CN114134147A - 一种调控fzd9的非编码rna及其应用 - Google Patents
一种调控fzd9的非编码rna及其应用 Download PDFInfo
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- CN114134147A CN114134147A CN202111359068.5A CN202111359068A CN114134147A CN 114134147 A CN114134147 A CN 114134147A CN 202111359068 A CN202111359068 A CN 202111359068A CN 114134147 A CN114134147 A CN 114134147A
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Abstract
本发明属于基因工程技术领域,涉及一种调控FZD9的非编码RNA及其应用,所述调控FZD9的非编码RNA命名为lncRNA MSTRG.24062.2,其编码区核苷酸序列如SEQ ID NO.1所示,该非编码RNA可以通过结合miRNA‑125a‑5p或miRNA‑125b‑5p调控靶基因FZD9,进而对骨髓间充质干细胞的成骨分化及种植体骨结合起到明显调控作用;本发明首次对lncRNAMSTRG.24062.2进行功能表征,证实lncRNA具有作为骨生物标志物的潜在价值,为进一步以此为靶点来研究骨质疏松症以及相关患者的临床诊疗提供了新的途径。
Description
技术领域
本发明属于基因工程学领域,涉及一种调控FZD9的非编码RNA及其应用。
背景技术
高脂血症是以血液中总胆固醇、三酰甘油等血脂指标异常为特点的代谢性疾病,在全球具有较高的发病率。临床研究结果表明,高脂血症患者发生骨质疏松和骨质减少的风险很高。高脂血症对骨骼的再生和修复起着破坏性的作用,削弱了骨骼的机械强度。高脂饮食引起的血清脂质氧化产物增加,刺激脂肪生成和破骨细胞生成的过程,抑制成骨。种植体周围良好的骨结合与种植体的长期存活率有着密不可分的联系。高脂血症患者的骨密度降低,骨-种植体结合强度降低,导致种植体植入的失败率增加。
卷曲受体蛋白9(Frizzled-9,FZD9)是Wnt通路上细胞膜FZD受体家族中的一员。FZD是一种7次跨膜蛋白,在对果蝇表皮细胞的研究中首次发现FZD基因,其基因序列的突变会造成年成果蝇表皮细胞的去极性化甚至是产生平面化。FZD基因通过影响细胞发育的三条主要信号通路:Wnt/β-catenin信号通路;Wnt/钙通路和平面细胞极性通路,来对脊椎动物的基础进化产生重要影响。FZD9基因的表达对于骨代谢有重要意义,包括Wnt信号通路在内的多条信号通路在骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向成骨细胞分化的过程中起到重要作用。前期研究已证实FZD9对高脂血症大鼠种植体骨结合有重要意义。
近年来,随着科学研究方法和科学技术的不断向前发展,在对生物学研宄中,它们已不仅仅局限于蛋白及基因等水平,过去曾经被认定为“暗物质”或者“噪音”的无生物学功能的许多种非编码RNA(non-coding RNA,ncRNA)亦被发现为具有许多种调节功能的重要的调节因子。LncRNA是长度大于200nt的ncRNA,具有保守的二级结构,使其能够与调节各种生物过程的蛋白质;LncRNA通过多种机制参与蛋白质编码和表观遗传基因的调控,例如表观遗传修饰,选择性剪接、转录后和翻译调控。LncRNA通过介导染色质修饰,进而导致基因沉默;同时研究还显示它们可以控制蛋白质合成,RNA成熟和转运。除此之外,LncRNA还有一个重要功能是它可能起“海绵”/“诱饵”的作用,与其他具有相同miRNA应答元件的转录物(LncRNA、mRNA、circRNA、假基因转录物等)基因对靶标miRNA的共同结合位点的竞争,从而改变miRNA的功能,降低了miRNA对靶mRNA的调节作用,这也是Pier教授提出竞争性内源RNA(Competing endogenous RNA,ceRNA)理念。越来越多的研究表明,LncRNA-miRNA的共表达调控网络在细胞分化中发挥重要功能。
