lncRNAs transactivate Staufen1-mediated mRNA decay by duplexing with 3'UTRs via Alu elements

延边大学医学学报　２０２２年３月　第４５卷　第１期与疾病报告２０２０概要［Ｊ］．中国循环杂志，２０２１，３６（６）：５２１－５４５．［２］　ＭＥＤＩＮＡ－ＬＥＹＴＥ　ＤＪ，ＺＥＰＥＤＡ－ＧＡＲＣíＡ　Ｏ，ＤＯＭíＮＧ－ＵＥＺＰéＲＥＺ　Ｍ，ｅｔ　ａｌ．．Ｅｎｄｏｔｈｅｌｉａｌ　ｄｙｓｆｕｎｃｔｉｏｎ，ｉｎｆｌａｍ－ｍａｔｉｏｎ　ａｎｄ　ｃｏｒｏｎａｒｙ　ａｒｔｅｒｙ　ｄｉｓｅａｓｅ：ｐｏｔｅｎｔｉａｌ　ｂｉｏｍａｒｋ－ｅｒｓ　ａｎｄ　ｐｒｏｍｉｓｉｎｇ　ｔｈｅｒａｐｅｕｔｉｃａｌ　ａｐｐｒｏａｃｈｅｓ［Ｊ］．Ｉｎｔ　ＪＭｏｌ　Ｓｃｉ，２０２１，２２（８）：３８５０．［３］　ＨＵＡＮＧ　Ｃ，ＨＵＡＮＧ　ＷＨ，ＷＡＮＧ　Ｒ，ｅｔ　ａｌ．．Ｕｌｉｎａｓｔａｔｉｎｉｎｈｉｂｉｔｓ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ，ｉｎｖａｓｉｏｎ　ａｎｄ　ｐｈｅｎｏｔｙｐｉｃｓｗｉｔｃｈｉｎｇ　ｏｆ　ＰＤＧＦ－ＢＢ－ｉｎｄｕｃｅｄ　ＶＳＭＣｓ　ｖｉａ　Ａｋｔ／ｅＮＯＳ／ＮＯ／ｃＧＭＰ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ［Ｊ］．Ｄｒｕｇ　Ｄｅｓ　Ｄｅｖｅｌ　Ｔｈｅｒ，２０２０，１４：５５０５－５５１４．［４］　赵高峰．延边地区朝鲜族ＳＭＡＲＣＡ４基因ＳＮＰｒｓ１１２２６０８与冠心病相关性研究［Ｄ］．延吉：延边大学，２０２０．［５］　ＩＭ　ＰＫ，ＭＩＬＬＷＯＯＤ　ＩＹ，ＫＡＲＴＳＯＮＡＫＩ　Ｃ，ｅｔ　ａｌ．．Ａｌ－ｃｏｈｏｌ　ｄｒｉｎｋｉｎｇ　ａｎｄ　ｒｉｓｋｓ　ｏｆ　ｔｏｔａｌ　ａｎｄ　ｓｉｔｅ－ｓｐｅｃｉｆｉｃ　ｃａｎｃ－ｅｒｓ　ｉｎ　Ｃｈｉｎａ：Ａ　１０－ｙｅａｒ　ｐｒｏｓｐｅｃｔｉｖｅ　ｓｔｕｄｙ　ｏｆ　０．５ｍｉｌｌｉｏｎａｄｕｌｔｓ［Ｊ］．Ｉｎｔ　Ｊ　Ｃａｎｃｅｒ，２０２１，１４９（３）：５２２－５３４．［６］　ＲＯＳＣＨ　ＰＪ．Ｃｏｕｌｄ　ｔｈｅ　ｂｅｎｅｆｉｔｓ　ｏｆ　ｄｒｉｎｋｉｎｇ　ａｌｃｏｈｏｌ　ｏｕｔ－ｗｅｉｇｈ　ａｌｌ　ｉｔｓ　ｒｉｓｋｓ［Ｊ］．Ｓｔｒｅｓｓ　Ｈｅａｌｔｈ，２０１３．［７］　ＷＡＬＬＥＲＡＴＨ　Ｔ，ＰＯＬＥＯ　Ｄ，ＬＩ　Ｈ，ｅｔ　ａｌ．．Ｒｅｄ　ｗｉｎｅ　ｉｎ－ｃｒｅａｓｅｓ　ｔｈｅ　ｅｘｐｒｅｓｓｉｏｎ　ｏｆ　ｈｕｍａｎ　ｅｎｄｏｔｈｅｌｉａｌ　ｎｉｔｒｉｃ　ｏｘｉｄｅｓｙｎｔｈａｓｅ：ａ　ｍｅｃｈａｎｉｓｍ　ｔｈａｔ　ｍａｙ　ｃｏｎｔｒｉｂｕｔｅ　ｔｏ　ｉｔｓ　ｂｅｎｅ－ｆｉｃｉａｌ　ｃａｒｄｉｏｖａｓｃｕｌａｒ　ｅｆｆｅｃｔｓ［Ｊ］．ＪＡＣＣ，２００３，４１（３）：４７１－４７８．［８］　ＺＨＡＯ　Ｆ，ＪＩ　Ｚ，ＣＨＩ　Ｊ，ｅｔ　ａｌ．．Ｅｆｆｅｃｔｓ　ｏｆ　Ｃｈｉｎｅｓｅ　ｙｅｌｌｏｗｗｉｎｅ　ｏｎ　ｎｉｔｒｉｃ　ｏｘｉｄｅ　ｓｙｎｔｈａｓｅ　ａｎｄ　ｉｎｔｅｒｃｅｌｌｕｌａｒ　ａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ－１ｅｘｐｒｅｓｓｉｏｎｓ　ｉｎ　ｒａｔ　ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｃｅｌｌｓ［Ｊ］．Ａｃｔａ　Ｃａｒｄｉｏｌｏｇｉｃａ，２０１６，７１（１）：２７－３４．［９］　金春花，刘文博，关宏锏，等．一氧化氮合酶各种亚型的信号通路对心力衰竭的作用机制与治疗的研究进展［Ｊ］．中国循环杂志，２０１７，３２（８）：８３０－８３２．［１０］ＷＡＧＮＥＲ　ＭＣ，ＹＥＬＩＧＡＲ　ＳＭ，ＬＯＵ　ＡＢ，ｅｔ　ａｌ．．ＰＰＡＲγｌｉｇａｎｄｓ　ｒｅｇｕｌａｔｅ　ＮＡＤＰＨ　ｏｘｉｄａｓｅ，ｅＮＯＳ，ａｎｄ　ｂａｒｒｉｅｒｆｕｎｃｔｉｏｎ　ｉｎ　ｔｈｅ　ｌｕｎｇ　ｆｏｌｌｏｗｉｎｇ　ｃｈｒｏｎｉｃ　ａｌｃｏｈｏｌ　ｉｎｇｅｓｔｉｏｎ［Ｊ］．Ａｌｃｏｈｏｌ　Ｃｌｉｎ　Ｅｘｐ　Ｒｅｓ，２０１２，３６（２）：１９７－２０６．［１１］ＴＩＲＡＰＥＬＬＩ　ＣＲ，ＬＥＯＮＥ　ＡＦ，ＹＯＧＩ　Ａ，ｅｔ　ａｌ．．Ｅｔｈａｎｏｌｃｏｎｓｕｍｐｔｉｏｎ　ｉｎｃｒｅａｓｅｓ　ｂｌｏｏｄ　ｐｒｅｓｓｕｒｅ　ａｎｄ　ａｌｔｅｒｓ　ｔｈｅ　ｒｅ－ｓｐｏｎｓｉｖｅｎｅｓｓ　ｏｆ　ｔｈｅ　ｍｅｓｅｎｔｅｒｉｃ　ｖａｓｃｕｌａｔｕｒｅ　ｉｎ　ｒａｔｓ［Ｊ］．Ｊ　Ｐｈａｒｍ　Ｐｈａｒｍａｃｏｌ，２００８，６０（３）：３３１－３４１．［１２］ＣＨＥＮ　ＳＳ，ＷＡＮＧ　ＺＹ，ＺＨＯＵ　Ｈ，ｅｔ　ａｌ．．Ｉｃａｒｉｉｎ　ｒｅ－ｄｕｃｅｓ　ｈｉｇｈ　ｇｌｕｃｏｓｅ－ｉｎｄｕｃｅｄ　ｅｎｄｏｔｈｅｌｉａｌ　ｐｒｏｇｅｎｉｔｏｒ　ｃｅｌｌｄｙｓｆｕｎｃｔｉｏｎ　ｖｉａ　ｉｎｈｉｂｉｔｉｎｇ　ｔｈｅ　ｐ３８／ＣＲＥＢ　ｐａｔｈｗａｙ　ａｎｄａｃｔｉｖａｔｉｎｇ　ｔｈｅ　Ａｋｔ／ｅＮＯＳ／ＮＯ　ｐａｔｈｗａｙ［Ｊ］．Ｅｘｐ　ＴｈｅｒＭｅｄ，２０１９，１８（６）：４７７４－４７８０．■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■［基金项目］　国家自然科学基金项目（编号：８１５６０１７９）．［收稿日期］　２０２２－０１－０３［作者简介］　黄银珠（１９９５—），女，硕士，研究方向为口腔颌面外科．［通信作者］　金成日，Ｅｍａｉｌ：ｊｃｒ１１０５＠１６３．ｃｏｍ．初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路影响口腔鳞状细胞癌增殖研究黄银珠，李京旭，金成日（延边大学附属医院口腔科，吉林延吉１３３０００）［摘要］　［目的］探讨初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对口腔鳞状细胞癌增殖能力产生的影响．［方法］选择人舌鳞癌Ｔｃａ８１１３细胞株作为研究对象，分为ＫＩＦ３Ａ－ｓｉＲＮＡ转染组和对照组．采用蛋白印迹实验和实时荧光定量实验检测各组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白及ｍＲＮＡ的相对表达水平；利用ＭＴＴ细胞增殖实验观察Ｔｃａ８１１３细胞增殖能力的变化．［结果］蛋白印迹实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１蛋白表达水平均明显下调（Ｐ＜０．０５）；实时荧光定量实验结果显示，与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组ＫＩＦ３Ａ、Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平均明显下调（Ｐ＜０．０１）；ＭＴＴ细胞增殖实验结果显示，细胞培养２４ｈ时两组细胞增殖间差异无统计学意义（Ｐ＞０．０５），细胞培养４８、７２ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组增殖率较对照组明显降低（Ｐ＜０．０１）．［结论］沉默初级纤毛相关基因ＫＩＦ３Ａ可能通过调控Ｈｅｄｇｅｈｏｇ信号通路抑制口腔鳞癌的增殖能力．［关键词］　口腔鳞状细胞癌；初级纤毛；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ信号通路ＤＯＩ：１０．１６０６８／ｊ．１０００－１８２４．２０２２．０１．００２［中图分类号］　Ｒ　７３９．８ ［文献标志码］　Ａ ［文章编号］　１０００－１８２４（２０２２）０１－０００７－０４·７·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１Ｅｆｆｅｃｔｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｙ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙＨＵＡＮＧ　Ｙｉｎｚｈｕ，ＬＩ　Ｊｉｎｇｘｕ，ＪＩＮ　Ｃｈｅｎｇｒｉ（Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｓｔｏｍａｔｏｌｏｇｙ，Ａｆｆｉｌｉａｔｅｄ　Ｈｏｓｐｉｔａｌ　ｏｆ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ，Ｙａｎｊｉ　１３３０００，Ｊｉｌｉｎ，Ｃｈｉｎａ）ＡＢＳＴＲＡＣＴ：ＯＢＪＥＣＴＩＶＥＴｏ　ｅｘｐｌｏｒｅ　ｔｈｅ　ｅｆｆｅｃｔ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｏｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｔｈｒｏｕｇｈ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．ＭＥＴＨＯＤＳ　Ｈｕｍａｎ　ｔｏｎｇｕｅ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ　Ｔｃａ８１１３ｃｅｌｌ　ｌｉｎｅ　ｗａｓ　ｓｅｌｅｃｔｅｄ　ａｎｄ　ｄｉｖｉｄｅｄ　ｉｎｔｏ　ＫＩＦ３Ａ－ｓｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ａｎｄ　ｃｏｎｔｒｏｌｇｒｏｕｐ．Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ａｎｄ　ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｗｅｒｅ　ｕｓｅｄ　ｔｏ　ｄｅｔｅｃｔ　ｔｈｅ　ｒｅｌａｔｉｖｅ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｐｒｏｔｅｉｎｓ　ａｎｄ　ｍＲＮＡ　ｉｎ　ｅａｃｈ　ｇｒｏｕｐ．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｗａｓ　ｕｓｅｄ　ｔｏ　ｏｂｓｅｒｖｅ　ｔｈｅｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ．ＲＥＳＵＬＴＳ　Ｗｅｓｔｅｒｎ　ｂｌｏｔ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ，ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０５）．Ｒｅａｌ－ｔｉｍｅ　ｑＰＣＲ　ｒｅｓｕｌｔｓ　ｓｈｏｗｅｄ　ｔｈａｔ　ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｔｈｅ　ｃｏｎｔｒｏｌｇｒｏｕｐ，ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈ　ａｎｄ　Ｇｌｉ１ｉｎ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙ　ｄｏｗｎ－ｒｅｇｕｌａｔｅｄ（Ｐ＜０．０１）．ＭＴＴ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ａｓｓａｙ　ｓｈｏｗｅｄ　ｔｈａｔ　ｔｈｅｒｅ　ｗａｓ　ｎｏ　ｓｉｇｎｉｆｉｃａｎｔｄｉｆｆｅｒｅｎｃｅ　ｉｎ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｅｔｗｅｅｎ　ｔｈｅ　ｔｗｏ　ｇｒｏｕｐｓ　ａｆｔｅｒ　２４ｈｃｅｌｌ　ｃｕｌｔｕｒｅ（Ｐ＞０．０５）．Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎｒａｔｅ　ｏｆ　ｔｈｅ　ＫＩＦ３Ａ－ＳｉＲＮＡ　ｔｒａｎｓｆｅｃｔｉｏｎ　ｇｒｏｕｐ　ｗａｓ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ｌｏｗｅｒ　ｔｈａｎ　ｔｈａｔ　ｏｆ　ｔｈｅ　ｃｏｎｔｒｏｌ　ｇｒｏｕｐ　ａｆｔｅｒ　ｃｅｌｌｃｕｌｔｕｒｅ　ｆｏｒ　４８ａｎｄ　７２ｈ（Ｐ＜０．０１）．ＣＯＮＣＬＵＳＩＯＮＳｉｌｅｎｃｉｎｇ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ－ａｓｓｏｃｉａｔｅｄ　ｇｅｎｅ　ＫＩＦ３Ａｍａｙｉｎｈｉｂｉｔ　ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ　ｂｙ　ｒｅｇｕｌａｔｉｎｇ　Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ．Ｋｅｙｗｏｒｄｓ：ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ；ｐｒｉｍａｒｙ　ｃｉｌｉａ；ＫＩＦ３Ａ；Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｐａｔｈｗａｙ 口腔癌人数约占全世界罹患恶性肿瘤人数的３％，其中鳞状细胞癌比例超过９０％．２００５年，ＷＨＯ将口腔鳞状细胞癌（ＯＳＣＣ）定义为一种具有不同分化程度和侵袭性的肿瘤，早期可出现广泛的淋巴结转移，好发于４０～７０岁的烟酒爱好者［１］．口腔癌患者５年生存率约为５０％［２］，患者术后预后差和５年生存率低的主要原因为患者未及时就诊、淋巴结转移及复发［３］．控制ＯＳＣＣ的侵袭转移能力对预后至关重要，认为可偿试从分子水平出发找出更好的治疗方法［４］．初生纤毛属于一种细胞表面的细胞器，在脊椎动物发育和人类遗传疾病中发挥着重要作用．纤毛对发育信号的响应是必需的，而越来越多的证据表明初级纤毛是专属于Ｈｅｄｇｅｈｏｇ信号传导的［５］，而Ｈｅｄｇｅｈｏｇ信号传导对肿瘤的发生发展起着重要作用．研究［６］结果显示，多种肿瘤组织中均存在Ｈｅｄｇｅｈｏｇ信号的异常激活，如肺小细胞癌、胰腺癌、前列腺癌、乳腺癌、恶性胶质瘤、髓母细胞瘤及多发性骨髓瘤等．ＫＩＦ３Ａ属于初级纤毛内运输系统，具有维持和发挥初级纤毛正常功能的作用［７］．