显差异表达。结论:蛋白质芯片方法有助于鉴定大肠癌转移相关的差异蛋白,PI3K-AKT-mTOR细胞信号通路活性的变化可能与大肠癌转移有关。展开更多
基金National Youth Science Fund Project(No.8180140119).
文摘Objective:To investigate the mechanism of MiR-34a-mediated mTOR/4E-BP1 signaling pathway on apoptosis of esophageal cancer cells.Methods:Esophageal cancer cell lines were purchased from the ATCC cell bank and randomly divided into three groups. The first group, miR-34a group (miR-34a group): transfected miR-34a mimics into esophageal cancer cells by transfection;The second group, esophageal cancer group (EC group): simple esophageal cancer cell line without other treatment, normal culture;third group, idling group (ID group): transfected miR-34a negative control into esophageal cancer cell line . To explore MiR by qRT-PCR, TUNEL, cck-8 and Westernblot in the detection of miR-34a mRNA expression, apoptosis, proliferation, and protein content of mTOR/4E-BP1 in esophageal cancer cells. -34a mediates the mechanism of mTOR/4E-BP1 signaling pathway on apoptosis of esophageal cancer cells.Results:The results of qRT-PCR showed that the expression of miR-34a mRNA was the lowest in esophageal cancer cells in EC group, and the highest in miR-34a group in miR-34a group, miR-34a group and ID group, EC Compared with the group, the miR-34a mRNA content increased significantly, indicating successful transfection (allP<0.05). The results of TUNEL showed that the number of apoptosis of esophageal cancer cells in EC group was the least, and the apoptosis of esophageal carcinoma cells in miR-34a group was significantly increased. The apoptosis rate of miR-34a group was significantly higher than that of EC group and ID group (P<0.05). There was no significant difference in apoptosis between EC group and ID group. The OD value of cell proliferation in ID group and EC group was higher than that in miR-34a group, and there was significant difference between groups (P<0.05). The proliferation of esophageal cancer cells in EC group was the highest, and the number of esophageal cancer cells in miR-34a group was the least. There was no significant difference in the proliferation of esophageal cancer cells in the ID group compared with the EC group (P>0.05). The mTOR/4E-BP1 protein in esophageal cancer cells of EC group, ID group and miR-34a group was immunoblotted by Western blot. The gray scale showed that the mTOR/4E-BP1 protein content of EC group was the highest, EC group and Compared with the ID group, the protein content was similar, no significant (P>0.05), and the miR-34a group had the lowest protein content. Compared with the EC group and the ID group, the miR-34a group had lower protein content in esophageal cancer cells (allP< 0.05).Conclusions:MiR-34a may promote the apoptosis of esophageal cancer cells by inhibiting the expression of mTOR/4E-BP1 signaling pathway.
文摘Cells encountering hypoxic stress conserve resources and energy by downregulating the protein synthesis. Here we demonstrate that one mechanism in this response is the translational repression of TOP mRNAs that encode components of the translational apparatus. This mode of regulation involves TSC and Rheb, as knockout of TSC1 or TSC2 or overexpression of Rheb rescued TOP mRNA translation in oxygen-deprived celts. Stress-induced translational repression of these mRNAs closely correlates with the hypophosphorylated state of 4E-BP, a translational repressor. However, a series of 4E-BP loss- and gain-of-function experiments disprove a cause-and- effect relationship between the phosphorylation status of 4E-BP and the translational repression of TOP mRNAs under oxygen or growth factor deprivation. Furthermore, the repressive effect of anoxia is similar to that attained by the very efficient inhibition of mTOR activity by Torin 1, but much more pronounced than roptor or rictor knockouL Likewise, deficiency of raptor or rictor, even though it mildly downregulated basal translation efficiency of TOP mRNAs, failed to suppress the oxygen-mediated translational activation of TOP mRNAs. Finally, co-knockdown of TIA-1 and TIAR, two RNA-binding proteins previously implicated in translational repression of TOP mRNAs in amino acid-starved cells, failed to relieve TOP mRNA translation under other stress conditions. Thus, the nature of the proximal translational regulator of TOP m RNAs remains elusive.
基金supported by the National Key Research and Development Program of China(2018YFC2000100)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16000000)+9 种基金the National Natural Science Foundation of China(8190143281921006,82125011,92149301,92168201,91949209,92049304,92049116,32121001,82192863,82122024,82071588,81861168034,81922027,81870228,32100937,31900524,82201727)the National Key Research and Development Program of China(2020YFA0804000,2020YFA0113400,2020YFA0112200,2018YFA0107203,the STI2030-Major Projects-2021ZD0202400,2021YFF1201005,2022YFA1103700,2022YFA1103800)CAS Project for Young Scientists in Basic Research(YSBR-076,YSBR-012)the Program of the Beijing Natural Science Foundation(Z190019,JQ20031)K.C.Wong Education Foundation(GJTD-2019-06,GJTD-2019-08)Young Elite Scientists Sponsorship Program by CAST(YESS20200012)Youth Innovation Promotion Association of CAS(EiCAZW0401)the Pilot Project for Public Welfare Development and Reform of Beijing-affliated Medical Research Institutes(11000022T000000461062)the Informatization Plan of Chinese Academy of Sciences(CAS-WX2021SF-0301,CASWX2022SDC-XK14)CAS Special Research Assistant(SRA)Program,and the Tencent Foundation(2021-1045).
文摘Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders,the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown.Here,we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem celis(hMSCs).Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration,increases mitochondrial reactive oxygen species(Ros)production,and accelerates cellular senescence.Mechanistically,the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes,especially several key subunits of complex III including UQCRC2.Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs.These findings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis,particularly for the mitochondrial respiration complex Il,thus providing a new potential target to counteract human stem cell senescence.
文摘目的:筛选不同转移潜能人大肠癌细胞株的差异信号通路,探讨大肠癌转移的分子机制。方法:利用Full Moon抗体芯片(Phospho Explorer Array PEX100)对31条信号通路、432个信号蛋白的679个磷酸化位点进行高通量检测,分析来自同一病人的原位和转移大肠癌细胞株SW480和SW620信号通路蛋白磷酸化水平的变化谱。通过生物信息学分析,了解大肠癌转移相关信号通路并进行Western-blot检测。结果:SW480和SW620两个细胞株存在169个差异磷酸化蛋白(31个上调,138个下调)。差异表达的蛋白质中,大部分与基因转录、信号转导、细胞凋亡等通路相关。Western-blot证实PI3K-AKT-mTOR信号通路分子在原位和转移性大肠癌中存在明显差异表达。结论:蛋白质芯片方法有助于鉴定大肠癌转移相关的差异蛋白,PI3K-AKT-mTOR细胞信号通路活性的变化可能与大肠癌转移有关。