miRNA是长度为21-22个核苷酸的非编码RNA,可与靶基因的mRNA相互作用,促进其降解或抑制被编码蛋白的翻译。miRNA是在调节多个信号网络中起重要作用的小分子,作为发育和细胞稳态的关键调节剂控制着多种生物过程。本发明发明人的的前期研究已证实,miRNA-125a-5p、miRNA-125b-5p可靶向结合FZD9,抑制高脂环境下BMSCs的成骨分化,但是是否存在影响miRNA-125a-5p、miRNA-125b-5p的相关因素,对于本领域技术人员而言是未知的,也是本申请发明人进一步研究的主要方向。
发明内容
针对现有技术中的上述情况,本发明的发明人提供了一种通过调控FZD9的非编码基因在调控种植体骨结合中的应用,所述的调控FZD9的非编码基因为LncRNAMSTRG.24062.2,其编码区核苷酸序列如所示,应用该基因可以通过结合miRNA-125a-5p或miRNA-125b-5p位点调控FZD9对种植体骨结合起到明显促进作用,首次对LncRNAMSTRG.24062.2进行功能表征,证实LncRNA作为骨生物标志物的潜在价值,为进一步以此为靶点来开展骨质疏松症患者的临床诊疗的可能性。
本发明的具体原理如下:
发明人首次发现FZD9对高脂血症大鼠种植体骨结合具有重要意义,miRNA-125a-5p和miRNA-125b-5p通过结合FZD9-3’-UTR,进而抑制BMSCs成骨分化。在此基础上,利用RNA测序结果及生物信息学软件分析,预测出与miRNA-125a-5p、miRNA-125b-5p竞争性结合的LncRNA MSTRG.24062.2,首次对LncRNA MSTRG.24062.2进行功能表征。本发明人进一步发现通过调控LncRNA MSTRG.24062.2表达,与miRNA-125a-5p、miRNA-125b-5p竞争性结合,可以实现对FZD9的调控的作用,进而对BMSCs的成骨分化产生重要影响,从而更优异地成为促进种植体早期骨结合。
本发明所述的调控FZD9的非编码RNA为LncRNA MSTRG.24062.2,其编码区核苷酸序列如SEQ ID NO.1所示;该序列为本领域首次提供,填补了本领域的空白。
为了验证该基因与miRNA-125a-5p、miRNA-125b-5p及FZD9的相互关系,并验证他们之间对于种植体骨结合的关系,发明人选择BMSCs及高脂血症大鼠为研究对象,进行了如下的实验:
1.LncRNA MSTRG.24062.2在高脂环境下BMSCs成骨分化中表达受到抑制
利用大鼠骨髓间充质干细胞进行高脂或普通成骨诱导,诱导3、5、7、14天后收集细胞。使用实时定量PCR检测两组BMSCs中LncRNA MSTRG.24062.2、ALP、Runx2、PPAR-γ和FZD9mRNA水平。研究结果表明成骨相关因子Runx2和ALP的表达减少,与成脂相关因子PPAR-γ的表达增加,同时LncRNA MSTRG.24062.2和FZD9的表达量减少。(如图1所示)
形态学检测碱性磷酸酶(ALP)、茜素红和油红O染色,评价高脂环境下BMSCs成骨分化能力。研究结果表明普通、高脂成骨诱导28d,茜素红染色显示普通组的细胞矿化结节的数量明显多于高脂组,且红染较深。油红O染色液染色结果显示,高脂成骨诱导组的细胞形态改变,逐渐从长梭形改变为椭圆和圆形,同时有小脂滴的出现,并有很强的折射特性。随着诱导时间的增加,小脂滴逐渐融合形成大脂滴,检测到红色珠状脂肪颗粒。普通组未见脂肪颗粒形成。ALP染色结果显示,与普通组相比,高脂组的ALP的染色程度较低,高脂血症环境明显抑制了BMSCs早期的成骨分化能力,影响了BMSCs向成骨细胞的分化过程。(如图2所示)
2.LncRNA MSTRG.24062.2促进BMSCs的成骨分化