本研究探讨了初级纤毛相关基因ＫＩＦ３Ａ通过Ｈｅｄｇｅｈｏｇ信号通路对ＯＳＣＣ增殖能力产生的影响．１　材料与方法１．１　细胞　人舌鳞癌细胞株Ｔｃａ８１１３购自美国菌种保藏中心（ＡＴＣＣ），置于延边大学附属医院中心实验室封存．１．２细胞培养与转染　取Ｔｃａ８１１３细胞置于温度３７℃、二氧化碳体积分数５％、饱和湿度９５％的恒温孵育箱中，用含１０％（质量分数）ＦＢＳ、青链霉素的ＤＭＥＭ高糖型培养基传代培养．在转染前１ｄ，将Ｔｃａ８１１３细胞分为对照组和ＫＩＦ３Ａ－ｓｉＲＮＡ转染组后接种至６孔板中，待生长至８０％时进行细胞转染．ＫＩＦ３Ａ－ｓｉＲＮＡ转染组转染ＫＩＦ３Ａ－ｓｉＲＮＡ，对照组未进行转染．转染操作按照ＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ２０００Ｒｅ－ａｇｅｎ转染说明书进行．ＫＩＦ３Ａ－ｓｉＲＮＡ序列（５′－３′）：ＵＡＡ　ＧＧＡ　ＡＵＧ　ＣＵＧ　ＡＡＧ　ＡＡＧ　ＡＣＧ　ＡＧＵ　Ｃ．１．３　蛋白印迹实验　转染２４ｈ后分别收集两组细胞，并加入ＲＩＰＡ与ＰＭＳＦ混合（１００:１）冰上裂解细胞，提取细胞总蛋白，采用ＢＣＡ试剂盒测定总蛋白水平．取各组标本行８％（质量分数）十二烷基硫酸钠聚丙烯酰胺凝胶（ＳＤＳ－ＰＡＧＥ）电泳，转膜，室温下用５％（质量分数）脱脂奶粉封闭２ｈ．ＴＢＳＴ洗涤３·８·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■延边大学医学学报　２０２２年３月　第４５卷　第１期次，每次１０ｍｉｎ，加入兔抗人Ｋｉｆ３ａ一抗（１:１　０００）、兔抗人Ｓｈｈ一抗（１:２　０００）、兔抗人Ｇｌｉ１（１:１　０００）及兔抗人ＧＡＰＤＨ（１:１　０００）后置于湿盒中，于４℃冰箱中孵育过夜；次日ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，分别加入山羊抗兔ＩｇＧ二抗（１:１０　０００）后置于湿盒中室温孵育２ｈ；ＴＢＳＴ洗涤３次，每次１０ｍｉｎ，加入增强型化学发光剂ＥＣＬ显影．以ＧＡＰＤＨ为内参，采用Ｉｍａｇｉｎｅ　Ｊ软件分析蛋白条带灰度值．１．４　实时荧光定量实验　转染２４ｈ后分别收集两组细胞，采用ＴＲＮｚｏｌ法提取细胞中的总ＲＮＡ，反转录为ｃＤＮＡ，按照ＲＴ－ＰＣＲ试剂盒说明书以ｃＤＮＡ为模板进行扩增．测定循环阈值（Ｃｔ），采用２－△△Ｃｔ法进行统计．实验重复３次．引物序列，ＫＩＦ３Ａ－正向：５′－ＡＧＡ　ＧＣＧ　ＴＣＡ　ＡＣＧ　ＡＧＧ　ＴＧＴ　ＴＴ－３′；ＫＩＦ３Ａ－反向：５′－ＴＡＴ　ＴＧＡ　ＴＣＧ　ＧＣＡ　ＴＣＴ　ＴＧＧ　ＣＣＣ－３′；Ｓｈｈ－正向：５′－ＣＴＣ　ＧＣＴ　ＧＣＴ　ＧＧＴ　ＡＴＧ　ＣＴＣ　Ｇ－３′；Ｓｈｈ－反向：５′－ＡＴＣ　ＧＣＴ　ＣＧＧ　ＡＧＴ　ＴＴＣ　ＴＧＧＡＧＡ－３′；Ｇｌｉ１－正向：５′－ＡＧＣ　ＧＴＧ　ＡＧＣ　ＣＴＧ　ＡＡＴＣＴＧ　ＴＧ－３′；Ｇｌｉ１－反向：５′－ＣＡＧ　ＣＡＴ　ＧＴＡ　ＣＴＧＧＧＣ　ＴＴＴ　ＧＡＡ－３′；ＧＡＰＤＨ－正向：５′－ＧＧＡ　ＧＣＧＡＧＡ　ＴＣＣ　ＣＴＣ　ＣＡＡ　ＡＡＴ－３′；ＧＡＰＤＨ－反向：５′－ＧＧＣ　ＴＧＴ　ＴＧＴＣＡＴＡＣＴ　ＴＣＴＣＡＴＧＧ－３′．１．５　ＭＴＴ细胞增殖实验　转染２４ｈ后分别收集两组细胞，以５　０００个／孔接种于９６孔板中，每组设置５个复孔．培养２４、４８、７２ｈ后弃去旧培养液，每孔加入１００μＬ　ＭＴＴ（５　ｇ／Ｌ），于３７℃、５％（体积分数）二氧化碳、９５％饱和湿度的恒温孵育箱中培养４　ｈ，弃去上清液终止反应，每孔加入１００μＬ　ＤＭＳＯ，置于振荡器３　ｍｉｎ溶解结晶后在酶标仪波长４９０　ｎｍ处测定各孔的光密度（ＯＤ）值，计算相对细胞增殖率．相对细胞增殖率（％）＝（实验组ＯＤ值／对照组ＯＤ值）×１００％．１．６　统计学分析　采用Ｇｒａｐｈｐａｄ　Ｐｒｉｓｍ　７．０软件进行数据分析．计量数据以均数±标准偏差（ ｘ±ｓ）表示，进行两个样本均数的ｔ检验，以Ｐ＜０．０５为差异有统计学意义．２　结果２．１　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中Ｓｈｈ、Ｇｌｉ１蛋白表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组２４ｈ时ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１蛋白表达水平均明显降低，差异均具有统计学意义（Ｐ＜０．０５），见Ｆｉｇｕｒｅ　１．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊Ｐ＜０．０５．Ｆｉｇｕｒｅ　１　Ｔｈｅ　ｐｒｏｔｅｉｎ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．２　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞中Ｈｅｄｇｅｈｏｇ信号通路Ｓｈｈ、Ｇｌｉ１ｍＲＮＡ表达水平的影响　与对照组比较，ＫＩＦ３Ａ－ｓｉＲＮＡ转染２４ｈ组ＫＩＦ３Ａ、Ｓｈｈ及Ｇｌｉ１ｍＲＮＡ表达水平均显著降低，差异均具有统计学意义（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　２．ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　２　Ｔｈｅ　ｍＲＮＡ　ｅｘｐｒｅｓｓｉｏｎ　ｌｅｖｅｌｓ　ｏｆ　ＫＩＦ３Ａ，Ｓｈｈａｎｄ　Ｇｌｉ１ｄｅｃｒｅａｓｅｄ　ｓｉｇｎｉｆｉｃａｎｔｌｙ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ２．３　沉默ＫＩＦ３Ａ基因后对Ｔｃａ８１１３细胞增殖能力的影响　ＭＴＴ检测结果显示，细胞培养２４ｈ时ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组细胞增殖间差异无统计学意义（Ｐ＞０．０５）；细胞培养４８、７２ｈ时，ＫＩＦ３Ａ－ｓｉＲＮＡ转染组与对照组比较增殖显著受到抑制（Ｐ＜０．０１），见Ｆｉｇｕｒｅ　３．·９·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃａｌ　Ｓｃｉｅｎｃｅ　Ｙａｎｂｉａｎ　Ｕｎｉｖｅｒｓｉｔｙ　Ｍａｒ．２０２２　Ｖｏｌ．４５　Ｎｏ．１ｃｏｍｐａｒｅｄ　ｗｉｔｈ　ｃｏｎｔｒｏｌ，＊＊Ｐ＜０．０１．Ｆｉｇｕｒｅ　３　Ｔｈｅ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｒａｔｅ　ｏｆ　Ｔｃａ８１１３ｃｅｌｌｓ　ａｆｔｅｒｔｒａｎｓｆｅｃｔｉｏｎ３　讨论初级纤毛为多数分化型细胞表面上的感觉附属物，具有化学感觉和机械感觉功能，在细胞周期控制中具有重要的作用．以往的研究认为初级纤毛在快速增殖的细胞上是找不到的，如癌症细胞，但ＫＯＷＡＬ等［８］通过一种初级纤毛标志物Ａｒｌ１３ｂ在ＨｅＬａ和ＭＧ６３细胞中发现了初级纤毛．未在ＯＳＣＣ中查看是否存在初级纤毛是本研究的不足之处．ＫＩＦ３Ａ属于初级纤毛内运输系统，在维持和发挥初级纤毛功能中起重要作用．ＨＯＡＮＧ－ＭＩＮＨ等［９］的研究结果显示，沉默ＫＩＦ３Ａ基因表达后可降低胶质母细胞瘤中初级纤毛的数量，最终促进肿瘤的发生．玄云泽等［１０］首次在ＯＳＣＣ中发现有初级纤毛，后来有学者亦在口腔白斑与ＯＳＣＣ组织中发现了初级纤毛，ＥＧＦＲ－Ａｕｒｏｒａ　Ａ异常信号被激活后，口腔黏膜癌变过程中原发性纤毛逐渐减少［１１］．本研究结果显示，利用ｓｉＲＮＡ技术沉默ＫＩＦ３Ａ基因后ｍＲＮＡ和蛋白表达水平明显降低，提示初级纤毛的检出率降低．Ｈｅｄｇｅｈｏｇ信号通路对胚胎发育及肿瘤的发生发展具有重要意义．研究［１２－１３］结果显示，Ｈｅｄｇｅｈｏｇ信号通路的异常激活与皮肤、乳腺、肺及消化道等多种恶性肿瘤的发生、增殖密切相关．程志芬等［１２］利用Ｈｅｄｇｅｈｏｇ信号通路特异性抑制剂环巴胺处理Ｔｃａ８１１３细胞株后，Ｓｍｏｏｔｈｅｎｅｄ的ｍＲＮＡ和蛋白表达明显减少，且细胞增殖受到抑制．ＫＵＲＯＤＡ等［１４］的研究结果显示，给予Ｈｅｄｇｅｈｏｇ信号通路抑制剂可抑制肿瘤前沿血管内皮细胞的增殖、迁移，进而抑制肿瘤的增殖和迁移．在小细胞肺癌中，沉默ＫＩＦ３Ａ后初级纤毛明显减少，且Ｓｈｈ信号通路介导的Ｇｌｉ１ｍＲＮＡ表达水平明显降低［１５］，此结果与本研究结果相符．研究［１６］结果显示，Ｈｅｄｇｅｈｏｇ信号通路转录因子Ｇｌｉ１的激活是恶性肿瘤的一个不良预后因素，利用环巴胺阻断Ｈｅｄｇｅｈｏｇ信号通路可抑制Ｇｌｉ１激活，并增强头颈部鳞癌对放疗的敏感性，提示Ｈｅｄｇｅｈｏｇ信号通路影响头颈部鳞癌的放疗预后．总之，本研究揭示了初级纤毛相关基因ＫＩＦ３Ａ在人舌鳞癌Ｔｃａ８１１３细胞Ｈｅｄｇｅｈｏｇ信号通路中的作用，初步解释了沉默ＫＩＦ３Ａ后可能通过抑制Ｈｅｄｇｅｈｏｇ信号通路影响ＯＳＣＣ细胞的增殖能力，为ＯＳＣＣ患者的靶向治疗提供了理论依据．［参　考　文　献］［１］　ＤＩ　ＰＡＲＤＯ　ＢＪ，ＢＲＯＮＳＯＮ　ＮＷ，ＤＩＧＧＳ　ＢＳ，ｅｔ　ａｌ．．Ｔｈｅｇｌｏｂａｌ　ｂｕｒｄｅｎ　ｏｆ　ｅｓｏｐｈａｇｅａｌ　ｃａｎｃｅｒ：ａ　ｄｉｓａｂｉｌｉｔｙ－ａｄｊｕｓｔｅｄｌｉｆｅ－ｙｅａｒ　ａｐｐｒｏａｃｈ［Ｊ］．Ｗｏｒｌｄ　Ｊ　Ｓｕｒｇ，２０１６，４０（２）：３９５－４０１．［２］　ＢＲＯＣＫＬＥＨＵＲＳＴ　ＰＲ，ＢＡＫＥＲ　ＳＲ，ＳＰＥＩＧＨＴ　ＰＭ．Ｏｒａｌｃａｎｃｅｒ　ｓｃｒｅｅｎｉｎｇ：ｗｈａｔ　ｈａｖｅ　ｗｅ　ｌｅａｒｎｔ　ａｎｄ　ｗｈａｔ　ｉｓ　ｔｈｅｒｅｓｔｉｌｌ　ｔｏ　ａｃｈｉｅｖｅ［Ｊ］．Ｆｕｔｕｒｅ　Ｏｎｃｏｌ，２０１０，６（２）：２９９－３０４．［３］　ＢＡＶＬＥ　ＲＭ，ＶＥＮＵＧＯＰＡＬ　Ｒ，ＫＯＮＤＡ　Ｐ，ｅｔ　ａｌ．．Ｍｏ－ｌｅｃｕｌａｒ　ｃｌａｓｓｉｆｉｃａｔｉｏｎ　ｏｆ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ［Ｊ］．ＪＣＤＲ，２０１６，１０（９）：Ｚ１８－Ｚ２１．［４］　陈新，徐文华，周健，等．口腔鳞状细胞癌现状［Ｊ］．口腔医学，２０１７，３７（５）：４６２－４６５．［５］　ＧＯＥＴＺ　ＳＣ，ＡＮＤＥＲＳＯＮ　ＫＶ．Ｔｈｅ　ｐｒｉｍａｒｙ　ｃｉｌｉｕｍ：ａ　ｓｉｇｎａｌｌｉｎｇ　ｃｅｎｔｒｅ　ｄｕｒｉｎｇ　ｖｅｒｔｅｂｒａｔｅ　ｄｅｖｅｌｏｐｍｅｎｔ［Ｊ］．Ｎａｔ　Ｒｅｖ　Ｇｅｎｅ，２０１０，１１（５）：３３１－３４４．［６］　那玉岩，刘万林．纤毛相关疾病：细胞学机制及转化应用进展［Ｊ］．中国组织工程研究，２０１６，２０（２４）：３６４２－３６４８．［７］　ＮＩＥＷＩＡＤＯＭＳＫＩ　Ｐ，ＮＩＥＤＺＩółＫＡ　ＳＭ，ＭＡＲＫＩＥＷＩＣＺŁ，ｅｔ　ａｌ．．Ｇｌｉ　ｐｒｏｔｅｉｎｓ：ｒｅｇｕｌａｔｉｏｎ　ｉｎ　ｄｅｖｅｌｏｐｍｅｎｔ　ａｎｄｃａｎｃｅｒ［Ｊ］．Ｃｅｌｌｓ，２０１９，８（２）：１４７．［８］　ＫＯＷＡＬ　ＴＪ，ＦＡＬＫ　ＭＭ．Ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｆｏｕｎｄ　ｏｎ　ＨｅＬａａｎｄ　ｏｔｈｅｒ　ｃａｎｃｅｒ　ｃｅｌｌｓ［Ｊ］．Ｃｅｌｌ　Ｂｉｏｌ　Ｉｎｔ，２０１５，３９（１１）：１３４１－１３４７．［９］　ＨＯＡＮＧ－ＭＩＮＨ　ＬＢ，ＤＥＬＥＹＲＯＬＬＥ　ＬＰ，ＳＩＥＢＺＥＨ－ＮＲＵＢＬ　Ｄ，ｅｔ　ａｌ．．Ｄｉｓｒｕｐｔｉｏｎ　ｏｆ　ＫＩＦ３Ａｉｎ　ｐａｔｉｅｎｔ－ｄｅ－ｒｉｖｅｄ　ｇｌｉｏｂｌａｓｔｏｍａ　ｃｅｌｌｓ：ｅｆｆｅｃｔｓ　ｏｎ　ｃｉｌｉｏｇｅｎｅｓｉｓ，ｈｅｄｇｅ－ｈｏｇ　ｓｅｎｓｉｔｉｖｉｔｙ，ａｎｄ　ｔｕｍｏｒｉｇｅｎｅｓｉｓ［Ｊ］．Ｏｎｃｏｔａｒｇｅｔ，２０１６，７（６）：７０２９－７０４３．［１０］玄云泽，李书进，金成日，等．初级纤毛相关基因ＫＩＦ３Ａ通过调控ＥＭＴ过程在口腔鳞状细胞癌中的作用机制研究［Ｊ］．重庆医学，２０２０，４９（１）：７－１２．［１１］ＹＩＮ　Ｆ，ＣＨＥＮ　Ｑ，ＳＨＩ　Ｙ，ｅｔ　ａｌ．．Ａｃｔｉｖａｔｉｏｎ　ｏｆ　ＥＧＦＲ－Ａｕ－ｒｏｒａ　Ａ　ｉｎｄｕｃｅｓ　ｌｏｓｓ　ｏｆ　ｐｒｉｍａｒｙ　ｃｉｌｉａ　ｉｎ　ｏｒａｌ　ｓｑｕａｍｏｕｓ　ｃｅｌｌｃａｒｃｉｎｏｍａ［Ｊ］．Ｏｒａｌ　Ｄｉｓ，２０２２，２８（３）：６２１－６３０．［１２］程志芬，崔演，玄延花．Ｐｔｃｈ和Ｓｍｏ在舌鳞状细胞癌Ｔｃａ８１１３细胞中的表达及其意义［Ｊ］．口腔医学研究，２０１５，３１（５）：４３３－４３６．［１３］ＫＯＮＳＴＡＮＴＩＮＯＵ　Ｄ，ＢＥＲＴＡＵＸ－ＳＫＥＩＲＩＫ　Ｎ，ＺＡＶＲＯＳＹ．Ｈｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ　ｉｎ　ｔｈｅ　ｓｔｏｍａｃｈ［Ｊ］．Ｃｕｒｒ　ＯｐｉｎＰｈａｒｍａｃｏｌ，２０１６，３１：７６－８２．［１４］ＫＵＲＯＤＡ　Ｈ，ＫＵＲＩＯ　Ｎ，ＳＨＩＭＯ　Ｔ，ｅｔ　ａｌ．．Ｏｒａｌ　ｓｑｕａｍｏｕｓｃｅｌｌ　ｃａｒｃｉｎｏｍａ－ｄｅｒｉｖｅｄ　ｓｏｎｉｃ　ｈｅｄｇｅｈｏｇ　ｐｒｏｍｏｔｅｓ　ａｎｇｉｏｇｅｎｅ－ｓｉｓ［Ｊ］．Ａｎｔｉｃａｎｃｅｒ　Ｒｅｓ，２０１７，３７（１２）：６７３１－６７３７．［１５］ＣＯＣＨＲＡＮＥ　ＣＲ，ＶＡＧＨＪＩＡＮＩ　Ｖ，ＳＺＣＺＥＰＮＹ　Ａ，ｅｔ　ａｌ．．Ｔｒｐ５３ａｎｄ　Ｒｂ１ｒｅｇｕｌａｔｅ　ａｕｔｏｐｈａｇｙ　ａｎｄ　ｌｉｇａｎｄ－ｄｅｐｅｎｄｅｎｔＨｅｄｇｅｈｏｇ　ｓｉｇｎａｌｉｎｇ［Ｊ］．Ｊ　Ｃｌｉｎ　Ｉｎｖｅｓｔ，２０２０，１３０（８）：４００６－４０１８．［１６］ＧＡＮ　ＧＮ，ＥＡＧＬＥＳ　Ｊ，ＫＥＹＳＡＲ　ＳＢ，ｅｔ　ａｌ．．Ｈｅｄｇｅｈｏｇｓｉｇｎａｌｉｎｇ　ｄｒｉｖｅｓ　ｒａｄｉｏｒｅｓｉｓｔａｎｃｅ　ａｎｄ　ｓｔｒｏｍａ－ｄｒｉｖｅｎｔｕｍｏｒ　ｒｅｐｏｐｕｌａｔｉｏｎ　ｉｎ　ｈｅａｄ　ａｎｄ　ｎｅｃｋ　ｓｑｕａｍｏｕｓ　ｃａｎｃｅｒｓ［Ｊ］．Ｃａｎｃｅｒ　Ｒｅｓ，２０１４，７４（２３）：７０２４－７０３６．■·０１·■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■。

