CN116430048A - BTF3L4基因及其RNAi干扰系统的应用 - Google Patents
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Abstract
本发明公开了BTF3L4基因及其RNAi干扰系统的应用,涉及生物医药技术领域。本发明的BTF3L4基因和BTF3L4基因的RNAi干扰系统用于制备治疗胶质瘤的药物,该RNAi干扰系统可高效敲低胶质瘤细胞中BTF3L4基因/蛋白,抑制癌细胞的侵袭与迁移,操作简单,效率高;本发明还公开了将BTF3L4基因/蛋白作为精准治疗的靶点,制备用于胶质瘤等高表达BTF3L4基因/蛋白的诊断或预后判断的试剂盒,也用于制备靶向药物。
Description
技术领域
本发明属于生物医药技术领域,更具体地说,涉及BTF3L4基因(基因ID:91408和蛋白ID:Q96K17)及其RNAi干扰系统在制备用于治疗胶质瘤的药物中的应用。
背景技术
胶质瘤是全球最常见的脑部恶性肿瘤之一,确诊一年内死亡率接近80%,即使经过靶向治疗,患者预后仍不理想。我国脑胶质瘤年发病率为5~8/10万,患者5年生存率低于5%,其5年病死率仅次于胰腺癌和肺癌。近年,全球恶性肿瘤发病率呈持续升高趋势下,给社会经济带来严重的负担。
虽然胶质瘤分子病理学诊断的进展已经明确了多种分子病理学靶点,但针对这些分子突变的临床治疗仍没有显著效果。目前普遍认为,胶质瘤微环境由不同种类的细胞组成,通过多种机制逃避免疫系统监测和靶向治疗。最近的研究也表明,脑胶质瘤对治疗的反应发生了显著的变化,多种类型的分子调控异常在原发性胶质瘤中引起广泛的肿瘤间和肿瘤内异质性,因此,临床诊断和治疗生物标志物的开发对脑胶质瘤患者提升的生存机会至关重要。随着二代、三代测序、单细胞测序、蛋白组学,代谢组学等高通量技术的应用,靶向治疗已成为除手术、放疗、化疗之外的治疗恶性肿瘤的新方法,靶向治疗通过干预肿瘤的发生、进展所需的特定靶向分子来阻止癌细胞的生长,特异性强、疗效明显、不良反应小,但靶向治疗不可避免的需要我们寻找特异性的分子标记物作为治疗靶点,脑胶质瘤的发展是一个多基因参与的过程,存在众多潜在的治疗靶点,目前,还没有明确效果的胶质瘤诊断试剂盒上市,因而,迫切需要研发针对有效靶点的检测试剂及针对性药物。
碱性转录因子3样蛋白4(BTF3L4)是一种新的软骨形成相关蛋白,最初被报道为一种促进软骨形成的转录因子。转录因子(Transcription factor,TF)是一类与基因特定序列专一性结合,从而调控目的基因以特定强度在特定的时间与空间表达的蛋白质分子,作为基因表达调控的重要组成部分,TFs广泛参与体内生理与病理过程。转录因子在癌症等疾病中具有关键的生物学作用,因此它被认为是极具潜力的治疗靶点。据估计,人类基因组中至少有1600个转录因子,其中约19%与包括癌症在内的多种疾病密切相关,鉴于TFs对靶基因的直接调控作用,它们具有更高特异性的疾病调节能力,因而,转录因子可作为新的治疗靶点和脑胶质瘤进展的驱动因素。同时,BTF3L4也是一种致癌基因,可以通过提高核迁移率促进甲状腺肿瘤、结直肠癌的生长和转移,也可以抑制胃癌细胞生长。此外,在中枢神经系统中,BTF3L4是神经元形态所必需的基因,参与了脑恶性肿瘤的形态构成,然而,BTF3L4与胶质瘤的关系目前还不明确,其在胶质瘤中的表达和具体调节机制也还不清楚。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供BTF3L4基因及其RNAi干扰系统的应用,用于制备治疗胶质瘤的药物。
为了解决上述技术问题,本发明所采用的技术方案如下:
BTF3L4基因在制备用于治疗胶质瘤的药物中的应用。
进一步地,所述的治疗胶质瘤为抑制细胞的迁移。
进一步地,所述的治疗胶质瘤为抑制细胞的侵袭。
用于检测肿瘤组织中BTF3L4蛋白表达量的生物标志物为BTF3L4基因。
用于检测肿瘤组织中BTF3L4蛋白表达量的试剂盒含有BTF3L4基因。