在体外研究LncRNA MSTRG.24062.2成骨中的功能上,使用大鼠骨髓间充质干细胞建立了LncRNA MSTRG.24062.2高表达细胞模型。在BMSCs中过表达LncRNA MSTRG.24062.2后高脂诱导,通过RT-qPCR及western blot结果显示,与对照组相比,过表达组LncRNAMSTRG.24062.2、ALP、Runx2和FZD9的表达升高,而miRNA-125a-5p、miRNA-125b-5p表达降低。(如图3所示)
另外ALP和茜素红染色表明LncRNA MSTRG.24062.2过表达后,红染程度增加,矿化结节的形成增多。(如图4所示)证实BMSCs向成骨分化增强。LncRNA MSTRG24062.2促进BMSCs成骨分化。
3.LncRNA MSTRG.24062.2通过结合miRNA-125a-5p和miRNA-125b-5p调控FZD9基因表达
在上述研究的基础上,发明人为了进一步验证LncRNA MSTRG.24062.2对下游信号的影响。采用靶基因预测软件对LncRNA MSTRG.24062.2与miRNA-125a-5p和miRNA-125b-5p的结合区域进行预测(如图5所示)。将miRNA-125a-5p和miRNA-125b-5p与LncRNAMSTRG.24062.2结合位点的序列克隆到pmirGLO载体构成野生型(WT)荧光素酶报告质粒;将miRNA-125a-5p和miRNA-125b-5p与LncRNA MSTRG.24062.2结合位点突变的序列克隆到pmirGLO构成突变型荧光素酶报告质粒(Mut)。
分为以下组:(1)NC mimic+MSTRG.24062.2WT in pmirGLO;(2)rno-miRNA-125b-5pmimics+MSTRG.24062.2WT in pmirGLO;(3)rno-miRNA-125a-5p mimics+MSTRG.24062.2WT in pmirGLO(4)NC mimic+MSTRG.24062.2MUT in pmirGLO;(5)rno-miRNA-125b-5p mimics+MSTRG.24062.2MUT in pmirGLO;(6)rno-miRNA-125a-5p mimics+MSTRG.24062.2MUT in pmirGLO;
将miR-125a-5p和miR-125b-5p mimics分别与野生型萤光素酶报告质粒(WT)、突变型荧光素酶质粒(Mut)共转染进入293T细胞。
双荧光素酶报告基因系统检测结果显示,相对NC+WT组,mimics+WT组荧光素酶活性显著降低,miRNA-125a-5p组和miRNA-125b-5p组的荧光素酶活性分别降低了65%和50%。相反,NC+Mut组和mimics+Mut组,荧光素酶活性无显著差异(如图6所示)。表明LncRNAMSTRG.24062.2可以直接与miRNA-125a-5p和miRNA-125b-5p结合并抑制其表达。
4.LncRNA MSTRG.24062.2在高脂血症大鼠中表达受到抑制
通过对4周龄成年雄性Wistar大鼠给予普通饲料和高脂饲料喂养8w构建正常及高脂血症大鼠血症模型。在大鼠双侧股骨干骺端分别植入种植体,2w后获取种植体周1mm骨组织。Micro-CT结果显示,高脂大鼠组种植体周围新骨形成较正常组减少,BV/TV为正常组0.73倍。(如图7所示)RT-PCR结果分析显示,高脂组大鼠种植体周围骨组织中LncRNAMSTRG.24062.2、Runx2、ALP、FZD9的RNA水平降低,miRNA-125a-5p和miRNA-125b-5p的表达量升高。(如图8所示)
5.LncRNA MSTRG.24062.2促进高脂血症大鼠种植体骨结合
发明人为了进一步研究未表征功能的LncRNA MSTRG.24062.2在种植体骨结合的作用。在高脂血症大鼠模型建成后,种植术前3天,通过在双侧股骨干骺端邻近部位以1×108个病毒数目滴度分别肌肉注射LncRNA MSTRG.24062.2和LV5-nc慢病毒载体,实现LncRNA MSTRG.24062.2的过表达。