LncRNAs在细胞衰老中的作用和治疗靶标戴凯琴;刘俊;凌霜;许锦文【摘要】长链非编码RNA(long non-coding RNAs,LncRNAs)是一类转录本长度超过200 bp的非编码RNA.细胞衰老是一种稳定的增殖性停滞状态,这种不可逆的细胞停滞状态可能由多种因素引起,如端粒缩短、癌基因诱导或氧化应激.多因素参与细胞衰老,但p53/p21和p16/Rb是不同细胞类型中细胞衰老的主要调节途径.细胞衰老作为一种强大的肿瘤抑制机制已经被广泛研究,以抵抗致癌基因的出现.该文将深入探讨LncRNAs在细胞衰老中的作用机制和治疗靶标.【期刊名称】《中国药理学通报》【年(卷),期】2019(035)004【总页数】4页(P464-467)【关键词】长链非编码RNA;细胞衰老;p53;p16;衰老相关性疾病;治疗靶标【作者】戴凯琴;刘俊;凌霜;许锦文【作者单位】上海中医药大学交叉科学研究院穆拉德中药现代研究中心,上海201203;上海中医药大学交叉科学研究院穆拉德中药现代研究中心,上海 201203;上海中医药大学交叉科学研究院穆拉德中药现代研究中心,上海 201203;上海中医药大学交叉科学研究院穆拉德中药现代研究中心,上海 201203【正文语种】中文【中图分类】R-05;R339.38;R341;R342.2;R394.2;R977.6细胞衰老是一种稳定的增殖性停滞状态，这种不可逆的细胞停滞状态可能由多种因素引起，如端粒缩短、癌基因诱导或氧化应激。