BTF3L4基因的RNAi干扰系统在制备用于治疗胶质瘤的药物中的应用,所述的BTF3L4基因的RNAi干扰系统的序列如下:
BTF3L4-sh1:5′-CCTGATGTTACAGTTTGGTAGATTT-3′,
BTF3L4-sh2:5′-CAAACTGGAATAGCTAGCATGTGCT-3′。
进一步地,所述的BTF3L4基因的RNAi干扰系统为敲低胶质瘤组织细胞中BTF3L4基因。
进一步地,所述的BTF3L4基因的RNAi干扰系统为抑制胶质瘤组织细胞中BTF3L4蛋白表达。
相比于现有技术,本发明的有益效果为:
1)本发明RNAi干扰可高效敲低胃癌细胞中BTF3L4基因/蛋白,抑制胶质瘤组织细胞的侵袭与迁移,该系统操作简单,效率高。
2)BTF3L4基因/蛋白是精准治疗的一个靶点,制备用于胶质瘤等高表达BTF3L4基因/蛋白的诊断或预后判断的试剂盒,也用于制备靶向药物。
附图说明
图1为多重免疫荧光定量分析BTF3L4在脑胶质瘤及对照非瘤脑组织芯片中的差异表达情况图;
图2为免疫荧光检测BTF3L4在胶质瘤及对照非瘤脑组织中的表达情况图(A、A1为BTF3L4在胶质瘤组织中的荧光染色图;B、B1为BTF3L4在非瘤脑组织中的免疫荧光染色图)(标尺=50um);
图3为BTF3L4蛋白差异表达与胶质瘤患者预后的关系图;
图4为Western Blot检测BTF3L4在6对手术切除胶质瘤组织及癌旁组织中的表达情况及定量分析图(A)与BTF3L4在U87mg细胞、U251细胞、SHG44细胞及T98G细胞的情况及定量分析图(B);
图5为干扰BTF3L4基因后U251细胞中蛋白表达及定量分析情况图;
图6为干扰BTF3L4基因后对胶质瘤细胞的迁移影响图;
图7为干扰BTF3L4基因后对胶质瘤细胞的侵袭影响图;
图8为干扰BTF3L4基因后对胶质瘤细胞周期的影响图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例对本发明进一步进行描述。以下实施例中如无特殊说明,所用的技术手段均为本领域技术人员所熟知的常规手段。
从南通大学附属医院收集2012.3-2018.3年间胶质瘤组织样本,其中新鲜冰冻胶质瘤组织197例,对照非瘤脑组织28例。这些组织样本均是由福尔马林固定,石蜡包埋,并根据最新的WHO诊断标准分级。所有的病例均是由两名病理学专家进行病理组织学确定,患者术前没有接受过免疫治疗、化疗或放疗,临床病例资料详细完整。
以下实施例中使用的主要试剂为:
Opal7色免疫组化试剂盒:美国Perkin Elmer公司。
兔抗人BTF3L4单克隆抗体:Biorbyt公司。
辣根过氧化物酶标记鼠兔二抗(免疫荧光实验用):美国Perkin Elmer公司。
抗体稀释液/封闭液:美国AKOYA公司。
AR6修复液:美国AKOYA公司。
胶质瘤细胞株购自中科院上海细胞库。
DMEM培养基、胎牛血清:美国Gibco公司。
BCA蛋白测定试剂盒:碧云天生物技术公司。
PVDP膜:Merk-Millipore公司。
GAPDH抗体:美国Proteintech公司。
ECL显影液:Vazyme公司。
细胞冻存液:新赛美公司。
DMEM完全培养液:分别加入DMEM与胎牛血清,配成终浓度为含有10%FBS的完全培养基,4℃保存。
1×TBST 1L:取Tris 2.42g、NaCl8.0g、Tween-200.5mL,混合溶解,定容至1L,常温保存。
1×转膜Buffer1L:甘氨酸14.4g、Tris3.03g,加适量双蒸水搅拌溶解,再加200mL无水甲醇,定容至1L,混合均匀(用时配制)。
封闭液100mL:取脱脂奶粉5g,加入100mL 1×TBS,混合溶解即可(需用时配制)。
洗涤液PBST:NaCl 0.8g,KH2PO4 0.02g,Na2HPO4·12H2O 0.29g,KCl 0.02g,Tween20 0.05mL,叠氮钠0.01g,加双蒸水至100mL,调至pH7.4。
蛋白质裂解试剂:1mL解试液中加入10μL蛋白酶抑制剂混合液,5μL PMSF和5μL磷酸酶抑制剂。