Micro-CT结果显示:与LV5-nc组相比,LncRNA MSTRG.24062.2-overexpressed组可看到较多的新骨形成,BV/TV升高1.29倍。(如图9所示)
在证实LncRNA MSTRG.24062.2能通过慢病毒载体被高表达后,RT-PCR及Western--blot检测种植体周骨组织结果显示,与lv5-nc组相比,LncRNA MSTRG.24062.2-enhancer组FZD9、Runx2的表达升高,而miRNA-125a-5p、miRNA-125b-5p的表达均降低。(如图10所示)
LncRNA MSTRG.24062.2过表达通过负调控miRNA-125a-5p和miRNA-125b-5p促进高脂血症大鼠的种植体骨结合,新骨形成增加,BV/TV、BIC%升高,提示高脂环境下种植体骨结合率降低与LncRNA MSTRG.24062.2表达降低与有着密切联系,揭示LncRNA作为骨生物标志物的潜在价值。
综合上述各种验证实验可知,本申请所提供的LncRNA MSTRG.24062.2可以通过结合miRNA-125a-5p和miRNA-125b-5p调控FZD9基因表达,从根本上解释了LncRNAMSTRG.24062.2的作用机理,为进一步临床诊疗和新型的靶向药物的研发提供理论和实验依据,同时该LncRNA MSTRG.24062.2还可以作为诊断早期骨质疏松的一个可行生物标志物,提高检测的准确性和速度。
附图说明
图1为利用大鼠骨髓间充质干细胞进行高脂或普通成骨诱导后,使用实时定量PCR检测两组BMSCs中LncRNA MSTRG.24062.2、ALP、Runx2、PPAR-γ和FZD9 mRNA水平结果示意图,其中control group:普通组;experimental group:高脂组,结果表明成骨相关因子Runx2和ALP的表达减少,与成脂相关因子PPAR-γ的表达增加,同时LncRNA MSTRG.24062.2和FZD9的表达量减少;
图2为利用形态学检测碱性磷酸酶(ALP)、茜素红和油红O染色,评价高脂环境下BMSCs成骨分化能力结果示意灰度图,结果证实高脂血症环境明显抑制了BMSCs早期的成骨分化能力,影响了BMSCs向成骨细胞的分化过程;
图3为LncRNA MSTRG.24062.2过表达后RT-PCR和Western Blot实验结果示意图;
其中NC group:阴性对照组,LncRNA overexpressed:LncRNA MSTRG.24062.2过表达组;
图4为LncRNA MSTRG.24062.2过表达后ALP及茜素红染色结果示意灰度图;
结果证实LncRNA MSTRG24062.2促进BMSCs成骨分化;
图5为软件预测LncRNA MSTRG.24062.2与miRNA-125a-5p、miRNA-125b-5p的结合片段示意图;
图6为双荧光素酶报告基因结果柱状图,
结果显示,相对NC+WT组,mimics+WT组荧光素酶活性显著降低;
图7为大鼠种植体植入后2w后micro-CT分析结果示意图,
图中normal group:正常组;HF group:高脂组,结果显示,高脂大鼠组种植体周围新骨形成较正常组减少;
图8为大鼠种植体植入后2w后高脂组与普通组的相关分子表达RT-PCR结果示意图;
图9为利用慢病毒载体过表达LncRNA MSTRG.24062.2后,种植体植入2w后micro-CT分析结果示意图,
图10利用慢病毒载体过表达LncRNA MSTRG.24062.2后RT-PCR及Western Blot检测结果示意图;
图11为pmirGLO载体构建示意图。
具体实施方式
以下结合附图对本发明的上述发明内容作进一步的详细描述。应理解,这些实施例仅为了说明本发明而不是为了限制本发明的保护范围。实施例中所采用的具体技术均为本领域的常规技术,所采用的生物材料均为发明人在研究过程中从正规途径和合法渠道获得的已知生物材料,发明人列举相关技术如下:但是其他未被列入的具体技术也均为已知技术,发明人不再赘述。