除此之外，不同的细胞类型中，细胞衰老的形态和分子特征有所不同，例如明显的扁平状态，溶酶体β-半乳糖苷酶的活性增加，laminB1表达降低以及炎性因子的表达增加。

虽然许多因素参与细胞衰老，但p53/p21和p16/Rb是不同细胞类型中细胞衰老的两个主要调节途径。

衰老是一个复杂的生物过程，引发与年龄有关的诸多疾病，如心血管疾病、糖尿病、神经退行性疾病、癌症。

分子生物学问答题1什么是中心法则？答：是指遗传信息从DNA传递给RNA，再从RNA传递给蛋白质的转录和翻译的过程，以及遗传信息从DNA 传递给DNA的复制过程。

这是所有有细胞结构的生物所遵循的法则。

在某些病毒中的RNA自我复制和在某些病毒中能以RNA为模板逆转录成DNA的过程是对中心法则的补充。

2什么是分子生物学？答：广义——在分子水平研究生命的现象与规律的学科。

狭义——核酸化学（DNA，RNA）。

在分子水平上研究生命现象的科学。

研究生物大分子(核酸、蛋白质)的结构、功能和生物合成等方面来阐明各种生命现象的本质。

3试举出20世纪三例分子生物学发展中的重大发现答：1950 Chargaff提出Chargaff法则：A+G=T+C1953 Waston&Crick提出：DNA双螺旋模型1954 Crick提出：中心法则1958 Meselson等提出：DNA的半保留复制1961 Brener等提出三联体密码假说1961 Jacob&Monod提出操纵子模型1972 Berg第一次实现体外DNA的重组第二章1、简述DNA复制的基本法则及复制过程中涉及的酶和蛋白质（以E.coli为例）。