以下实施例中使用的主要仪器如下:
组织芯片制作仪:韩国Quick Ray(UNITMA)公司;倒置显微镜:日本Olympus公司;凝胶成像系统:中国天能公司;多功能酶标仪:美国Thermo公司;多光谱病理扫描系统:美国Perkin Elmer公司。
实施例1
1、制作组织芯片
1)病理组织切片
取手术切除新鲜组织块(厚度0.5cm),放入预先配好的10%福尔马林溶液固定,随后,梯度浓度酒精脱水至二甲苯透明;将透明的组织块置于溶化的石蜡中,待石蜡完全浸入组织块后进行包埋;冷却凝固后连续切片(厚度5-8um),随后置于45℃恒温箱中烘干。
2)HE染色
将水化后的切片放入苏木精水溶液中染色数分钟;盐酸酒精及氨水分色;流水冲洗1小时后,置入蒸馏水;随后,在70%,75%,90%酒精中梯度脱水各5分钟;放入伊红染色液染色2-3分钟;染色后的切片经梯度酒精脱水,再经二甲苯使切片透明,滴上中性树胶,盖上盖玻片封固后,在显微镜下观察,确定肿瘤区域,在供体病理组织蜡块上选取有代表性的癌巢区域做好标记。
3)胶质瘤组织芯片制作
1∶1混合石蜡与蜂蜡,制作空白受体蜡块;在蜡块上设计10×7孔,共350点组织阵列,然后用组织芯片仪制成TMA空白蜡块;在标记的供体蜡块上选取最有代表性的肿瘤区域,取直径2mm的组织块,每例各取1个芯;将取好的组织芯转移到受体蜡块的孔中,并取相应非瘤脑组织作为对照;组织阵列块在55℃的恒温烤箱中加热融合10分钟,在快融化之前放至室温冷却,使受体蜡块与供体组织融为一体;将组织芯片置于4℃条件下冷冻4小时左右,随后用全自动组织切片机对组织阵列块进行修正,速度为20mm/转,等修到所有组织芯完全曝露;用切片机对组织阵列块进行切片,将连续切片分别漂在凉水中,使其自然展开,再将切片转移至45℃温水中展片2分钟左右,待展开后将其贴在经过防脱片处理的载玻片上晾干;将切片置于60℃的环境下烤片3分钟,58℃继续烤片16h;将做好的组织芯片保存于切片盒,置于冰箱-20℃保存备用。
2、免疫荧光染色
将切好的石蜡组织芯片放置在烘片仪上70℃烤片1h,60℃烘片1h;将已干燥的组织芯片浸于二甲苯中5分钟,重复2次;取出后进行梯度酒精脱水:100%乙醇浸泡5分钟,95%乙醇浸泡5分钟,75%乙醇浸泡5分钟,最后用蒸馏水冲洗组织芯片;将组织芯片置于耐高温切片架上,置于pH为6.0的AR6修复液中,100%功率加热2.5min,接着20%功率加热15min,进行高温抗原修复;自然冷却至室温后,取出蒸馏水中的芯片,用PBS冲洗3次,每次2分钟;用免疫组化笔在组织芯片上画出大概的组织范围,接着滴加一抗封闭液200μL,封闭10分钟;滴加200μL的兔抗人BTF3L4单克隆抗体工作液(稀释比例为1∶100)于组织芯片上,在4℃条件下过夜;第二天,取出组织芯片,复温半小时,回收一抗后用PBS冲洗2分钟,重复3次,之后取出甩干;在组织芯片上滴加200μL的二抗工作液,于室温孵育10分钟,接着用PBS冲洗2分钟,重复3次,之后取出甩干;配制所需的波长的荧光染料,向组织芯片上滴加配好的荧光染料,避光室温孵育10min,接着用PBS冲洗2分钟,重复3次;切片干燥透明后,用DAPI封片。
显微镜下观察免疫荧光染色结果,细胞相应部位出现染色作为阳性表现。使用Vectra 3成像软件在20倍放大倍率下捕获每个样本。使用inForm 26.1.0(Perkin Elmer)对图像进行分析和评分,并为每个细胞设置阳性或阴性细胞的阈值。计算每个区域的细胞百分比并进行评分(0-100)。BTF3L4的最终染色得分为染色强度与阳性细胞染色面积的乘积。BTF3L4表达分数的分界点由X-tile软件根据生存时间及生存状态得出。评分如下:0-50为低表达或无表达,51-100为高表达。所有数据均用统计软件SPSS V.25.0处理,计量资料以均数±标准差表示,组间比较采用单因素方差分析,BTF3L4表达与胶质瘤患者的预后关系分析用Kaplan-Meier生存分析,所有检验结果以P<0.05为差异有统计学意义。结果如图1-3所示,BTF3L4在胶质瘤组织中表达稍低于对照非瘤脑组织,在肿瘤患者中,高表达BTF3L4的患者胶质瘤级别高,组织病理学分级恶性程度更高,患者生存期短,预后差。
