目的探讨miRNA-423-5p与干扰素调节因子1(IRF1)在肺腺癌中的作用及机制。方法体外培养肺腺癌细胞A549、H1299和H1975。实验分组:(1)在过表达miRNA-423-5p实验中,mimics NC作为对照组,miRNA-423-5p作为过表达组;(2)在敲降miRNA-423-5p实...目的探讨miRNA-423-5p与干扰素调节因子1(IRF1)在肺腺癌中的作用及机制。方法体外培养肺腺癌细胞A549、H1299和H1975。实验分组:(1)在过表达miRNA-423-5p实验中,mimics NC作为对照组,miRNA-423-5p作为过表达组;(2)在敲降miRNA-423-5p实验中,ASO-NC作为对照组,ASO-miRNA-423-5p作为敲降组;(3)过表达IRF1实验中,pcDNA3.1作为对照组,pcDNA3.1-IRF1作为过表达组;(4)同时过表达miRNA-423-5p和IRF1实验中,mimics NC+pcDNA3.1作为对照组,miRNA-423-5p+pcDNA3.1作为只过表达miRNA-423-5p组,miRNA-423-5p+pcDNA3.1-IRF1作为同时过表达miRNA-423-5p和IRF1组。采用CCK-8、克隆形成、5-乙炔基-2′-脱氧尿嘧啶核苷(EDU)、蛋白质印迹法、Transwell和划痕实验分别检测各组细胞增殖凋亡、侵袭和迁移能力的影响。通过Star Base数据库预测miRNA-423-5p与IRF1的结合位点,双荧光素酶实验验证miRNA-423-5p与IRF1之间的结合作用。裸鼠皮下成瘤实验检测稳定敲降miRNA-423-5p和稳定过表达IRF1细胞对移植瘤生长的影响。多组比较采用单因素方差分析,两组间比较采用独立样本t检验。结果CCK-8、克隆形成、EDU、蛋白质印迹法、划痕和Transwell实验结果示,过表达miRNA-423-5p会促进H1299细胞的增殖、迁移和侵袭能力,抑制H1299细胞的凋亡能力,均P<0.001。敲低miRNA-423-5p会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P<0.001。过表达IRF1会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P<0.001。过表达IRF1可以挽救过表达miRNA-423-5p对H1299细胞增殖、凋亡、迁移和侵袭能力的作用,均P<0.001。Star Base数据库预测显示,miRNA-423-5p与IRF1之间存在结合位点。双荧光素酶实验证明miRNA-423-5p与IRF1可以结合,t=26.470,P<0.001。qRT-PCR(t=7.085,P=0.001)和蛋白质印迹法(t=28.660,P<0.001)实验表明,miRNA-423-5p可以调节IRF1的表达。裸鼠皮下成瘤实验结果提示,稳定敲降miRNA-423-5p的H1299细胞瘤体体积[(491.40±94.34)mm^(3)vs(382.20±16.04)mm^(3),t=2.825,P=0.037]、瘤组织Ki-67(0.40±0.10 vs 0.24±0.02,t=3.843,P=0.012)和PCNA表达(0.45±0.09 vs 0.25±0.04,t=4.974,P=0.004)均显著降低,稳定过表达IRF1的H1299细胞瘤体体积[(291.50±109.1)mm^(3)vs(125.46±98.52)mm^(3),t=2.766,P=0.040]、瘤组织Ki-67(0.44±0.04 vs 0.25±0.02,t=10.407,P<0.001)和PCNA表达(0.39±0.03 vs 0.15±0.02,t=16.305,P<0.001)均显著降低。结论miRNA-423-5p可能通过调控IRF1促进肺腺癌的恶性表型。展开更多
MicroRNAs(miRNAs)have been demonstrated to control chicken skeletal muscle growth,however,the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown.Here,our study identified the two...MicroRNAs(miRNAs)have been demonstrated to control chicken skeletal muscle growth,however,the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown.Here,our study identified the two chicken(Gallus gallus;Gga)miR-181-5p family members widely expressed in various tissues,specifically miR-181a-5p and miR-181b-5p.Besides,the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers.Functionally,miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including myogenin(MyoG),myogenic differentiation 1(MyoD1),and myosin heavy chain(MyHC),meanwhile accelerating the myotube formation of skeletal muscle satellite cells(SMSCs).Mechanistically,miR-181a-5p and miR-181b-5p directly bind to the 3′untranslated region(UTR)of the transforming growth factor beta receptor 1(TGFBR1)mRNA,further reducing the expression of TGFBR1.TGFBR1 is a key Transforming growth factor beta(TGF-β)signaling transduction receptor and had a negative function in muscle cell differentiation.Furthermore,knockdown of TGFBR1 facilitated the expression of chicken myogenic factors,boosted myotube formation,and decreased the SMAD family member 2/3(SMAD2/3)phosphorylation in chicken SMSCs.SMAD2/3 are downstream of TGF-βsignaling,and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation.Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-βsignaling transduction,which resulted in promoting chicken skeletal muscle development.展开更多