答：1）○1DNA的半保留复制：DNA复制是产生的新链中一条单链来自母链（模板链），另一条是新合成的（新生链有一半的母链被保留下来）即半保留复制；○2DNA复制的半不连续性：DNA复制时其中一条单链（3’—5’）先复制，是连续的，即先导链，另一条链的复制滞后一步且是先合成一段段的冈崎片段，通过连接酶形成完整子代单链。

2）酶和蛋白质：DNApol包括DNApolI、DNApolII、DNApolIII三类TopI，解旋酶、SSB、RNA聚合酶、引发酶、DNA连接酶。

2、基因有哪些存在形式、真核生物DNA序列有哪些种类？答：1）割裂基因，重叠基因，跳跃基因，假基因，重组基因等；2）高度重复序列，中度重复序列，单拷贝序列。

细胞与分子免疫学杂志（Chin J Cell Mol ImmU n〇l)2021, 37(2)185 .综述. 文章编号：1007-8738(2021 )02*0185>06长链非编码R N A(ln cR N A)参与天然免疫应答调控机制的研究进展滕培英，亢涛，辛斯琪，陈伟*(昆明理工大学医学院病原学微生物实验室，云南昆明650500)[摘要]长链非编码RNA(lnC R N A)长度大于200个核苷酸，具有多种生物学功能。

我们主要总结了 IncRNA在单核巨噬细胞 和树突状细胞中的天然免疫应答机制以及IncRNA导向、海绵以及与蛋白质相互作用等生物学功能。

同时，也重点叙述了其在 天然免疫应答核因子k B(N F-k B)信号通路中的调控作用和IncRNA在病毒与宿主相互作用中的重要作用。

强调了宿主抗病毒 反应的调控网络，IncRNA在生物学与免疫学领域进行跨学科研究的需求，以加深对病毒发病机制的理解。

[关键词]长链非编码R N A(lncR N A);天然免疫；核因子k B(N F-k B)信号通路；病毒；综述[中图分类号]R392.12, R293. 11, G353.l l[文献标志码]A长链非编码R N A (long non-coding R N A s,I n c R N A)是转录本长度大于200核苷酸且不具备蛋 白编码功能的非编码R N A(n o n-coding R N A,n c R N A)的总称[1]，作为哺乳动物基因组功能注释(Functional Annotation O f the M a m m a l i a n G e n o m e s，F A N T0M)项目的一部分，最初是由日本科学家在对小鼠全长 c D N A文库进行大规模测序中发现[2]。

目前，在G E N C0D E(ver.29)人类基因组数据库中记录了16 066个I n c R N A基因和29 566个I n c R N A转录本。

LncRNA SNHG8对乳腺癌细胞MDA-MB-231的细胞生物学功能的影响通信作者：李杰华,1978年5月，男，博士学位，主任医师，普通外科*******************基金项目：Rab25通过转录因子Snail调控三阴性乳腺癌上皮间充质转化的研究（2018GXNSFAA281255）摘要目的：探讨长链非编码RNA（LncRNA）SNHG8对乳腺癌细胞MDA-MB-231的细胞生物学功能的影响。

方法：收集乳腺癌患者的癌及癌旁标本，提取组织中的总RNA，实时荧光定量聚合酶链反应（qRT-PCR）技术检测乳腺癌组织和癌旁组织中SNHG8的相对表达量；体外培养乳腺癌细胞株MDA-MB-231，设置三组对照组，分别是不转入任何载体的正常对照组，转入空载慢病毒的阴性对照组，以及转入过表达慢病毒的SNHG8过表达组；CCK-8检测细胞增殖情况；划痕实验检测细胞迁移情况，transwell实验检测细胞侵袭情况，使用IBM SPSSStatitics25进行统计学分析。

结果：SNHG8在乳腺癌组织中表达量低于癌旁组织，差异具有统计学意义（P<0.05）；正常对照组和阴性对照组的细胞在OD 值，细胞迁移率和穿膜细胞数无明显差异（P>0.05），正常组的凋亡率要低于阴性对照组（P<0.05）；SNHG8过表达组的细胞OD值，细胞迁移率和穿膜细胞数明显低于空白对照组和阴性对照组（P<0.05）。

结论： LncRNA SNHG8抑制乳腺癌细胞MDA-MB-231的增值，迁移和侵袭。

关键词乳腺癌;长链非编码RNA;SNHG8背景据2021年GLOBOCAN全球癌症统计数据显示，全球乳腺癌每年新发病例高达226万例，乳腺癌已取代肺癌成为全球第一大癌症[1]。

