CN109536610B - Setdb1在调控胆固醇途径中的应用 - Google Patents
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Abstract
本发明公开了SETDB1在调控胆固醇途径中的应用，下调SETDB1表达可下调胆固醇含量和下调胆固醇途径相关基因的表达，过表达SETDB1可以增加神经母细胞瘤软琼脂克隆形成能力中和胆固醇途径相关基因的表达，SREBF2是Cholesterol途径的转录激活因子，过表达SREBF2能显著促进Cholesterol途径相关基因的表达，表明SETDB1调控胆固醇代谢途径，可作为胆固醇相关疾病的诊断和治疗靶点。
Description
技术领域
本发明涉及生物医学领域，具体涉及SETDB1在调控胆固醇途径中的应用。
背景技术
神经母细胞瘤是儿童常见的颅外实体肿瘤之一，约占儿童肿瘤的6-10％，年发病率大约为1/10万，仅低于白血病和中枢神经系统肿瘤，占儿童癌症死亡的15％。神经母细胞瘤临床表现为恶性程度高、异质性高、易转移、易复发以及预后差等特点，因此其治疗比较困难，迫切需要新的途径和新方法来治疗。
肿瘤细胞表观遗传学是当前研究热点之一，诸多实验证据证实组蛋白甲基化对肿瘤的发生发展有着十分重要的影响。表观遗传经常导致癌症中的肿瘤抑制基因的沉默，为癌症生物学中一个关键的调节作用。组蛋白修饰主要有甲基化，乙酰化，磷酸化等多种形式。组蛋白甲基化转移酶SETDB1,其功能是常染色质主要的H3K9三催化甲基化和二甲基化的甲基化转移酶，在多个生物过程中都具有重要的作用。
选择性的改变新陈代谢以满足生物合成和细胞生长和增殖的能量需求是肿瘤细胞的显著特征，胆固醇合成途径是肿瘤维持生长增殖所必须，以胆固醇合成通路作为研究对象在肿瘤防治中具有重要的科学价值。胆固醇广泛分布于各种细胞与组织，在细胞的正常生命活动中发挥重要的生理生化功能。众所周知，肿瘤细胞代谢重编程不仅改变了细胞命运，还改变了细胞的代谢水平：如有氧糖酵解取代了正常的有氧呼吸和糖酵解途径，脂肪酸和胆固醇合成途径增强等，从而满足肿瘤细胞生长和增殖的合成代谢需要。胆固醇的摄取和合成大大增加的状况在多种肿瘤中被证实。因此以肿瘤胆固醇代谢为靶点研究具有重要的理论和现实意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种下调SETDB1表达或下调SETDB1表达的试剂在制备下调胆固醇含量的药物中的应用；本发明的目的之二在于提供下调SETDB1表达或下调SETDB1表达的试剂在制备降低胆固醇途径相关基因表达量的药物中的应用；本发明的目的之三在于提供上调SETDB1表达或上调SETDB1表达的试剂在制备增加神经母细胞瘤软琼脂克隆形成能力的药物中的应用；本发明的目的之四在于提供上调SETDB1表达或上调SETDB1表达的试剂在制备上调胆固醇途径相关基因表达量的药物中的应用；本发明的目的之五在于提供下调SREBF2表达或下调SREBF2表达的试剂在制备抑制胆固醇途径相关基因表达量的药物中的应用；本发明的目的之六在于提供活性炭/葡聚糖处理胎牛血清在作为神经母细胞瘤胆固醇诱导剂中的应用。
为达到上述目的，本发明提供如下技术方案：
1、下调SETDB1表达或下调SETDB1表达的试剂在下调胆固醇含量中的应用。
2、下调SETDB1表达或下调SETDB1表达的试剂在降低胆固醇途径相关基因表达量中的应用。
优选的，所述胆固醇途径相关基因为ACAT2、HMGCR、HMGCS1、SREBF2或SREBF1。
3、上调SETDB1表达或上调SETDB1表达的试剂在增加神经母细胞瘤软琼脂克隆形成能力中的应用。
4、上调SETDB1表达或上调SETDB1表达的试剂在上调胆固醇途径相关基因表达量中的应用，所述胆固醇途径相关基因为ACAT2、HMGCR、HMGCS1、SREBF2或SREBF1。
5、下调SREBF2表达或下调SREBF2表达的试剂在抑制胆固醇途径相关基因表达量中的应用。
优选的，所述胆固醇途径相关基因为ACAT2、HMGCR、HMGCS1、SREBF2或SREBF1。
