BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found t...BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.展开更多
旨在探究鸡骨骼肌中VGLL2基因天然反义链转录本VGLL2 AS lncRNA(VGLL2-AS)与VGLL2的关系。本研究首先采用PCR和测序技术验证VGLL2-AS是否存在;之后分别采集不同周龄固始蛋鸡(1日龄、6周龄、16周龄、22周龄、30周龄,每个周龄各6只)组织样...旨在探究鸡骨骼肌中VGLL2基因天然反义链转录本VGLL2 AS lncRNA(VGLL2-AS)与VGLL2的关系。本研究首先采用PCR和测序技术验证VGLL2-AS是否存在;之后分别采集不同周龄固始蛋鸡(1日龄、6周龄、16周龄、22周龄、30周龄,每个周龄各6只)组织样,采用荧光定量PCR分析VGLL2基因和其反义链转录本VGLL2-AS的表达谱;采用双向转录试验和核酸酶保护试验检测VGLL2和VGLL2-AS是否可以双向转录且两者之间关系;体外分离培养原代成肌细胞(11胚龄固始鸡胚),然后用2μg·mL^(-1)的放线菌素D处理成肌细胞(对照组不做处理),收取处理不同时间点细胞(0~8 h,每组各个时间点各做3个重复),检测VGLL2-AS与VGLL2的半衰期;分离鸡成肌细胞的细胞核和细胞质,通过RT-qPCR方法确定两者的细胞定位;利用PCR扩增及测序对VGLL2的转录本进行验证;最后利用RT-qPCR检测它们在固始蛋鸡胸肌组织(1日龄、6周龄、16周龄、22周龄、30周龄每个周龄各6只)中的时空表达规律和相关性。结果表明,VGLL2-AS在鸡的转录组中真实存在;VGLL2与VGLL2-AS均在肌肉中特异高表达(P<0.05);VGLL2-AS与VGLL2可以进行双向转录且二者之间可以形成双链RNA;VGLL2-AS半衰期较VGLL2长;在成肌细胞中,VGLL2-AS和VGLL2主要定位于细胞质中(P<0.001);VGLL2只存在VGLL2-mRNA、VGLL2-X2和VGLL2-X3转录本;VGLL2-mRNA和VGLL2-X3与VGLL2-AS表达趋势一致,且VGLL2-mRNA、X3和VGLL2-AS的表达呈现极强的正相关(r分别为0.943和0.935,P<0.01)。综上所述,VGLL2-AS作为VGLL2的反义链编码的lncRNA定位于细胞质中,可能通过与VGLL2形成的双链RNA之间的相互作用,然后参与调节VGLL2的表达并维持其稳定性,最终在鸡的早期肌肉发育中发挥重要作用。本研究的结果扩展了鸡中关于NATs的研究,并为鸡VGLL2基因与其天然反义链转录本VGLL2-AS在鸡骨骼肌发育中的生物学功能的研究奠定了基础,对于提高禽类的生长发育具有一定的意义。展开更多
文摘BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.
文摘旨在探究鸡骨骼肌中VGLL2基因天然反义链转录本VGLL2 AS lncRNA(VGLL2-AS)与VGLL2的关系。本研究首先采用PCR和测序技术验证VGLL2-AS是否存在;之后分别采集不同周龄固始蛋鸡(1日龄、6周龄、16周龄、22周龄、30周龄,每个周龄各6只)组织样,采用荧光定量PCR分析VGLL2基因和其反义链转录本VGLL2-AS的表达谱;采用双向转录试验和核酸酶保护试验检测VGLL2和VGLL2-AS是否可以双向转录且两者之间关系;体外分离培养原代成肌细胞(11胚龄固始鸡胚),然后用2μg·mL^(-1)的放线菌素D处理成肌细胞(对照组不做处理),收取处理不同时间点细胞(0~8 h,每组各个时间点各做3个重复),检测VGLL2-AS与VGLL2的半衰期;分离鸡成肌细胞的细胞核和细胞质,通过RT-qPCR方法确定两者的细胞定位;利用PCR扩增及测序对VGLL2的转录本进行验证;最后利用RT-qPCR检测它们在固始蛋鸡胸肌组织(1日龄、6周龄、16周龄、22周龄、30周龄每个周龄各6只)中的时空表达规律和相关性。结果表明,VGLL2-AS在鸡的转录组中真实存在;VGLL2与VGLL2-AS均在肌肉中特异高表达(P<0.05);VGLL2-AS与VGLL2可以进行双向转录且二者之间可以形成双链RNA;VGLL2-AS半衰期较VGLL2长;在成肌细胞中,VGLL2-AS和VGLL2主要定位于细胞质中(P<0.001);VGLL2只存在VGLL2-mRNA、VGLL2-X2和VGLL2-X3转录本;VGLL2-mRNA和VGLL2-X3与VGLL2-AS表达趋势一致,且VGLL2-mRNA、X3和VGLL2-AS的表达呈现极强的正相关(r分别为0.943和0.935,P<0.01)。综上所述,VGLL2-AS作为VGLL2的反义链编码的lncRNA定位于细胞质中,可能通过与VGLL2形成的双链RNA之间的相互作用,然后参与调节VGLL2的表达并维持其稳定性,最终在鸡的早期肌肉发育中发挥重要作用。本研究的结果扩展了鸡中关于NATs的研究,并为鸡VGLL2基因与其天然反义链转录本VGLL2-AS在鸡骨骼肌发育中的生物学功能的研究奠定了基础,对于提高禽类的生长发育具有一定的意义。