在我国，乳腺癌是女性最常见的恶性肿瘤,发病率位居中国女性恶性肿瘤首位[2]。

乳腺在各种内外因素的作用和影响下，比如：激素[3]、遗传因素[4]、机体免疫功能下降[5]；病毒[6]、放射线[7]等发生发展为乳腺癌。

UNIT19.20 Strep/FLAG Tandem Affinity Purification(SF-TAP)to Study Protein InteractionsChristian Johannes Gloeckner,1Karsten Boldt,1,2and Marius Ueffing1,21Helmholtz Zentrum M¨u nchen,Neuherberg,Germany2Technical University of Munich,Munich,GermanyABSTRACTIn recent years,several methods have been developed to analyze protein-protein interac-tions under native conditions.One of them,tandem affinity purification(TAP),combinestwo affinity-purification steps to allow isolation of high-purity protein complexes.Thisunit presents a methodological workflow based on an SF-TAP tag comprising a doubletStrep-tag II and a FLAG moiety optimized for rapid as well as efficient tandem affinitypurification of native proteins and protein complexes in higher eukaryotic cells.Depend-ing on the stringency of purification conditions,SF-TAP allows both the isolation ofa single tagged-fusion protein of interest and purification of protein complexes undernative conditions.Curr.Protoc.Protein Sci.57:19.20.1-19.20.19.C 2009by John Wiley&Sons,Inc.Keywords:SF-TAP r tandem affinity purification r protein complexesINTRODUCTIONThe analysis of protein-protein interactions under native conditions has been a challengeever since immunoprecipitation(IP)became a common methodology.Low yields andnonspecific binding of proteins have been associated with IP.On the other hand,IPfacilitates targeted analysis of protein interactions with respect to a predefined proteinof interest,given that a suitable antibody is available that features monospecificity andselectivity for this protein.Tandem affinity purification(TAP;UNIT19.19)can significantly reduce the backgroundcaused by nonspecific binding of proteins,as it combines two affinity purifications basedon two different affinity matrices(Rigaut et al.,1999).TAP has been widely used topurify protein complexes from different species(Collins and Choudhary,2008).The TAPtechnique was originally developed to analyze the yeast protein interactome(Gavin et al.,2002).Although the original TAP tag,consisting of a Protein A-tag,a TEV(tobacco etchvirus)protease cleavage site,and a calmodulin binding peptide(CBP)tag,has alreadybeen successfully used in mammalian cells(Bouwmeester et al.,2004),several featuresof thisfirst-generation tag remain suboptimal,such as its high molecular mass(21kDa),the dependency on proteolytic cleavage,and CBP,which may interfere with calciumsignaling within eukaryotic cells.This unit presents an alternative TAP protocol for theisolation of protein complexes from higher eukaryotic cells.The Strep/FLAG tandemaffinity purification(SF-TAP)tag(Gloeckner et al.,2007)combines a tandem Strep-tagII(Skerra and Schmidt,2000;Junttila et al.,2005)and a FLAG tag,resulting in a small4.6-kDa tag.Both moieties have a medium affinity and avidity to their immobilizedbinding partners.Therefore,the tagged fusion proteins and their binding partners canbe recovered under native conditions without the need for time-consuming proteolyticcleavage.In thefirst step,desthiobiotin is used for elution of the SF-TAP fusion proteinfrom the Strep-Tactin matrix.In the second step,the FLAG octapeptide is used for elutionof the SF-TAP fusion protein from the anti-FLAG M2affinity matrix.An overview of the Current Protocols in Protein Science19.20.1-19.20.19,August2009Published online August2009in Wiley Interscience().DOI:10.1002/0471140864.ps1920s57Copyright C 2009John Wiley&Sons,Inc.Identification of Protein Interactions19.20.1 Supplement57Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.2Supplement 57Current Protocols in Protein Science A B 1. purification 2. purification binding to Strep-Tactin binding to FLAG matrix elution with desthiobiotin elution with FLAG peptide Key:SF-TAP desthiobiotin FLAG peptide Figure 19.20.1The S trep/FLAG ta n dem affin ity p u rificatio n .(A )N-a n d C-termi n al S F-T AP ta gs (POI,protei n of i n tere s t).(B )Overview of both p u rificatio n s tep s .(1)P u rificatio n by the ta n dem S trep-ta g II moiety:bi n di ng to S trep-T acti n matrix followed by el u tio n with de s thiobioti n .(2)P u rificatio n by the FLAG-ta g moiety:bi n di ng to a n ti-FLAG M2affin ity matrix followed by el u -tio n with FLAG peptide.Abbreviatio ns :s p.,s pecific i n teractor s (s how n a s g ray circle s );n .s p.,n o ns pecific protei ns (co n tami n a n t s ;s how n a s white circle s ).SF-TAP technique and the tag sequence is shown in Figure 19.20.1.The SF-TAP protocol represents an efficient,fast and straightforward purification of protein complexes from mammalian cells within 2hr.This unit describes the full workflow,starting with the cell culture work needed for recombinant expression of the SF-TAP fusion proteins,followed by the SF-TAP protocol (see Basic Protocol 1)and ending with mass spectrometric analysis of the samples (see Basic Protocol 4).Special focus is given to the crucial step of sample preparation for mass spectrometry.For the identification of associated proteins following SF-TAP,the volume of the SF-TAP eluates is reduced by ultrafiltration using centrifugal units with a low molecular weight cut-off or by chloroform/methanol precipitation (see Support Protocol 2).The samples are then directly subjected to proteolytic digestion (see Basic Protocol 2)for analysis on a nano liquid chromatography (LC)–coupled electron sprayIdentification of Protein Interactions 19.20.3Current Protocols in Protein Science Supplement 57Figure 19.20.2Flow chart of a S F-T AP approach i n cl u di ng M S ide n tificatio n of cop u rified pro-tei ns .Thi s figu re co nn ect s all protocol s pre s e n ted i n thi s un it.tandem mass spectrometer.For complex samples,which contain many proteins,an alternative procedure for SDS-PAGE pre-fractionation is provided,including a method for sensitive MS-compatible Coomassie protein staining (see Support Protocol 3)followed by in-gel proteolytic digestion (see Basic Protocol 3).By reducing sample complexity,pre-fractionation helps to increase the number of protein identifications on state-of-the-art LC-coupled tandem mass spectrometers.Representative MS-analysis protocols are provided for an Orbitrap mass spectrometer (Thermo Fisher Scientific),a fast and sensitive system allowing high identification rates from SF-TAP purifications even with low amounts of protein in the sample (see Basic Protocol 4).Finally,a strategy for meta analysis of mass spectrometric data sets using the Scaffold software is provided (see Support Protocol 4).It can generally be used for the analysis of large MS/MS data sets.Figure 19.20.2provides a flowchart of the entire analytical process.Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.4Supplement 57Current Protocols in Protein ScienceBASICPROTOCOL 1STREP/FLAG TANDEM AFFINITY PURIFICATION (SF-TAP)OF PROTEIN COMPLEXES FROM HEK293CELLS A flowchart of the SF-TAP procedure is shown in Figure 19.20.3.Materials HEK293cells (ATCC no.CRL-1573)Complete DMEM containing 10%FBS (APPENDIX 3C )SF-TAP vectors with appropriate insert,and empty control plasmid (see Critical Parameters)Negative control (see annotation to step 3,below)Transfection reagent of choice (see UNIT 5.10)Phosphate-buffered saline (PBS;APPENDIX 2E ),prewarmed Lysis buffer (see recipe)Strep-Tactin Superflow resin (IBA GmbH,cat.no.2-1206-10)Tris-buffered saline (TBS;see recipe)Wash buffer (see recipe)Desthiobiotin elution buffer:dilute 10×buffer E (IBA GmbH,cat.no.2-1000-025)1:10in H 2O (final concentration,2mM desthiobiotin)Anti–FLAG M2agarose (Sigma-Aldrich)FLAG elution buffer (see recipe)14-cm tissue culture plates Cell scraper Millex GP 0.22-μm syringe-driven filter units (Millipore)End-over-end rotator Microspin columns (GE Healthcare,cat.no.27-3565-01)End-over-end rotator Microcon YM-3centrifugal filter devices (Millipore)Additional reagents and equipment for transfection of mammalian cells (UNIT 5.10)Transfect HEK293cells 1.Seed HEK293cells on 14-cm plates at ∼1–2×107cells per dish in complete DMEM medium containing 10%FBS.The amount of cells used for SF-TAP purification can be varied depending on the ex-pression levels of the bait ually,four 14-cm dishes,corresponding to a final amount of ∼4×108HEK293cells,is a good starting point.Strong overexpression of the bait protein usually increases copurification of heat-shock proteins such as HSP70.For in-depth analysis,it is therefore recommended to generate cell lines stably expressing the bait protein.See Support Protocol 1for a stable transfection method.2.Grow cells overnight.3.Transfect cells with the SF-TAP plasmids using a transfection reagent of choice (according to manufacturer’s protocols).HEK293cells can be easily transfected with lipophilic transfection reagents.The trans-fection efficiency is usually >80%.For a typical SF-TAP experiment,1to 4μg plasmid per 14-cm dish is used.Depending on the cell type other transfection reagents may be favorable (also see UNIT 5.10).Although SF-TAP purifications typically exhibit low background caused by nonspecific binding of proteins to the affinity matrix,a suitable negative control should be used in every experiment.Cells transfected with the empty expression vectors may be used in the same amount as for the SF-TAP-tagged bait protein.However,the tag is quite small and expressed at low levels if not fused to a protein.Thus,the untransfected cell line is an acceptable,simple,and inexpensive alternative for a negative control.Identification of Protein Interactions 19.20.5Current Protocols in Protein Science Supplement 571-4 × 108 HEK293 cell s(1-4 co n fl u e n t 14-cm plate s )expre ss i ng S F-TAP f us io n protei nly s i s(15 mi n 4C)vol u mered u ctio nce n trif ug atio n (10 mi n 10,000 × g )a n aly s i sretai n su per n ata n t fi n alel u atei n c u batio n with50 μl/plate S trep-Tacti n matrix (1 hr)el u tio n with200 μl FLAGel u tio n b u ffer(10 mi n )wa s h 3 time s with 500 μl wa s h b u ffer (s pi n 5 s ec, 100 × g )wa s h 3 time s with500 μl wa s h b u ffer(s pi n 5 s ec, 100 × g )el u tio n with 500 μl de s thiobioti n el u tio n b u ffer (10 mi n )i n c u batio n with25 μl/platea n ti-FLAG M2a g aro s e(1 hr)Figure 19.20.3Flow chart for the S F-T AP proced u re.4.Let cells grow for 48hr.If necessary,cells can be starved in DMEM without FBS for 12hr prior to harvesting.Starving might be desirable if cell signaling is to be analyzed,especially prior to differ-ential treatment with growth factors,to eliminate effects of serum growth factors.Lyse cells5.Remove medium from the plates.6.Optional:Rinse cells in warm PBS.Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.6Supplement 57Current Protocols in Protein Science7.Scrape off cells in 1ml lysis buffer per 14-cm plate on ice using a cell scraper,and combine lysates from each experimental condition in a 1.5-ml microcentrifuge tube.8.Lyse cells by incubating 15min on ice with mixing by hand from time to time.9.Pellet cell debris,including nuclei,by centrifuging 10min at 10,000×g ,4◦C.10.Clear lysate supernatant by filtration through a 0.22-μm syringe filter.Perform SF-TAP 11.Wash Strep-Tactin Superflow resin twice,each time with 4resin volumes TBS and once with 4resin volumes lysis buffer.12.Incubate lysates with 50μl per 14-cm plate of settled Strep-Tactin Superflow resin for 1hr at 4◦C (use an end-over-end rotator to keep the resin evenly distributed).Note that a maximum of 200μl settled resin per spin column should not be exceeded.If more than four 14-cm plates (∼4×108HEK293cells)are used,reduce the volume per plate or use additional spin columns in step 13.13.Centrifuge for 30sec at 7000×g ,4◦C,remove the supernatant until 500μl remains,and transfer resin to a microspin column.Snap off bottom closure of the spin column prior to use.The maximum volume of the spin columns is 650μl.Alternatively,centrifugations for wash and elution steps can be performed at room temperature if no cooled centrifuge is available.14.Remove remaining supernatant by centrifugation in the spin column for 5sec at 100×g ,then wash resin three times,each time with 500μl wash buffer (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.Replug spin columns with inverted bottom closure prior to adding the elution buffer in step 15.IMPORTANT NOTE:Do not allow the resin to run dry.Depending on the bait protein,this markedly reduces the yield.15.Add 500μl desthiobiotin elution buffer and gently mix the resin by hand for 10min on ice.16.Remove the plug of the spin column,transfer the column to a new collection tube,and collect the eluate by centrifuging 10sec at 2000×g ,4◦C.If spin columns were closed by the top screw cap during incubation with elution buffer,the cap needs to be removed prior to centrifugation,to allow the pressure to balance out.17.Wash anti–FLAG M2agarose resin three times,each time with 4resin volumes TBS.Suspend resin in TBS and transfer it to microspin columns,then remove the buffer by centrifuging 5sec at 100×g .25μl settled resin per 14-cm plate will be needed.18.Transfer eluate from step 16corresponding to each 14-cm plate to a microspin column containing 25μl settled anti-FLAG M2agarose prepared as in step 17.19.Plug columns,close columns with top screw caps,and incubate for 1hr at 4◦C (on an end-over-end rotator).20.Wash once with 500μl wash buffer,and then twice,each time with 500μl TBS (centrifuge 5sec at 100×g each time to remove the supernatant)at 4◦C.21.For elution,incubate with 4bead volumes (at least 200μl)FLAG elution buffer for 10min,keeping the columns plugged and gently mixing the resin several times.22.After incubation,remove the plugs and top screws of the spin columns,transfer to new collection tubes,and collect the eluate(s)by centrifugation (10sec at 2000×g ).Identification of Protein Interactions 19.20.7Current Protocols in Protein Science Supplement 5723.Depending on downstream method to be used,either precipitate protein (see SupportProtocol 2)or concentrate the eluate by Microcon YM-3centrifugal filter units according to manufacturer’s protocols.SUPPORT PROTOCOL 1GENERATION OF HEK293CLONES STABLY EXPRESSINGSF-TAP-TAGGED PROTEINSIn Basic Protocol 1,SF-TAP-tagged