6、活性炭/葡聚糖处理胎牛血清在作为神经母细胞瘤胆固醇诱导剂中的应用。
此外，SETDB1还可以诊断胆固醇含量的靶点，其通过检测SETDB1表达可以检测胆固醇的含量。
本发明的有益效果在于：本发明首次公开了SETDB1参与调控胆固醇途径，下调SETDB1表达可下调胆固醇含量和下调胆固醇途径相关基因的表达，过表达SETDB1可以增加神经母细胞瘤软琼脂克隆形成能力中的应用和胆固醇途径相关基因的表达，SREBF2是Cholesterol途径的转录激活因子，过表达SREBF2能显著促进Cholesterol途径相关基因的表达，因此可以通过调控或检测SETDB1调控胆固醇代谢途径或诊断胆固醇相关疾病。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为下调SETDB1抑制神经母细胞瘤的Cholesterol途径基因表达情况(A：BE2-C细胞下调SETDB1表达后Cholesterol途径相关基因表达；B：KCNR细胞下调SETDB1表达后Cholesterol途径相关基因表达；C：SHEP1细胞下调SETDB1表达后Cholesterol途径相关基因表达；D：FilipinIII阳性标记检测下调SETDB1对胆固醇的影响)。
图2为上调SETDB1激活神经母细胞瘤的增殖、软琼脂克隆形成能力和Cholesterol途径基因表达情况(A：上调SETDB1神经母细胞瘤的增殖结果；B：上调SETDB1软琼脂克隆形成能力；C：上调SETDB1表达Cholesterol途径基因表达情况)。
图3为SETDB1对神经母细胞瘤相应Charcoal Stripped FBS的作用(A：BE2-C和KCNR细胞中固醇途径相关基因显著上调；B：BE2-C和KCNR细胞中SETDB1表达；C：KCNR细胞下调SETDB1之后再进行Charcoal Stripped FBS处理固醇途径相关基因表达情况)。
图4为下调SETDB1对SREBF2调控神经母细胞瘤Cholesterol途径有显著的抑制作用(A-B:过表达SREBF2能显著增加Cholesterol途径ACAT2、HMGCR、HMGCS1、SREBF2、SREBF1等基因的表达；C-D：下调SETDB1能显著抵制SREBF2引起的Cholesterol途径的激活)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明实施例中SETDB1shRNA载体构建方法如下：
根据RNA干扰序列设计原则，针对目的基因的靶序列，利用Sigma在线设计4条RNA干扰靶序列，由华大基因合成干扰序列DNA Oligio,退火后形成两端含有酶切位点AgeI和EcoRI的粘性末端，和AgeI和EcoRI双酶切的慢病毒载体pLKO.1-puro连接过夜，转化大肠杆菌感受态细胞，挑选抗性菌落并进行用NcoI和EcoRI酶切鉴定，将酶切验证后的重组子测序并进行序列比对。具体步骤如下：
(1)在http://www.sigmaaldrich.com/china-mainland/zh/life-science/functional-genomics-and-rnai/shrna/individual-genes.html网站输入SETDB1(GeneID:9869)，查找相应的靶序列，利用addgene上pLKO.1的protocol设计合成DNA序列。基本原则是：
正向寡核苷酸：5’CCGG-21个碱基(靶序列)-CTCGAG(loop)-21个反向互补碱基序列-TTTTTG 3’；
反向寡核苷酸(Reverse oligo)：5’AATTCAAAAA-21个碱基(靶序列)-CTCGAG(loop)-21个反向互补碱基序列3’，如表1所示：
表1、SETDB1干扰序列设计原则
(2)根据上面的设计原则及靶序列，由华大基因合成的DNA片段如下：
表2、SETDB1干扰合成序列
将合成好的Oligo溶解，终浓度20μM(高压灭菌水溶解核酸，水的体积＝(合成得到的核酸nmol数×50)μL)，按以下反应体系在1.5ml微型离心管内配制反应。水浴锅加热至95-100℃，将EP管置入水浴锅，关闭水浴锅加热开关，缓慢降温至室温，该过程大约需要3-4小时。