实施例中所采用的各种现有技术具体如下:
骨髓间充质干细胞(BMSCs)的培养与成骨诱导
(1)BMSCs的获取、培养
采用全骨髓细胞贴壁培养法:取20天60g左右的Wistar大鼠,脱颈处死,浸泡消毒10min(放置于装有75%乙醇的烧杯中),擦干,并移入超净工作台。剪开大鼠后腿区域皮肤,剥离组织,暴露骨面,取出双侧股骨,PBS缓冲液(含双抗)冲洗备用;提前准备体积分数为15%胎牛血清(FBS)的α-MEM完全培养基(含链霉素100mg/L,100U/mL),用以冲洗股骨和胫骨骨髓腔,多次缓慢冲洗后,可观察到髓腔内发白,得原代BMSCs,置于5%CO2、37℃恒温培养箱中传代培养。
(2)高脂成骨及普通成骨诱导
高脂及普通成骨诱导液配置①在避光的条件下称取地塞米松0.00393g置于10mL离心管中、β-甘油磷酸钠0.206g和维生素C 0.005g同时置于另一个10mL离心管中;②将离心管移动至超净工作台内(避光操作),加入2mL无水乙醇溶解地塞米松,再加入3mL 10%(体积分数)FBS的α-MEM培养液,并使用0.22μm过滤器过滤除菌,得到5mL的2×10-3mol/L的地塞米松溶液;再取5μL置于15mL离心管中,并添加5mL 10%(体积分数)FBS的α-MEM培养液制得2×10-6mol/L的地塞米松溶液;所配制的地塞米松溶液置于4℃冰箱避光保存,2周内用完;③用1mL移液器取②中所得避光保存的地塞米松溶液250μL加入到47.25mL 10%(体积分数)FBS的α-MEM培养液中;④取5mL 10%(体积分数)FBS的完全培养液加入装有β-甘油磷酸钠及维生素C的离心管中,轻轻摇晃直至完全溶解,并用0.22μm过滤器过滤除菌,然后使用5mL移液器取2.5mL加入到③中所得的47.50mL溶液中,最后得到50mL普通成骨诱导液;⑤配制的普通成骨诱导液放置于4℃冰箱储藏,不超过1周。使用高脂培养基替代α-MEM培养液重复上述步骤即可得到高脂成骨诱导液。高脂培养基为PYTHONBI品牌,货号AAPR156。
(3)BMSCs成骨诱导
取步骤(1)中传代培养的第三代细胞,将细胞随机分为实验组(高脂组)、对照组(正常组),分别用步骤(2)配制的高脂成骨诱导液和普通成骨诱导液进行成骨诱导。
(4)BMSCs成骨成脂能力鉴定
成骨诱导第7、14、28天,分别进行ALP染色、茜素红染色和油红O染色,观察两组细胞成骨分化情况。
RNA提取、cDNA合成和实时定量PCR检测
(1)待细胞成骨诱导至3d、5d、7d、10d、14d时,使用Trizol试剂提取总RNA,OD比值在1.8-2.0之间视为合格。
(2)用试剂盒合成第一链cDNA。按照配置体系1μl总RNA、2μl 5×gDNA EraserBuffer,1μl gDNA Eraser混合物加水至总体积10μl混合均匀,逆转录仪反应条件设定为:42℃2min,4℃停止。按照产品说明书要求的体系,配置cDNA合成所需要的混合物,将10μl的上述混合物,4μl 5×PrimeScript Buffer 2;4μl RNase Free dH2O;1μl Prime ScriptRT Enzyme MixⅠ;1μl RT Primer Mix加到每一管RNA/引物混合物中轻柔混合均匀,PCR仪反应条件设定为在37℃15min;温度升高至85℃5s,之后降温至4℃停止。逆转录后样本置于-20℃保存。
(3)使用cDNA做实时定量RT-PCR扩增模版,按照试剂盒使用说明,依次加入DEPC水、引物、dNTP混合物以及模版进行实时定量PCR。使用GAPDH组作为内部参照。
LncRNA MSTRG.24062.2转染BMSCs细胞
(1)以lipofectamineTM3000(简称lipo3000)为载体,在24孔板内,以MOI值为20、40、50转染LncRNA MSTRG.24062.2,进行预实验,得出MOI值为40时,转染效率最好;
