RESUMO
Objective To establish a method of LC-MS/MS for determining cordycepin(Cor)and 3′-deoxyinosine(3′-Deo)concentration in rat plasma,and to study their pharmacokinetics in rats.Methods Protein was precipitated with methanol using 2-chloadenosine(2-Chl)as an internal standard.The chromatography was performed on Kinetex C18(3 mm×100 mm,2.6 μm,Phenomenex,USA)with gradient elution in aqueous(5 mmol·L-1 ammonium acetate)-methanol solution as mobile phase.ESI ion source was used for mass spectrometry,and positive ion multiple reaction monitoring(MRM)was used for scanning detection.The pharmacokinetics of Cor and 3′-Deo after oral administration of Cor(10 mg·kg-1)were studied in rats.Results Cor at 0.5-100 ng·mL-1 and 3′-Deo at 1-200 ng·mL-1 had good linearity,and the lower limits of quantification were 0.5 and 1 ng·mL-1,respectively.After oral administration of Cor in rats,the plasma concentration of Cor was low,which was mainly converted into the metabolite 3′-Deo.The Cmax of Cor and 3′-Deo were(5.4±3.4)and(142.0±50.0)ng·mL-1,and AUC0-360min min were(658.4±459.3)and(18 034.9±4 981.1)ng·min·mL-1,respectively.Conclusion The method is simple,sensi-tive,and accurate,which is suitable for determining Cor and 3′-Deo concentration in plasma and the pharmacokinetic study.
RESUMO
Objective:To study the influences of circular RNA ras homolog family member T1(CircRHOT1)on the prolifera-tion,apoptosis and immune escape of breast cancer cells by regulating the miR-187-3p/sex-determining region Y-box protein 4(SOX4)axis.Methods:Real-time quantitative PCR and Western blot were used to detect the expressions of CircRHOT1,miR-187-3p and SOX4 in human normal breast epithelial cells MCF-10A and breast cancer cells MCF-7,Hs-578T and MDA-MB-231 in vitro;MCF-7 cells cultured in vitro were randomly separated into control group,CircRHOT1 knockdown(transfected with CircRHOT1 siRNA plasmid)group,miR-187-3p mimics(transfected with miR-187-3p mimics)group,co-transfection negative control(transfected with empty plasmid and miR-187-3p inhibitor negative control)group,and CircRHOT1 knockdown+miR-187-3p inhibitor(transfected with CircRHOT1 siRNA plasmid and miR-187-3p inhibitor)group.After grouping and transfection,real-time quantitative PCR and Western blot were used to detect the expressions of CircRHOT1,miR-187-3p and SOX4 in each group;cell proliferation in each group was detected by EdU staining and plate colony formation assay;cell apoptosis in each group was detected by flow cytometry;im-munofluorescence staining was used to detect the ratio of apoptosis-related proteins Bax and Bcl-2(Bax/Bcl-2)expressions in each group.The cells of each group were co-cultured with human peripheral blood lymphocytes and transfected into groups,flow cytometry was used to detect the proportion of activated CD8+T cells in human peripheral blood lymphocytes in each group;the killing rate of human peripheral blood lymphocytes to MCF-7 cells in each group was detected by MTT assay.Dual-luciferase reporter assay was used to detect the targeted regulation of miR-187-3p and miR-187-3p on SOX4 by CircRHOT1 in MCF-7 cells.Results:Compared with human normal breast epithelial cells MCF-10A,human breast cancer MCF-7,Hs-578T and MDA-MB-231 cells had obviously higher protein and mRNA expression of CircRHOT1 and SOX4(P<0.05),and obviously lower expression of miR-187-3p(P<0.05).Com-pared with control group,the protein and mRNA expression of SOX4,EdU positive rate and colony formation rate of cells in Circ-RHOT1 knockdown group and miR-187-3p mimics group decreased(P<0.05),the expression of miR-187-3p,apoptosis rate,Bax/Bcl-2,the proportion of activated CD8+T cells in human peripheral blood lymphocytes and the killing rate on MCF-7 cells increased(P<0.05).Compared with CircRHOT1 knockdown group,there was no obvious difference in the expression of CircRHOT1 in the CircRHOT1 knockdown+miR-187-3p inhibitor group(P>0.05),the protein and mRNA expression of SOX4,EdU positive rate and colony formation rate of cells increased(P<0.05),the expression of miR-187-3p,apoptosis rate,Bax/Bcl-2,the proportion of activated CD8+T cells in human peripheral blood lymphocytes and the killing rate on MCF-7 cells decreased(P<0.05);there was no obvious difference in the indexes of cells in co-transfection negative control group(P>0.05).Conclusion:Knockdown of CircRHOT1 can reduce the expression of SOX4 by up-regulating miR-187-3p,thereby attenuating the proliferative activity of breast cancer cells and promoting their apoptosis.It can also inhibit the activation of CD8+T cells and enhance their cancer cell cytotoxicity,thereby weakening immune evasion of breast cancer cells.