实施例2
1、shRNA设计
首先,使用Invitrogen Block-iT RNAi Designer设计针对BTF3L4基因序列,并委托北京Oligobi0生物科技有限公司(中国北京)合成shRNA,特异性靶向BTF3L4基因的shRNA对应的DNA序列如下所示:
BTF3L4-sh1:5′-CCTGATGTTACAGTTTGGTAGATTT-3′,
BTF3L4-sh2:5′-CAAACTGGAATAGCTAGCATGTGCT-3′。
2、胶质瘤细胞系的培养
胶质瘤细胞系,包含:T98G细胞、U87mg细胞、U251细胞、SHG44细胞,用含10%胎牛血清的DMEM完全培养基培养,培养箱内保持温度37℃、5%的CO2湿饱和度,常规传代培养,2-3天换液一次,选取处于对数生长期的细胞进行实验。
3、BTF3L4在胶质瘤组织及细胞系中的表达
1)胶质瘤组织蛋白提取
每250mg组织中加入1mL预冷的含蛋白质裂解液;放入组织超声裂解仪中充分裂解,每次裂解间隔冰浴1分钟,至组织完全裂解;将组织匀浆置于预冷的离心机中12000g离心15分钟后,将上清液立刻转移入新的离心管中保存待用。
2)细胞总蛋白提取
从37℃培养箱取出相应细胞;弃培养基,用预冷的PBS洗涤细胞2次,弃去PBS并将残留的PBS溶液吸干净,以免稀释细胞蛋白;根据细胞培养瓶的大小和细胞的生长密度,加入RIPA细胞裂解液,后用细胞刮子收集细胞,转移至干净的EP管中;将刮下的细胞蛋白在冰上充分裂解20-30分钟;4℃,12000rpm离心15min;取上清,用BCA法测细胞蛋白的浓度,后加入loading buffer吹打混匀,95℃煮沸5分钟,分装,保存在-80℃冰箱备用。
3)蛋白免疫印迹(Western blot)
制备聚丙烯酰胺凝胶(5%浓缩胶,12.5%分离胶);将玻璃板清洗干净,倾斜晾干,组装玻璃板,加入分离胶,加至距玻璃板上端2cm处,立即加异丙醇液封,放置30min,等分离胶凝固后轻轻倒掉上层异丙醇,再加入浓缩胶至玻璃板顶部,立即插入梳子,静置30min,至浓缩胶凝固;将配置好的胶放入电泳槽内,用电泳缓冲液将内部加满,将Protein Maker及提取的蛋白样品上样后,将剩余的电泳液加入,接通电源,调节电压80V,Protein marker分开后,再将电压调至100V,结束后将胶取出,切取目的条带;剪取大小合适的PVDF膜,先在甲醇中极化30s左右,再放入转膜液中;同时将海绵及滤纸放入转膜液中浸泡20min,安装转膜装置,排放顺序为:阴极碳板+海绵+滤纸+胶+PVDF膜+滤纸+海绵+阳极碳板;将转膜装置放入转移槽内,加入冰袋,并加满转膜液;接通电源,300mA恒流调节湿转0.5h,转膜需全程在冰盒中进行;转膜结束后,将PVDF膜放入封闭液(脱脂奶粉5g溶解在100mL TBST)中,在摇床上80r/min室温封闭2h;封闭结束后,按一抗稀释比例用封闭液配制一抗稀释液,PVDF膜上均匀滴加稀释的一抗,4℃孵育过夜;第二天,TBST洗膜3次,每次15min;洗膜结束后,按二抗稀释比例用TBST配制二抗稀释液,PVDF膜上均匀滴加稀释的二抗,室温1.5h;孵育结束后,TBST洗膜3次,每次15min;洗膜结束后将PVDF膜用滤纸吸干,平铺于显影仪相应位置,ECL发光液在使用前等比例混合A液和B液,均匀滴加于PVDF膜上,凝胶成像系统拍照、保存。
按上述方法分别提取6对手术切除胶质瘤组织和癌旁组织以及4种胶质瘤细胞的蛋白。用Western blot检测4种胶质瘤细胞中BTF3L4的表达情况,筛选出高低表达细胞。结果如图4所示,BTF3L4蛋白在胶质瘤组织中的表达高于其对应癌旁组织,且BTF3L4蛋白在U251胶质瘤细胞中表达相对较高,在U87mg胶质瘤细胞中表达相对较低。