(2)转染前24h,用胰酶消化细胞并用细胞计数板计数,6孔板每孔接种细胞数1×10^5,待细胞密度为40%左右时,避光条件下,每孔加入1750μl不含抗生素的opti-MEM培养基;①稀释lipo3000:将lipo3000:opti-MEM按照1:5比例稀释,每孔加入200μl;②将LncRNA与opti-MEM以1:50的比例稀释,混匀,室温静置;③将①和③混匀,室温静置20min;④将混合好液体加入6孔板内,将培养板放置于37℃培养箱中培养,6h后更换为10%FBS的完全培养基,然后24h、48h及72h将细胞放置于倒置显微镜下观察。
蛋白的提取
(1)提取蛋白及蛋白变性
①弃六孔板内旧培养液,PBS溶液冲洗;②将pmsf:RIPA裂解液以1:100的比例混合,每孔中加100μl混合液,吹打,放在冰盒内30min。用塑料刮刀刮下6孔板内细胞,将裂解细胞悬液移到预冷的1.5ml EP管中,超声裂解细胞,间隔进行,防止过热;③提前预冷离心机至4℃,12000r/min,4℃离心15min;④离心完毕后,将离心好的上清液移入新EP管,利用BCA试剂盒,按照说明书,测出所得蛋白的浓度;⑤根据测得各组样本的蛋白浓度,吸取相应量上清液,加入一定量三蒸水稀释蛋白样本获得体积与浓度相同的蛋白样品,每4μl蛋白样品加lμl 5×SDS-PAGE上样缓冲液,颠倒混匀。在100℃下加热10分钟使蛋白充分变性,然后放置在冰上迅速冷却。置于-20℃保存。
(2)配胶电泳
①清洗玻璃板,肥皂水→自来水→蒸馏水清洗,晾干后组装。两块玻璃板底边平齐后置于灌胶架上,用楔子压紧;②配胶:凝胶浓度的选择依据目的蛋白分子量大小,按照说明书配制相应的浓缩胶和分离胶,充分混匀后,沿玻璃板壁注入两组玻璃板之间的间隙,胶至矮板下2cm处,在上层轻慢加入三蒸水与矮板平齐,凝固后将三蒸水去除;③配制上层浓缩胶,沿玻璃板壁缓慢加入浓缩胶,平行插入梳子,勿产生气泡;④待浓缩胶完全凝固后,安装好电泳装置,加入部分电泳缓冲液,平行拔出梳子,防止胶断;⑤将蛋白样本从-20℃取出,解冻离心,用移液器吸取上层蛋白样本及蛋白marker加入加样孔中,加入剩余电泳缓冲液直至玻璃板被电泳液覆盖;⑥连接电泳槽、电泳仪,红对红、黑对黑,先将电压调节电压为60V,30min将浓缩胶跑完,到达分离胶上层,将电压设置为160v,根据目的蛋白分子量大小选择电泳时间。
(3)转膜
①三蒸水冲洗后,修剪凝胶,留下目的因子分子量区域的凝胶,全程置于转膜液中,保持湿润;②修剪PVDF膜,与凝胶大小相近,将PVDF膜用甲醇浸泡30s后,使其变软,在PVDF膜一侧剪角作标记;③将黑板、海绵、滤纸、凝胶、PVDF膜、滤纸、海绵、红板的顺序放置,电压100v,转膜75min,将转膜槽放置于冰水混合物中;
(4)封闭及抗体孵育
①在转膜结束之后,将PVDF膜取出放置于TBST溶液中,摇床上清洗,5%封闭剂封闭1h,封闭后用TBST清洗;②一抗孵育:封闭液稀释一抗,4℃过夜,1%TBST洗PVDF膜3次,10次/min;③按照说明书稀释二抗,室温摇床上孵育1h,1%TBST洗PVDF膜3次,10次/min。
(5)显影,根据说明书避光配制显影液,在膜上滴加显影液,放入显影设备曝光目的蛋白条带。
双荧光素酶报告基因检测
(1)根据试剂盒说明书进行试剂配制。主要试剂:5X Passive Lysis Buffer(PLB);Luciferase Assay Substrate;Luciferase Assay Buffer II;50×Stop&GloStop&Glo
(2)①去除24孔板中的旧培养基,PBS洗涤细胞。②将1×PLB按100μl/孔加入到24孔板中,在室温下轻轻摇动培养皿15分钟。将裂解液转移到试管或小瓶中,把裂解液转移到96孔检测试板中,每孔20μl,实验设计3复孔。③每孔加入100μl配制好的LARII,检测萤火虫荧光强度,然后再加入100μl Stop&Glo Reagent,再检测海肾荧光强度。
高脂血症大鼠实验模型及LncRNA MSTRG.24062.2过表达动物模型构建