proteins are transiently expressed.However,strong overexpression of the bait protein usually increases copurification of heat-shock proteins such as HSP70.For in-depth analysis,it is therefore recommended to generate cell lines stably expressing the bait protein.This protocol presents a quick method for generating stable HEK293lines.MaterialsHEK293cells (ATCC no.CRL-1573)Complete DMEM containing 10%FBS (APPENDIX 3C )SF-TAP vectors with appropriate insert,and empty control plasmid (see Critical Parameters)Transfection reagent of choice (see UNIT 5.10)Phosphate-buffered saline (PBS;APPENDIX 2E )Complete DMEM medium (APPENDIX 3C )G418(PAA Laboratories, )Freezing solution:90%fetal bovine serum (FBS;Invitrogen)/10%dimethylsulfoxide (DMSO;AR grade)Lysis buffer (see recipe)Blocking reagent:5%(w/v)nonfat dry milk in TBS (see recipe for TBS)containing 0.1%(v/v)Tween 20Anti-FLAG M2antibody (Sigma-Aldrich)10-cm tissue culture dishes12-well and 6-welll tissue culture platesCentrifuge2-ml cryovials (Nunc)Additional reagents and equipment for transfection of mammalian cells (UNIT 5.10),trypsinization and counting of cells (UNIT 5.10),and immunoblotting (UNIT 10.10)Grow and transfect cells1.Grow cells in complete DMEM containing 10%FBS.2.Transfect cells with expression plasmid using a transfection reagent of choice ac-cording to the manufacturer’s protocols.3.Change medium after 6hr.Select cells4.After 48hr,trypsinize and count cells (APPENDIX 3C )and seed them at low density (1×106cells per 10-cm dish)to allow formation of single colonies upon selection.5.Add G418(500to 1000μg/ml)for selection of the SF-TAP expression vectors,which are based on pcDNA3.0and contain a neomycin-resistance gene.6.Grow the cells under G-418selection for 2to 4weeks,changing the medium every second day.7.Collect single colonies with a 200-μl pipet into 12-well plates.8.Keep colonies under G418selection until the cell density is sufficient for expanding them to 6-well dishes (two wells per clone).Strep/FLAGTandem AffinityPurification (SF-TAP)19.20.8Supplement 57Current Protocols in Protein ScienceCryopreserve cells 9.Grow cells to >90%confluency and trypsinize (APPENDIX 3C )one well of each clone for generation of cryostocks.10.Generate cryostocks:a.Wash cells from one well once by adding 3ml PBS,centrifuging 5min at 800×g ,room temperature,and resuspending the pellet in 500μl freezing buffer.b.Transfer resuspended cells to 2-ml cryovials.c.Freeze cells slowly:keep cells for 1hr at −20◦C,then overnight at −80◦C,followed by storage in a liquid nitrogen tank.For cultivation and expansion of confirmed clones,thaw the cryostock at 37◦C,wash cells once with medium,and plate cells onto 10-cm culture dishes.Test for expression of bait protein 11.Lyse one well of each clone in 300μl lysis buffer and test for expression of the bait protein by immunoblotting (UNIT 10.10).SF-TAP proteins can be detected using the anti-FLAG M2antibody (Sigma-Aldrich)at a dilution of 1:1000to 1:5000in blocking reagent.SUPPORTPROTOCOL 2CHLOROFORM/METHANOL PRECIPITATION OF PROTEINS The chloroform/methanol precipitation method described by Wessel and Fl¨u gge (1984)precipitates proteins with high efficiency and yields samples containing low levels of salt contamination.Materials SF-TAP eluate (from Basic Protocol 1)Methanol (AR grade)Chloroform (AR grade)2-ml polypropylene sample tubes 1.Transfer 200μl SF-TAP eluate to a 2-ml sample tube.All steps are performed at ambient temperature.2.Add 0.8ml of methanol,vortex,and centrifuge for 20sec at 9000×g ,room temperature.3.Add 0.2ml chloroform,vortex,and centrifuge for 20sec at 9000×g ,room temperature.4.Add 0.6ml of deionized water,vortex for 5sec,and centrifuge for 1min at 9000×g ,room temperature.5.Carefully remove and discard the upper layer (aqueous phase).The protein precipitate (visible as white flocks)is in the interphase.6.Add 0.6ml of methanol,vortex,and centrifuge for 2min at 16,000×g ,room temperature.7.Carefully remove the supernatant and air dry the pellet.The pellet can be stored for several months at –80◦C.Identification of Protein Interactions 19.20.9Current Protocols in Protein Science Supplement 57BASIC PROTOCOL 2IN-SOLUTION DIGEST OF PROTEINS FOR MASS SPECTROMETRIC ANALYSISThe in-solution digest described here is a quick and efficient method to digest the SF-TAP eluate after protein precipitation (Support Protocol 2).The use of an MS-compatible surfactant helps to solubilize the precipitated proteins.In order to allow the identification of cysteine-containing peptides,random oxidation is prevented,rather than reverted,by applying a DTT/iodoacetamide treatment prior to digestion,leading to a defined-mass adduct.The digested protein sample can then be directly subjected to analysis on an LC-coupled tandem mass spectrometer.MaterialsPrecipitated protein (see Support Protocol 2)50mM ammonium bicarbonate (freshly prepared)RapiGest SF (Waters):prepare 2%(10×)stock solution in deionized water 100mM DTT (prepare from 500mM stock solution;store stock up to 6months at −20◦C)300mM iodoacetamide (prepare fresh)50×(0.5μg/μl)trypsin stock solution (Promega;store at −20◦C)Concentrated (37%)HCl60◦C incubatorPolypropylene inserts (Supelco,cat.no.24722)1to 200μl gel-loader pipet tips (Sorenson Bioscience,/contact.cfm )1.Dissolve the protein pellet in 30μl of 50mM ammonium bicarbonate by extensive vortexing.2.Add 3μl of 10×(2%)RapiGest stock solution (final concentration,0.2%).RapiGest (sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxyl]-1-propanesulfo-nate)is an acid-labile surfactant that helps to solubilize and denature proteins to make them accessible to proteolytic digestion (Yu et al.,2003).3.Add 1μl of 100mM DTT and vortex.4.Incubate 10min at 60◦C.5.Cool the samples to room temperature.6.Add 1μl of 300mM iodoacetamide and vortex.7.Incubate for 30min at room temperature.Samples should be protected from light,since iodoacetamide is light-sensitive.8.Add 2μl trypsin stock solution and vortex.9.Incubate at 37◦C overnight.10.Add 2μl of concentrated (37%)HCl to hydrolyze the RapiGest.For hydrolysis of the RapiGest reagent,the pH must be <2.11.Transfer samples to polypropylene inserts (remove spring).12.Incubate for 30min at room temperature.13.Place inserts in 1.5-ml microcentrifuge tubes and microcentrifuge 10min at 13,000×g ,room temperature.One hydrolysis product of the RapiGest reagent is water-immiscible and can be removed by centrifugation.After centrifugation,it is visible as faint film (oleic phase)on top of theStrep/FLAGTandem Affinity Purification (SF-TAP)19.20.10Supplement 57Current Protocols in Protein Science aqueous sample phase.The other hydrolysis product is an ionic water-soluble component which does not interfere with reversed phase LC or MS analysis.A white pellet might appear.14.Carefully recover the solution between the upper oleic phase and the pellet using gel-loader tips.The sample can now be directly subjected to C18HPLC separation prior to MS/MS-analysis (LC-MS/MS;Basic Protocol 4).Pre-fractionation (Basic Protocol 3)is optional.BASIC PROTOCOL 3PRE-FRACTIONATION VIA SDS-PAGE AND IN-GEL DIGESTION PRIOR TO LC-MS/MS ANALYSIS Pre-fractionation prior to MS analysis increases the number of peptides which can be an-alyzed,and therefore the peptide coverage of identified proteins.This benefit is achieved by overcoming the undersampling problem mainly caused by the limited capacity of the trapping columns used in nano–LC chromatography,or that occurs with high complexity.For these samples,SDS-PAGE pre-fractionation can be used to reduce the complexity.For less complex samples or samples with low protein content,the in-solution digest (Basic Protocol 2)is preferred.Materials Protein sample (e.g.,from Basic Protocol 1or Support Protocol 2)10%NuPAGE gels (Invitrogen)MOPS running buffer (Invitrogen)40%and 100%acetonitrile (AR grade;prepare fresh)5mM DTT (prepare from 500mM stock;store stock up to 6months at −20◦C)25mM iodoacetamide (prepare fresh)Digestion solution:dilute 50×trypsin stock solution (0.5μg/μl,Promega)1:50in 50mM ammonium bicarbonate (freshly prepared)1%and 0.5%(v/v)trifluoroacetic acid (TFA;prepare fresh from 10%v/v stock)50%(v/v)acetonitrile/0.5%(v/v)TFA (prepare fresh)99.5%(v/v)acetonitrile/0.5%(v/v)TFA (prepare fresh)2%(v/v)acetonitrile/0.5%(v/v)TFA Concentration units (e.g.,Microcon from Millipore)Scalpel Polypropylene 96-well microtiter plate:polystyrene material should be avoided since,depending on the product,polymers can be extracted from plastics which produce strong background signals in mass spectrometry 60◦C incubator or heating block Polypropylene 0.5-ml reaction tubes Microtiter plate shaker (e.g.,V ortex mixer equipped with microtiter-plate adaptor)HPLC sample tubes Additional reagents and equipment for SDS-PAGE (UNIT 10.1)and colloidal Coomassie blue staining of gels (Support Protocol 3)Prepare samples 1.Concentrate samples using concentration units (e.g.,Microcon).2.Supplement samples with Laemmli loading buffer (SDS-PAGE loading buffer;UNIT 10.1).A detailed description of the SDS gel electrophoresis and standard buffers can be found in UNIT 10.1or in the protocols supplied with the NuPAGE system.Identification of ProteinInteractions19.20.11Perform electrophoresis and stain gels3.Separate samples on 10%NuPAGE gels according to the manufacturer’s protocols,using MOPS running buffer.4.Stop electrophoresis after the gel front has travelled 1to 2cm.5.Stain gels with colloidal Coomassie blue (see Support Protocol 3).Avoid strong staining of the bands since it increases the time necessary for destaining.6.Excise desired gel pieces with a clean scalpel (three to ten slices,depending on the complexity of the sample).Destain and process gel slices7.Transfer gel pieces into individual wells of a 96-well plate.8.Wash by adding 100μl water to each well and incubating for 30min.9.For destaining:a.Wash twice,each time by incubating the gel slices for 10min in 100μl/well of 40%acetonitrile.b.Wash for 5min in 100μl/well of 100%acetonitrile (if gels are still blue,repeat de-staining).10.Add 100μl of 5mM DTT,then incubate 15min at 60◦C in an incubator or heatingblock.11.Remove DTT solution and cool the plate to room temperature.12.Add 100μl per well of freshly prepared 25mM iodoacetamide,then incubate 30minin the dark.13.Wash twice,each time for 10min with 100μl/well of 40%acetonitrile.14.Wash 5min with 100μl/well of 100%acetonitrile.15.Discard supernatant and air dry (or SpeedVac)the gel pieces to complete dryness.Digest and extract gel slices16.Add 20to 30μl per well of freshly prepared digestion solution (depending on the sizeof the gel plugs).Wrap plates in Parafilm to reduce evaporation during the overnight incubation (or use a humidified incubator in step 17).17.Digest overnight at 37◦C.18.For extraction of the peptides from the gel piece,add 10μl 1%TFA,then shake15min on a V ortex mixer with a microtiter plate adapter.The peptides are extracted in three steps with increasing acetonitrile concentrations (steps 18to 23).19.Transfer liquid (extract 1)to a 0.5-ml polypropylene tube.20.Add 50μl 50%acetonitrile/0.5%TFA to the gel piece and shake 15min on a V ortexmixer with a microtiter plate adapter.21.Remove the liquid (extract 2)and pool extracts 1and 2.22.Add 50μl 99.5%acetonitrile/0.5%TFA to the gel piece,then shake 15min on aV ortex mixer with a microtiter plate adapter.23.Remove the liquid (extract 3)and pool extract 3with 1and 2.Strep/FLAG Tandem AffinityPurification(SF-TAP)19.20.1224.Dry samples to complete dryness in a SpeedVac evaporator.25.Redissolve samples in50μl of2%acetonitrile/0.5%TFA by shaking(e.g.,on aV ortex mixer)for10to15min,then transfer the sample into HPLC sample tubes for LC-MS/MS analysis.SUPPORT PROTOCOL3QUICK MS-COMPATIBLE COLLOIDAL COOMASSIE STAIN OF PROTEINS AFTER SDS-PAGE SEPARATIONThe colloidal Coomassie stain(Kang et al.,2002)represents a fast and sensitive MS-compatible protein staining method.In contrast to the classical staining protocol,no intense and time-consuming destaining is needed to visualize protein bands.Therefore, this method is ideal for a quick staining of the protein bands and provides good orientation on how the gel can be fractionated without splitting predominant bands(see Basic Protocol3).MaterialsElectrophoresed SDS gel containing protein samples of interest(e.g.,from Basic Protocol3)Colloidal Coomassie staining solution(see recipe)Destaining solution:10%(v/v)ethanol/2%(v/v)orthophosphoric acidGel staining trays of appropriate size1.Wash gels twice,each time for10min in deionized water in a staining tray.The SDS must be removed before staining to reduce background signals.2.Incubate gels for10min in colloidal Coomassie staining solution.The incubation steps are kept short for the staining of gels used for pre-fractionation.The staining can be prolonged up to overnight.The maximum staining will be reached after ∼3hr incubation in the staining solution.3.Incubate gels for10min in destaining solution.4.Wash gels twice,each time for10min in deionized water.BASIC PROTOCOL4LC-MS/MS ANALYSIS OF DIGESTED SF-TAP SAMPLESThe following protocol describes MS analysis of digested protein samples on an LC-coupled ESI tandem mass spectrometer.The representative MS-analysis protocol is provided for an Orbitrap mass spectrometer(Thermo Fisher Scientific).The Orbitrap system combines fast data acquisition with high mass accuracy and is therefore ideal for the analysis of SF-TAP samples.Background information on mass spectrometric analysis can be found in UNIT16.11.MaterialsDigested protein sample,either from in-solution digest(Basic Protocol2)or in-gel digest(Basic Protocol3)Nano HPLC loading buffer:0.1%formic acid in HPLC-grade waterNano HPLC buffer A:2%acetonitrile/0.1%formic acid in HPLC-grade waterNano HPLC buffer B:80%acetonitrile/0.1%formic acid in HPLC-grade water HPLC vials(Dionex)Nano HPLC system(UltiMate3000,Dionex)equipped with a trap column (100μm i.d.×2cm,packed with Acclaim PepMap100C18resin,5μm,100◦A;Dionex)and an analytical column(75μm i.d.×15cm,packed with AcclaimPepMap100C18resin,3μm,100◦A;Dionex)Mass spectrometer:Oritrap XL with a nanospray ion source(ThermoFisher Scientific;also see UNIT16.11)。