文摘目的探讨miRNA-423-5p与干扰素调节因子1(IRF1)在肺腺癌中的作用及机制。方法体外培养肺腺癌细胞A549、H1299和H1975。实验分组:(1)在过表达miRNA-423-5p实验中,mimics NC作为对照组,miRNA-423-5p作为过表达组;(2)在敲降miRNA-423-5p实验中,ASO-NC作为对照组,ASO-miRNA-423-5p作为敲降组;(3)过表达IRF1实验中,pcDNA3.1作为对照组,pcDNA3.1-IRF1作为过表达组;(4)同时过表达miRNA-423-5p和IRF1实验中,mimics NC+pcDNA3.1作为对照组,miRNA-423-5p+pcDNA3.1作为只过表达miRNA-423-5p组,miRNA-423-5p+pcDNA3.1-IRF1作为同时过表达miRNA-423-5p和IRF1组。采用CCK-8、克隆形成、5-乙炔基-2′-脱氧尿嘧啶核苷(EDU)、蛋白质印迹法、Transwell和划痕实验分别检测各组细胞增殖凋亡、侵袭和迁移能力的影响。通过Star Base数据库预测miRNA-423-5p与IRF1的结合位点,双荧光素酶实验验证miRNA-423-5p与IRF1之间的结合作用。裸鼠皮下成瘤实验检测稳定敲降miRNA-423-5p和稳定过表达IRF1细胞对移植瘤生长的影响。多组比较采用单因素方差分析,两组间比较采用独立样本t检验。结果CCK-8、克隆形成、EDU、蛋白质印迹法、划痕和Transwell实验结果示,过表达miRNA-423-5p会促进H1299细胞的增殖、迁移和侵袭能力,抑制H1299细胞的凋亡能力,均P<0.001。敲低miRNA-423-5p会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P<0.001。过表达IRF1会抑制H1299细胞的增殖、迁移和侵袭能力,促进H1299细胞的凋亡能力,均P<0.001。过表达IRF1可以挽救过表达miRNA-423-5p对H1299细胞增殖、凋亡、迁移和侵袭能力的作用,均P<0.001。Star Base数据库预测显示,miRNA-423-5p与IRF1之间存在结合位点。双荧光素酶实验证明miRNA-423-5p与IRF1可以结合,t=26.470,P<0.001。qRT-PCR(t=7.085,P=0.001)和蛋白质印迹法(t=28.660,P<0.001)实验表明,miRNA-423-5p可以调节IRF1的表达。裸鼠皮下成瘤实验结果提示,稳定敲降miRNA-423-5p的H1299细胞瘤体体积[(491.40±94.34)mm^(3)vs(382.20±16.04)mm^(3),t=2.825,P=0.037]、瘤组织Ki-67(0.40±0.10 vs 0.24±0.02,t=3.843,P=0.012)和PCNA表达(0.45±0.09 vs 0.25±0.04,t=4.974,P=0.004)均显著降低,稳定过表达IRF1的H1299细胞瘤体体积[(291.50±109.1)mm^(3)vs(125.46±98.52)mm^(3),t=2.766,P=0.040]、瘤组织Ki-67(0.44±0.04 vs 0.25±0.02,t=10.407,P<0.001)和PCNA表达(0.39±0.03 vs 0.15±0.02,t=16.305,P<0.001)均显著降低。结论miRNA-423-5p可能通过调控IRF1促进肺腺癌的恶性表型。
基金supported by the National Key Research and Development Program of China(2022YFF10002020)Sichuan Science and Technology Program,China(2021YFYZ0007 and 2021YFYZ0031).
文摘MicroRNAs(miRNAs)have been demonstrated to control chicken skeletal muscle growth,however,the potential function of the miR-181-5p family in chicken myogenesis remains largely unknown.Here,our study identified the two chicken(Gallus gallus;Gga)miR-181-5p family members widely expressed in various tissues,specifically miR-181a-5p and miR-181b-5p.Besides,the breast muscles of fast-growing broilers expressed higher levels of miR-181a-5p and miR-181b-5p than those of slow-growing layers.Functionally,miR-181a-5p and miR-181b-5p both promote the expression level of myogenic factors including myogenin(MyoG),myogenic differentiation 1(MyoD1),and myosin heavy chain(MyHC),meanwhile accelerating the myotube formation of skeletal muscle satellite cells(SMSCs).Mechanistically,miR-181a-5p and miR-181b-5p directly bind to the 3′untranslated region(UTR)of the transforming growth factor beta receptor 1(TGFBR1)mRNA,further reducing the expression of TGFBR1.TGFBR1 is a key Transforming growth factor beta(TGF-β)signaling transduction receptor and had a negative function in muscle cell differentiation.Furthermore,knockdown of TGFBR1 facilitated the expression of chicken myogenic factors,boosted myotube formation,and decreased the SMAD family member 2/3(SMAD2/3)phosphorylation in chicken SMSCs.SMAD2/3 are downstream of TGF-βsignaling,and miR-181a-5p and miR-181b-5p could reduce the expression of TGFBR1 to further diminish the SMAD2/3 phosphorylation.Our findings revealed that the miR-181-5p family targets TGFBR1 to break the TGF-βsignaling transduction,which resulted in promoting chicken skeletal muscle development.
