Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classica...Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classically activated macrophage(M1)polarization.In this study,we established THP-1-derived testing state macrophages(M0),M1 macrophages,and alternately activated macrophages(M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins(CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated(PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence(IF)-RNA fluorescence in situ hybridization(FISH)double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.展开更多
To confirm the relationship between Circ_0003855 and EC,we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC,and the expression levels of Circ_0003855,miR-622,...To confirm the relationship between Circ_0003855 and EC,we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC,and the expression levels of Circ_0003855,miR-622,and FLOT1 were detected.The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells,while miR-622 was lowly expressed(p<0.05).Subsequently,Circ_0003855 small interfering RNA(si-Circ_0003855)and its negative control(si-NC)were used to detect changes in cellular biological behaviors.We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855,and miR-622 expression was elevated,while FLOT1 was decreased(p<0.05).Additionally,si-Circ_0003855 and miR-622 inhibitor sequence(miR-622-inhibition)were co-transfected into cells with miR-622-inhibition alone,and untreated Eca109 cells were used as a control to detect the expression of FLOT1.Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells(p>0.05).Synthesizing the results of these experiments above,we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells,and its mechanism is related to miR-622 and FLOT1.展开更多
Background:Hypertrophy of the ligamentumflavum(HLF)is a common contributor to spinal stenosis which results in significant neurological impairments.Circular RNA(circRNA)circ_0003609 has been linked to HLF;however,the ex...Background:Hypertrophy of the ligamentumflavum(HLF)is a common contributor to spinal stenosis which results in significant neurological impairments.Circular RNA(circRNA)circ_0003609 has been linked to HLF;however,the exact mechanism by which it causes this disease is unclear.Methods:Circ_0003609 expressions were regulated in HLF cells by overexpression vectors and RNA interference.Cell proliferation andfibrosis-related gene expression were checked by the Cell Counting Kit-8(CCK-8)assay and western blotting.CircBank’s prediction of the association between miR-155 and circ_0003609 was supported by a dual-luciferase reporter experiment.The function of the miR-155/sirtuin 1(SIRT1)axis in controlling HLFfibrosis was further examined.Results:Overexpression of circ_0003609 suppressed HLF cell propagation andfibrosis compared to its silencing.It was found that circ_0003609 served as the sponge for miR-155 and that the circ_0003609/miR-155 axis controlled thefibrosis of HLF cells.It was found that circ_0003609 acted as a sponge for miR-155,regulating thefibrosis of HLF cells.Further,miR-155 targets SIRT1,and the miR-155/SIRT1 axis promotes HLF cellfibrosis.Conclusion:Circ_0003609 ameliorates hypertrophied ligamentumflavum(LF)by modulating the miR-155/SIRT1 axis,indicating a potential treatment approach for HLF.展开更多
BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of canc...BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.展开更多
基金supported by the Natural Science Foundation of Shandong Province,No.ZR2020MH138(to XZ).
文摘Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classically activated macrophage(M1)polarization.In this study,we established THP-1-derived testing state macrophages(M0),M1 macrophages,and alternately activated macrophages(M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins(CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated(PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence(IF)-RNA fluorescence in situ hybridization(FISH)double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.
文摘To confirm the relationship between Circ_0003855 and EC,we purchased the Human esophageal carcinoma cell line Eca109 and normal human esophageal epithelial cells HEEC,and the expression levels of Circ_0003855,miR-622,and FLOT1 were detected.The results show that Circ_0003855 and FLOT1 were highly expressed in Eca109 cells,while miR-622 was lowly expressed(p<0.05).Subsequently,Circ_0003855 small interfering RNA(si-Circ_0003855)and its negative control(si-NC)were used to detect changes in cellular biological behaviors.We found that the activity of Eca109 cells was reduced after interfering with the expression of Circ_0003855,and miR-622 expression was elevated,while FLOT1 was decreased(p<0.05).Additionally,si-Circ_0003855 and miR-622 inhibitor sequence(miR-622-inhibition)were co-transfected into cells with miR-622-inhibition alone,and untreated Eca109 cells were used as a control to detect the expression of FLOT1.Co-transfection of si-Circ_0003855 and miR-622-inhibition showed no significant difference in FLOT1 expression compared to the control cells(p>0.05).Synthesizing the results of these experiments above,we believe that interfering with the expression of Circ_0003855 can inhibit the activity of EC cells,and its mechanism is related to miR-622 and FLOT1.
基金This research was supported by the Shanghai Natural Science Fund(No.21ZR1447500)Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Baoshan Branch Medical Key Specialty Construction Project(No.rbzdzk-2023-001).
文摘Background:Hypertrophy of the ligamentumflavum(HLF)is a common contributor to spinal stenosis which results in significant neurological impairments.Circular RNA(circRNA)circ_0003609 has been linked to HLF;however,the exact mechanism by which it causes this disease is unclear.Methods:Circ_0003609 expressions were regulated in HLF cells by overexpression vectors and RNA interference.Cell proliferation andfibrosis-related gene expression were checked by the Cell Counting Kit-8(CCK-8)assay and western blotting.CircBank’s prediction of the association between miR-155 and circ_0003609 was supported by a dual-luciferase reporter experiment.The function of the miR-155/sirtuin 1(SIRT1)axis in controlling HLFfibrosis was further examined.Results:Overexpression of circ_0003609 suppressed HLF cell propagation andfibrosis compared to its silencing.It was found that circ_0003609 served as the sponge for miR-155 and that the circ_0003609/miR-155 axis controlled thefibrosis of HLF cells.It was found that circ_0003609 acted as a sponge for miR-155,regulating thefibrosis of HLF cells.Further,miR-155 targets SIRT1,and the miR-155/SIRT1 axis promotes HLF cellfibrosis.Conclusion:Circ_0003609 ameliorates hypertrophied ligamentumflavum(LF)by modulating the miR-155/SIRT1 axis,indicating a potential treatment approach for HLF.
基金Supported by National Natural Science Foundation of China,No.82360329Inner Mongolia Medical University General Project,No.YKD2023MS047Inner Mongolia Health Commission Science and Technology Plan Project,No.202201275.
文摘BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.