(3)用AgeI和EcoRI酶切pLKO.1载体，用琼脂糖凝胶纯化回收试剂盒纯化酶切产物，30μL H2O洗脱回收5000bp左右大片段。
(4)将退火产物和载体酶切片段进行连接，连接反应，转化大肠杆菌，涂于氨苄抗性平板，每个平板挑选选3-5个克隆，进行酶切鉴定。将酶切验证后的载体进行测序验证。
过表达载体构建方法如下：取pCDH-CMV-MCS-EF1-Puro载体，通过设计含有XbalI和NotI的酶切位点的引物，克隆SETDB1的ORF片段，然后连接到经酶切以后的pCDH-CMV-MCS-EF1-Puro载体，连接后得到表达SETDB1的(pCDH-CMV-MCS-EF1-Puro-SETDB1)重组载体，经过酶切测序验证得到重组子，具体步骤如下：
(1)SETDB1cDNA的克隆
在NCBI中查找人类SETDB1基因的mRNA序列(NM_012432)，设计扩增CDS序列引物，然后以正常的人成纤维细胞cDNA为模板，扩增出人类全长SETDB1序列。引物如下：
SETDB1-F-XbalI:5’-gctctagaatgtcttcccttcctgggtgc-3’(SEQ ID NO.13)
SETDB1-R-NotI:5’-ataagaatgcggccgcctaaagaagacgtcctctgca-3’(SEQ IDNO.14)，采用Touchdown PCR方法进行扩增，
PCR反应条件如下：
将PCR产物用0.8％的琼脂糖凝胶电泳，检测是否扩增出目的片段。
(2)PCR产物T载体连接
PCR产物回收按照胶回收试剂盒说明书进行，按照全式金T-easy Zero载体说明书，将目的片段与T载体连接，37℃连接1～2h。
(3)目的片段与载体的转化
①将感受态细胞在冰上放置5-10min；
②加入5μl连接产物，冰上静止30min；
③42℃金属浴热击75s，冰上静止5min；
④加入400μl LB液体培养基(不含抗生素)，37℃，200rpm摇荡培养lh；
⑤取100μl的菌液，均匀涂布于平板上，37℃培养1h后，倒置培养12-16h。
(4)阳性克隆的筛选、验证和测序
取出37℃恒温培养箱中的平板，用白色10μL枪头挑取单菌落于500μl含有氨苄青霉素的LB培养基中，37℃恒温摇床培养8h。之后进行菌液电泳和菌液PCR，琼脂糖电泳检测，若菌液电泳条带滞后，菌液PCR产物大小与目的片段相符合，即推断该单菌落为阳性克隆，并送出华大基因测序。
实施例1、下调SETDB1抑制神经母细胞瘤的Cholesterol的途径基因表达和Cholesterol的含量
将神经母细胞瘤细胞BE2-C培养于含10％的胎牛血清(FBS)的DMEM/F12培养基中，和SHEP1细胞培养于含10％的胎牛血清(FBS)的DMEM培养基中，KCNR培养于含10％的胎牛血清(FBS)的RPMI 1640培养基。所有细胞中都加入1％的青霉素和链霉素，待细胞生长密度达到80-90％时可进行传代培养。
活性炭过滤血清的处理：将细胞于正常培养基条件下培养，待细胞密度为70％左右，更换培养基，选用活性炭过滤的血清代替正常的胎牛血清，于SETDB1shRNA载体转化后的8h、12h、24h取样，并检测Cholesterol途径相关基因ACAT2、HMGCR、HMGCS1、SREBF2、SREBF1基因的表达，结果如图1所示。结果显示，通过下调在神经母细胞瘤SETDB1的表达谱分析发现，SETDB1可以显著抑制固醇类代谢途径，具体表现为下调SETDB1显著降低Cholesterol途径相关基因ACAT2、HMGCR、HMGCS1、SREBF2、SREBF1基因的表达；说明下调SETDB1对Cholesterol途径有抑制作用。
FilipinIII可以用于标记生物膜上及非细胞结构内的自由胆固醇，具体如下：将贴壁细胞用PBS洗涤细胞三次，室温下用4％多聚甲醛固定30min，PBS洗涤细胞三次以后，使用1ml 1.5mg/ml甘氨酸培养10min，再用PBS洗涤细胞三次，室温下用50μg/ml FilipinIII染液给细胞染色2h，PBS清洗后，用荧光显微镜观察。研究结果发现下调SETDB1能显著降低FilipinIII的阳性标记，说明下调SETDB1能降低胆固醇的含量。
