Solid-phase extraction ultrahigh-performance liquid chromatography-tandem mass spectrometry(SPE-UHPLC-MS/MS)was used to evaluate the contamination of ochratoxin A(OTA)and assessed the human exposure risk of OTA in Ras...Solid-phase extraction ultrahigh-performance liquid chromatography-tandem mass spectrometry(SPE-UHPLC-MS/MS)was used to evaluate the contamination of ochratoxin A(OTA)and assessed the human exposure risk of OTA in Rasa roxburghii.A more suitable method for OTA extraction and purification of R.roxburghii was obtained.Treated 25 mL of R.roxburghii juice with enzymatic hydrolysis at a concentration of 0.06 mg/mL,filtered the resulting mixture and concentrated the filtrate to dry,then redissolved with 0.2 mL of methanol and diluted with 0.4 mL of ultra-pure water.Added sample solution to the activated hydrophilic-lipophilic balance(HLB)column,washed with 6 mL of ultra-pure water and purified by eluting with 6 mL of methanol.The eluent was collected and dried using nitrogen at 40℃,then redissolved in 1 mL of methanol and filtered for detection.The hazard quotient(HQ)values of all R.roxburghii fruit and juice,which were storage at room temperature,4 and−20℃from 0 day to 63 days,ranged from 0.077%to 35.792%,which were within the allowable limits for human consumption.From the perspective of OTA contamination,the results indicated that the maximal storage time of R.roxburghii fruit were 14 days at room temperature,35 days at 4℃and 63 days at−20℃.And the maximal storage time of R.roxburghii juice were 7 days for sealed storage and the same day for open storage at room temperature,14 days for sealed storage and 7 days for open storage at 4℃,and 63 days for sealed storage and 56 days for open storage at−20℃during the experiment period.And all thirty samples randomly sampled from the market were OTA negative.The results of this study can lay a foundation for the formulation of OTA limit standards in fruits and juice in the future,and provide a reference for consumers to consume R.roxburghii more healthily.展开更多
AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lin...AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lines was assessed by Western blot.Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased.RKO cells were transfected with vectors overexpressing or downregulating either mi R-21 or RASA1.Furthermore,a luciferase reporter assay was used to determine whether RASA1 is a gene target of mi R-21.Then,changes in m RNA and protein levels of RASA1,RASGTP,and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction,Western blot and immunoprecipitation.Finally,cell proliferation,apoptosis,invasion,and tumorformation ability w ere assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay,flow cytometry,transwell assay,and animal experiment,respectively.RESULTS:RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines,and RASA1 was confirmed as a target gene of mi R-21.Interestingly,RASA1 m RNA and protein levels in pre-mi R-21-LV(up-regulation of mi R-21) cells were lower than those in anti-mi R-21-LV(down-regulation of mi R-21) cells(P < 0.05).In addition,pre-mi R-21-LV or si RASA1(down-regulation of RASA1) cells showed higher cell proliferation,reduced apoptosis,increased expression of RAS-GTP,p-AKT,Raf-1,KRAS,and p-ERK1/2,and higher invasion and tumor formation ability,compared with control,antimi R-21-LV or pc DNA3.1-RASA1(up-regulation of RASA1) cells(P < 0.05).CONCLUSION:RASA1 is a target gene of mi R-21,which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.展开更多
基金supported by the Guizhou Province High-level Innovative Talent Project(Qiankehe Platform Talent-GCC[2022]027-1)the Science and Technology Project of Guizhou Province(Qiankehe Platform Support-[2021]193).
文摘Solid-phase extraction ultrahigh-performance liquid chromatography-tandem mass spectrometry(SPE-UHPLC-MS/MS)was used to evaluate the contamination of ochratoxin A(OTA)and assessed the human exposure risk of OTA in Rasa roxburghii.A more suitable method for OTA extraction and purification of R.roxburghii was obtained.Treated 25 mL of R.roxburghii juice with enzymatic hydrolysis at a concentration of 0.06 mg/mL,filtered the resulting mixture and concentrated the filtrate to dry,then redissolved with 0.2 mL of methanol and diluted with 0.4 mL of ultra-pure water.Added sample solution to the activated hydrophilic-lipophilic balance(HLB)column,washed with 6 mL of ultra-pure water and purified by eluting with 6 mL of methanol.The eluent was collected and dried using nitrogen at 40℃,then redissolved in 1 mL of methanol and filtered for detection.The hazard quotient(HQ)values of all R.roxburghii fruit and juice,which were storage at room temperature,4 and−20℃from 0 day to 63 days,ranged from 0.077%to 35.792%,which were within the allowable limits for human consumption.From the perspective of OTA contamination,the results indicated that the maximal storage time of R.roxburghii fruit were 14 days at room temperature,35 days at 4℃and 63 days at−20℃.And the maximal storage time of R.roxburghii juice were 7 days for sealed storage and the same day for open storage at room temperature,14 days for sealed storage and 7 days for open storage at 4℃,and 63 days for sealed storage and 56 days for open storage at−20℃during the experiment period.And all thirty samples randomly sampled from the market were OTA negative.The results of this study can lay a foundation for the formulation of OTA limit standards in fruits and juice in the future,and provide a reference for consumers to consume R.roxburghii more healthily.
基金Supported by National Natural Science Foundation of China,No.81272770Grants from Guangdong Natural Science Foundation,No.S2013020012746Foundation of Guangdong Provincial Department of Science and Technology,No.2012A030400018
文摘AIM:To determine how the oncogene mi R-21 regulates the RAS signaling pathways and affects colon cancer cell behaviors.METHODS:RAS p21 GTPase activating protein 1(RASA1) protein expression in six colon cancer cell lines was assessed by Western blot.Colon cancer RKO cells were chosen for transfection because they are KRAS wild type colon cancer cells whose RASA1 expression is significantly decreased.RKO cells were transfected with vectors overexpressing or downregulating either mi R-21 or RASA1.Furthermore,a luciferase reporter assay was used to determine whether RASA1 is a gene target of mi R-21.Then,changes in m RNA and protein levels of RASA1,RASGTP,and other components of the RAS signaling pathways were assessed in transfected RKO cells by real-time quantitative reverse transcription-polymerase chain reaction,Western blot and immunoprecipitation.Finally,cell proliferation,apoptosis,invasion,and tumorformation ability w ere assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay,flow cytometry,transwell assay,and animal experiment,respectively.RESULTS:RASA1 protein levels were significantly decreased in RKO cells compared with the other 5 colon cancer cell lines,and RASA1 was confirmed as a target gene of mi R-21.Interestingly,RASA1 m RNA and protein levels in pre-mi R-21-LV(up-regulation of mi R-21) cells were lower than those in anti-mi R-21-LV(down-regulation of mi R-21) cells(P < 0.05).In addition,pre-mi R-21-LV or si RASA1(down-regulation of RASA1) cells showed higher cell proliferation,reduced apoptosis,increased expression of RAS-GTP,p-AKT,Raf-1,KRAS,and p-ERK1/2,and higher invasion and tumor formation ability,compared with control,antimi R-21-LV or pc DNA3.1-RASA1(up-regulation of RASA1) cells(P < 0.05).CONCLUSION:RASA1 is a target gene of mi R-21,which promotes malignant behaviors of RKO cells through regulation of RASA1 expression.