4周龄成年雄性Wistar大鼠随机分为普通组和高脂组,分别给予普通饲料和高脂饲料喂养。所有大鼠均置于灭菌、无病原体、湿度在50%-65%、温度在20-25℃的动物培养箱内,每天光暗交接循环12h。喂养8周后,禁食12小时,于大鼠内眦静脉处取0.5ml全血,置于1.5ml EP管中,在冰盒内静置1h后,预冷的4℃离心机(4000rpm,15min),离心完全后吸取上层血清至新的EP管中,提取过程中不能触碰下层沉淀物质,对血清中低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、甘油三酯(TG)和血清总胆固醇(TC)的表达水平进行检测。
表1大鼠血清脂质水平(单位mmol/L),*两组有统计学差异(*P<0.05)。
高脂血症大鼠模型建立后,将高脂血症大鼠随机分为LncRNA MSTRG.24062.2过表达组和LV5-nc对照组。预实验摸得最佳病毒浓度。种植术前3天,在双侧股骨干骺端邻近部位以1×108个病毒数目滴度分别肌肉注射LncRNA MSTRG.24062.2和LV5-nc慢病毒载体,以实现LncRNA MSTRG.24062.2的过表达。
种植体的植入
高温高压蒸汽消毒灭菌所有手术器械。术前,对大鼠进行称重,用10%水合氯醛按照0.35ml/100g剂量进行腹腔注射麻醉;全麻后,将大鼠头部及四肢固定,种植区域周围5cm范围备皮及消毒,在双侧股骨干骺端的前内侧用尖刀片切开皮肤形成约2cm长的切口,将肌层和骨膜剥离,使得股骨干骺端充分暴露,FG557钻针制备窝洞(深度2.5mm,直径1.2mm),在窝洞预备过程中要用冷却的生理盐水持续冲洗降温。手动将种植体旋入窝洞,使用4-0的缝线分层对位缝合伤口,缝合后消毒。在种植体植入位置附近肌肉再次注射相应的病毒载体。术后3天内每天每只大鼠通过肌肉注射头孢唑啉钠16万单位来预防感染。
Micro-CT检测分析
大鼠标本固定后,置于Inveon MM-CT机进行扫描,然后利用Inveon ResearchWorkplace软件对扫描结果进行分析。种植体周围0.5mm骨组织被选作感兴趣区域(ROI),对骨小梁相对体积(BV/TV)使用Inveon Research Workplace软件分析,对ROI区新生骨可利用CO_BRA Exxim软件进行三维重建可视化分呈现,观察ROI区新骨形成情况。
硬组织切片、HE染色
(1)先在浓度为50%、60%、70%、85%、95%、100%酒精对样本进行梯度脱水,然后使用浸塑液浸泡,之后用聚甲基丙烯酸树脂对样本进行包埋。
(2)用硬组织切片机沿着包埋好的种植体长轴切割,获得厚度约为25-30μm的硬组织切片,然后用砂纸进行打磨。
(3)苏木素—伊红染色,滴加苏木素染色8min,流水轻柔冲洗2min,可于显微镜下观察着色效果。伊红染色3min,流水轻柔冲洗1min,去除多余染液。用75%、85%、90%、95%、100%酒精梯度浸泡,二甲苯进行透明,中性树胶进行封片。
(4)染色结束后,放置于倒置显微镜下观察、拍照
统计学分析
采用SPSS17.0对所有数据进行统计学分析。高脂组和普通组,过表达LncRNAMSTRG.24062.2组和阴性对照组,用t检验进行差异分析,P<0.05表示有统计学意义。实验结果以3次独立重复实验的均数±标准差表示。
在上述具体技术方案的支持下,发明人进行了如下的实施例操作:
实施例1.LncRNA MSTRG.24062.2在高脂环境下BMSCs成骨分化中表达受到抑制
利用大鼠骨髓间充质干细胞进行高脂或普通成骨诱导,诱导3、5、7、14天后收集细胞。使用实时定量PCR检测两组BMSCs中LncRNA MSTRG.24062.2、ALP、Runx2、PPAR-γ和FZD9mRNA水平。研究结果表明成骨相关因子Runx2和ALP的表达减少,与成脂相关因子PPAR-γ的表达增加,同时LncRNA MSTRG.24062.2和FZD9的表达量减少。(如图1所示)