实施例2、SETDB1对神经母细胞瘤的Cholesterol途径的影响
为了进一步研究SETDB1对Cholesterol途径的影响，采用慢病毒包装系统采用pLP1，pLP2，pLP/VSVG(Invitrogene)三质粒包装系统进行说明书进行慢病毒的包装慢病毒载体pCDH-CMV-MCS-EF1-Puro-SETDB1及感染BE2-C和SHEP1细胞，对神经母细胞瘤进行过表达SETDB1，结果如图2所示。结果显示，过表达SETDB1不会显著增加神经母细胞瘤的增殖(图2，A)，但是能显著增加软琼脂克隆形成能力(图2，B)。通过对Cholestero途径相关基因进行检测，发现上调SETDB1能显著增加ACAT2、HMGCR、HMGCS1、SREBF2、SREBF1基因的表达(图2，C)。
实施例3、SETDB1在Charcoal Stripped FBS处理后神经母细胞瘤响应Cholesterol途径
早期研究表明，当用Charcoal处理后的FBS去培养细胞，由于缺乏固醇等脂类物质，导致细胞中的脂肪酸代谢途径显著增加，相应的胆固醇途径也被激活。本研究中，用Charcoal处理后的FBS去培养细胞，在8h、24h、48h取样检测，结果如图3所示。结果显示，在BE2-C和KCNR细胞中均发现固醇途径相关基因显著上调(图3，A)。同时在此过程中SETDB1的表达也显著升高，说明SETDB1可以被Cholesterol物质缺乏的培养基激活(图3，B)，暗示着SETDB1与Cholesterol有重要的联系。
为了明确SETDB1在Cholesterol途径中的作用，对KCNR细胞下调SETDB1之后再进行Charcoal Stripped FBS(活性炭/葡聚糖处理胎牛血清)处理。结果显示，下调SETDB1之后Charcoal Stripped FBS处理不能显著激活神经母细胞瘤的Cholesterol途径(图3，C)。说明SETDB1对神经母细胞瘤Charcoal StrippedFBS处理后的Cholesterol途径的激活扮演着不可缺少的地位。
实施例4、下调SETDB1对SREBF2调控神经母细胞瘤Cholesterol途径的作用
SREBF2是Cholesterol途径的转录激活因子，在BE2-C细胞和SHEP1细胞过表达SREBF2，然后检测过表达SREBF2在BE2-C细胞和SHEP1细胞对Cholesterol途径相关基因的表达。研究中发现，过表达SREBF2能显著增加Cholesterol途径ACAT2、HMGCR、HMGCS1、SREBF2、SREBF1等基因的表达(图4，A-B)。在KCNR细胞核293FT细胞下调SETDB1表达，结果显示下调SETDB1表达能显著抵制SREBF2引起的Cholesterol途径的激活(图4，C-D)，进一步说明了SETDB1对SREBF2引起的Cholesterol途径的激活有显著的影响。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
富恩生物技术（成都）有限公司
<120> SETDB1在调控胆固醇途径中的应用
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Claims (3)
1.下调SETDB1表达的试剂在制备下调胆固醇含量的药物中的应用。
2.下调SETDB1表达的试剂在制备降低胆固醇途径相关基因表达量的药物中的应用，其特征在于：所述胆固醇途径相关基因为ACAT2、HMGCR、HMGCS1、SREBF2或SREBF1。
3.上调SETDB1表达的试剂在制备上调胆固醇途径相关基因表达量的药物中的应用，其特征在于：所述胆固醇途径相关基因为ACAT2、HMGCR、HMGCS1、SREBF2或SREBF1。
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