4、RNAi系统干扰胶质瘤细胞内BTF3L4基因表达
利用构建的RNAi干扰系统,特异性针对BTF3L4-sh1:5′-CCTGATGTTACAGTTTGGTAGATTT-3′序列转染U251胶质瘤细胞;在12孔板接种待转染的目的细胞,待细胞完全铺开,汇合度达到60-70%,且细胞状态较好时,即可开始转染细胞;感染6-8小时后,换液继续培养,48小时后收集细胞,裂解后提蛋白,使用Western blot检测干扰的U251细胞。
结果如图5所示,与未处理组相比,经RNAi干扰后的BTF3L4蛋白相对表达量明显降低,表明BTF3L4蛋白表达被有效地抑制了。
5、细胞迁移(Transwell小室法)
消化收集转染后48h的各组细胞,离心待用;用基础培养基重悬细胞,调整细胞密度至5×104/mL;在24孔板中加入800μL的完全培养基,放入小室,充分浸润,取100μL的细胞悬液加入上室;常规培养24h后取出,1×PBS洗2次,4%的多聚甲醛固定20min,1×PBS洗2次;在24孔板中加入500μL的结晶紫染液,放入小室,10min后取出,1×PBS洗2次,倒置小室,用棉签轻轻擦去上室内未穿过去的细胞。
结果如图6所示,干扰BTF3L4后U251细胞的迁移能力下降。
6、细胞侵袭(Transwell小室法)
先配置Matrigel基质胶(BD Biosciences,San Jose,CA),加入Transwell小室的上室,每个100μL,避免产生气泡;消化收集转染后48h的各组细胞,离心待用;用基础培养基重悬细胞,调整细胞密度至5×104/mL;在24孔板中加入800μL的完全培养基,放入小室,充分浸润,取100μL的细胞悬液至上室;常规培养24-48h后取出,1×PBS洗2次,4%的多聚甲醛固定20min,1×PBS洗2次;在24孔板中加入500μL的结晶紫染液,放入小室,10min后取出,1×PBS洗2次,倒置小室,用棉签轻轻擦去上室内未穿过去的细胞。
结果如图7所示,干扰BTF3I4后U251细胞的侵袭能力下降。
7、细胞周期检测
细胞汇合后,调整细胞浓度为105个/mL,接种于6孔培养板中,每孔3mL培养液,在37℃、5%CO2培养箱中培养24小时;转染siRNA-1后继续培养48小时;将板中细胞用胰酶消化,制备成单细胞悬液,固定后用细胞周期检测试剂盒(C1052,Beyotime)染色。随后,用BD-FACSVerse流式细胞仪检测不同分裂时期细胞所占百分比,最后用CELL Quest软件进行定量分析。
结果如图8所示,干扰BTF3L4后,定量结果显示,S期U251胶质瘤细胞数量显著增加,G0/G1期(2N)和G2/M期(4N)胶质瘤细胞生长受到明显阻滞,细胞分化能力受损。
Claims (8)
1.BTF3L4基因在制备治疗胶质瘤的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的治疗胶质瘤为抑制细胞的迁移。
3.根据权利要求1所述的应用,其特征在于,所述的治疗胶质瘤为抑制细胞的侵袭。
4.用于检测肿瘤组织中BTF3L4蛋白表达量的生物标志物,其特征在于,为BTF3L4基因。
5.用于检测肿瘤组织中BTF3L4蛋白表达量的试剂盒,其特征在于,含有BTF3L4基因。
6.BTF3L4基因的RNAi干扰系统在制备用于治疗胶质瘤的药物中的应用,其特征在于,所述的BTF3L4基因的RNAi干扰系统的序列如下:
BTF3L4-shl:5′-CCTGATGTTACAGTTTGGTAGATTT-3′,
BTF3L4-sh2:5′-CAAACTGGAATAGCTAGCATGTGCT-3′。
7.根据权利要求7所述的应用,其特征在于,所述的BTF3L4基因的RNAi干扰系统为敲低胶质瘤组织细胞中BTF3L4基因。
8.根据权利要求7所述的应用,其特征在于,所述的BTF3L4基因的RNAi干扰系统为抑制胶质瘤组织细胞中BTF3IA蛋白表达。
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