Objective:To study the mechanism of action of Yanghe Huayan Decoction on endocrine-resistant cells(MCF-7R/mTOR cells)with low expression of mTOR;Methods:CCK-8 assay and cell clone assay were used to detect cell prolif...Objective:To study the mechanism of action of Yanghe Huayan Decoction on endocrine-resistant cells(MCF-7R/mTOR cells)with low expression of mTOR;Methods:CCK-8 assay and cell clone assay were used to detect cell proliferation and clonal ability,and flow cytometry was used to detect cell apoptosis.The changes of cytokines in ER-PI3K/Akt/mTOR signaling pathway were analyzed by blot and QPCR.Results:Yanghe Huayan Decoction could significantly affect the proliferation(p<0.05)and cloning ability(p<0.05)of MCF-7R/mTOR cells,promote cell apoptosis(p<0.01),and downregulate the expression of ER,PI3K,AKT,mTOR and p-mTOR(p<0.05).Conclusion:Yanghe Huayan Decoction can regulate the ER-PI3K/Akt/mTOR signaling pathway with multiple targets,and the use of combined mTOR inhibitors can regulate the ER-PI3K/Akt/mTOR signaling pathway more significantly.展开更多
BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer...BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.展开更多
To detect the mammalian target of rapamycin （mTOR） expressed in Cashmere goat fetal fibroblasts （GFb）, mTOR gene was cloned from Inner Mongolia Cashmere goat （Capra hircus） and expressed in Escherichia coli foll...To detect the mammalian target of rapamycin （mTOR） expressed in Cashmere goat fetal fibroblasts （GFb）, mTOR gene was cloned from Inner Mongolia Cashmere goat （Capra hircus） and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an irnmunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay （ELISA）. The results showed that the recombinant polyclonal antibody had a titer 1：200000 and could react with the roTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR （Ser 2448） activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.展开更多
目的:构建哺乳类动物雷帕霉素靶蛋白(mTOR)的反义RNA真核表达载体,观察其对血管平滑肌细胞(VSMC)功能的影响。方法:提取人VSMC总RNA,反转录-聚合酶链反应(RT-PCR)扩增mTOR基因cDNA序列,经pGEM-T载体克隆后双酶切,将cDNA序列反向插入绿...目的:构建哺乳类动物雷帕霉素靶蛋白(mTOR)的反义RNA真核表达载体,观察其对血管平滑肌细胞(VSMC)功能的影响。方法:提取人VSMC总RNA,反转录-聚合酶链反应(RT-PCR)扩增mTOR基因cDNA序列,经pGEM-T载体克隆后双酶切,将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1,构建mTOR基因反义RNA真核表达载体。转染VSMC,采用W estern b lot法检验反义表达载体对mTOR蛋白表达的影响,流式细胞仪检测细胞周期的变化。结果:经RT-PCR获得664 bp产物,T载体克隆后,经DNA测序,确定该片段为mTOR基因cDNA,进而构建反义RNA真核表达载体pEGFP-C1-mTOR,测序证明序列正确后转染VSMC,证实其能够显著抑制mTOR蛋白产物表达,VSMC的分裂、增殖过程受阻。结论:已经成功构建mTOR基因的反义RNA真核表达载体。展开更多
基金National Natural Science Foundation of China(No.81573989)
文摘Objective:To study the mechanism of action of Yanghe Huayan Decoction on endocrine-resistant cells(MCF-7R/mTOR cells)with low expression of mTOR;Methods:CCK-8 assay and cell clone assay were used to detect cell proliferation and clonal ability,and flow cytometry was used to detect cell apoptosis.The changes of cytokines in ER-PI3K/Akt/mTOR signaling pathway were analyzed by blot and QPCR.Results:Yanghe Huayan Decoction could significantly affect the proliferation(p<0.05)and cloning ability(p<0.05)of MCF-7R/mTOR cells,promote cell apoptosis(p<0.01),and downregulate the expression of ER,PI3K,AKT,mTOR and p-mTOR(p<0.05).Conclusion:Yanghe Huayan Decoction can regulate the ER-PI3K/Akt/mTOR signaling pathway with multiple targets,and the use of combined mTOR inhibitors can regulate the ER-PI3K/Akt/mTOR signaling pathway more significantly.
基金Supported by the National Natural Science Funds of China,No.81974448Guangdong Medical Research Foundation,No.B2019126Shenzhen Science and Technology Innovation Commission,No.JCYJ20210324135005013.
文摘BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.
基金supported by the National Natural Science Foundation of China (30860191)the Graduate Student Research Projects of Inner Mongolia University and Major Projects for New Varieties of Genetically Modified Organisms of China (2008ZX08008-002)
文摘To detect the mammalian target of rapamycin （mTOR） expressed in Cashmere goat fetal fibroblasts （GFb）, mTOR gene was cloned from Inner Mongolia Cashmere goat （Capra hircus） and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an irnmunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay （ELISA）. The results showed that the recombinant polyclonal antibody had a titer 1：200000 and could react with the roTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR （Ser 2448） activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.
文摘目的:构建哺乳类动物雷帕霉素靶蛋白(mTOR)的反义RNA真核表达载体,观察其对血管平滑肌细胞(VSMC)功能的影响。方法:提取人VSMC总RNA,反转录-聚合酶链反应(RT-PCR)扩增mTOR基因cDNA序列,经pGEM-T载体克隆后双酶切,将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1,构建mTOR基因反义RNA真核表达载体。转染VSMC,采用W estern b lot法检验反义表达载体对mTOR蛋白表达的影响,流式细胞仪检测细胞周期的变化。结果:经RT-PCR获得664 bp产物,T载体克隆后,经DNA测序,确定该片段为mTOR基因cDNA,进而构建反义RNA真核表达载体pEGFP-C1-mTOR,测序证明序列正确后转染VSMC,证实其能够显著抑制mTOR蛋白产物表达,VSMC的分裂、增殖过程受阻。结论:已经成功构建mTOR基因的反义RNA真核表达载体。