IL-10通过上调socs3抑制衣原体感染细胞炎症因子表达罗玲艳杜昆（长江大学附属第一医院输血科，荆州 434000）中图分类号R374+1 文献标志码 A 文章编号1000-484X（2024）03-0530-04［摘要］目的：研究SOCS3蛋白在IL-10抑制衣原体感染细胞炎症因子表达中的作用及其机制。

方法：采用Western blot技术检测IL-10对衣原体感染细胞STAT3蛋白活化的影响，以及在socs3 siRNA作用下p38及ERK1/2活化情况；RT-PCR技术检测衣原体感染细胞在IL-10或Stattic作用下socs3基因表达情况；ELISA检测衣原体感染细胞在socs3 siRNA作用下，炎症因子IL-6和IL-12产生情况。

结果：IL-10可以通过激活STAT3蛋白上调socs3基因表达；衣原体能诱导IL-6及IL-12产生，但IL-10可以通过上调socs3基因表达抑制IL-6及IL-12产生；SOCS3蛋白能抑制p38和ERK1/2通路活化。

结论：IL-10通过诱导SOCS3蛋白抑制p38和ERK1/2通路活化，从而抑制炎症因子的产生。

［关键词］沙眼衣原体；炎症因子；白细胞介素10；细胞因子信号转导抑制因子IL-10 inhibits expression of inflammatory factors in Chlamydia-infected cells by up-regulating socs3LUO Lingyan， DU Kun. Department of Blood Transfusion， the First Affiliated Hospital of Yangtze University， Jing⁃zhou 434000， China［Abstract］Objective：To investigate the role and mechanisms of SOCS3 protein in IL-10 inhibiting the expression of inflam‑matory factors in Chlamydia-infected cells. Methods：The activation of STAT3 protein were examined in Chlamydia-infected cells by Western blot， and the activation of p38 and ERK1/2 were also examined in infected cells treated with socs3 siRNA. The expression of socs3 gene was examined in Chlamydia-infected cells treated with IL-10 or Stattic by RT-PCR. IL-6 and IL-12 were measured in infect‑ed cells treated with socs3 siRNA using ELISA kits. Results：Socs3 expression was up-regulated by IL-10 through activation of STAT3 protein. IL-6 and IL-12 induced by Chlamydia were down-regulated by IL-10 through induction of socs3. The activation of p38 and ERK1/2 signalling pathways were inhibited by SOCS3. Conclusion：IL-10 inhibits the production of pro-inflammatory cytokines through induction of SOCS3 and inhibition p38 and ERK1/2 signalling pathways.［Key words］Chlamydia trachomatis；Inflammatory factors；IL-10；SOCS衣原体是一种全球普遍流行的性传播疾病的病原体，常常感染生殖道、肛门直肠和口咽部的黏膜上皮细胞。

【lncRNA在转录水平上的调控】一、概述lncRNA（long non-coding RNA）是一类长度超过200个核苷酸的非编码RNA分子，近年来被广泛研究。

它们在转录水平上的调控作用备受关注，对基因表达调控、细胞命运决定和疾病发生发展等具有重要作用。

本文将从深度和广度两个方面对lncRNA在转录水平上的调控进行全面评估。

二、lncRNA的功能和作用机制1. 长链非编码RNA的分类lncRNA可根据其位置和功能分为多种类型，如染色质修饰相关lncRNA、转录调控lncRNA和mRNA的调控lncRNA等。

2. 转录水平上的调控机制lncRNA通过多种机制调控转录过程，包括染色质重塑、转录抑制和mRNA剪接等。

XIST（X-inactive specific transcript）是一种能够在X染色体失活中发挥关键作用的lncRNA，通过与染色质相互作用实现对基因的沉默。

三、lncRNA在细胞命运决定中的作用1. 干细胞命运决定lncRNA参与调控干细胞的自我更新、增殖和分化等过程，通过转录水平上的调控影响干细胞的命运。

2. 细胞凋亡和增殖一些lncRNA在调控细胞的凋亡和增殖中发挥重要作用，通过影响转录水平上的基因表达，调控细胞的命运选择。

四、lncRNA在疾病中的作用1. 癌症lncRNA在多种癌症的发生和发展中发挥重要作用，通过调控转录水平上的基因表达，影响癌细胞的增殖、凋亡和侵袭等功能。

2. 其他疾病lncRNA还参与调控多种其他疾病的发生和发展，如心血管疾病、神经系统疾病等，展现出广泛的生物学功能。

五、结语总结回顾，lncRNA在转录水平上的调控作用十分广泛，涉及干细胞命运决定、细胞凋亡和增殖以及各种疾病的发生和发展。

通过深入研究lncRNA的功能和作用机制，有望为解决许多疾病和进化等生物学问题提供新思路和方法。

个人观点：lncRNA在转录水平上的调控作用是一个前沿研究领域，其广泛的功能和作用机制为我们提供了更多认识基因表达调控的可能性。