形态学检测碱性磷酸酶(ALP)、茜素红和油红O染色,评价高脂环境下BMSCs成骨分化能力。研究结果表明普通、高脂成骨诱导28d,茜素红染色显示普通组的细胞矿化结节的数量明显多于高脂组,且红染较深。油红O染色液染色结果显示,高脂成骨诱导组的细胞形态改变,逐渐从长梭形改变为椭圆和圆形,同时有小脂滴的出现,并有很强的折射特性。随着诱导时间的增加,小脂滴逐渐融合形成大脂滴,检测到红色珠状脂肪颗粒。普通组未见脂肪颗粒形成。ALP染色结果显示,与普通组相比,高脂组的ALP的染色程度较低,高脂血症环境明显抑制了BMSCs早期的成骨分化能力,影响了BMSCs向成骨细胞的分化过程。(如图2所示)
实施例2.LncRNA MSTRG.24062.2促进BMSCs的成骨分化
在体外研究LncRNA MSTRG.24062.2成骨中的功能上,使用大鼠骨髓间充质干细胞建立了LncRNA MSTRG.24062.2高表达细胞模型。在BMSCs中过表达LncRNA MSTRG.24062.2后高脂诱导,通过RT-qPCR及western blot结果显示,与对照组相比,过表达组LncRNAMSTRG.24062.2、ALP、Runx2和FZD9的表达升高,而miRNA-125a-5p、miRNA-125b-5p表达降低。(如图3所示)
另外ALP和茜素红染色表明LncRNA MSTRG.24062.2过表达后,红染程度增加,矿化结节的形成增多。(如图4所示)证实BMSCs向成骨分化增强。LncRNA MSTRG24062.2促进BMSCs成骨分化。
实施例3.LncRNA MSTRG.24062.2通过结合miRNA-125a-5p和miRNA-125b-5p调控FZD9基因表达
在上述研究的基础上,发明人为了进一步验证LncRNA MSTRG.24062.2对下游信号的影响。采用靶基因预测软件对LncRNA MSTRG.24062.2与miRNA-125a-5p和miRNA-125b-5p的结合区域进行预测(如图5所示)。将miRNA-125a-5p和miRNA-125b-5p与LncRNAMSTRG.24062.2结合位点的序列克隆到pmirGLO载体构成野生型(WT)荧光素酶报告质粒;将miRNA-125a-5p和miRNA-125b-5p与LncRNA MSTRG.24062.2结合位点突变的序列克隆到pmirGLO构成突变型荧光素酶报告质粒(Mut)。
载体构建过程
基因名称:MSTRG.24062.2WT in pmirGLO
载体名称:pmirGLO
克隆位点:SacI--SalI
目的序列:如SEQ ID NO.2所示;
基因名称:MSTRG.24062.2MUT in pmirGLO
载体名称:pmirGLO
克隆位点:SacI--SalI
目的序列:如SEQ ID NO.3所示;
构建过程如图11所示;
分为以下组:(1)NC mimic+MSTRG.24062.2WT in pmirGLO;(2)rno-miRNA-125b-5pmimics+MSTRG.24062.2WT in pmirGLO;(3)rno-miRNA-125a-5p mimics+MSTRG.24062.2WT in pmirGLO(4)NC mimic+MSTRG.24062.2MUT in pmirGLO;(5)rno-miRNA-125b-5p mimics+MSTRG.24062.2MUT in pmirGLO;(6)rno-miRNA-125a-5p mimics+MSTRG.24062.2MUT in pmirGLO;
将miR-125a-5p和miR-125b-5p mimics分别与野生型萤光素酶报告质粒(WT)、突变型荧光素酶质粒(Mut)共转染进入293T细胞。
双荧光素酶报告基因系统检测结果显示,相对NC+WT组,mimics+WT组荧光素酶活性显著降低,miRNA-125a-5p组和miRNA-125b-5p组的荧光素酶活性分别降低了65%和50%。相反,NC+Mut组和mimics+Mut组,荧光素酶活性无显著差异(如图6所示)。表明LncRNAMSTRG.24062.2可以直接与miRNA-125a-5p和miRNA-125b-5p结合并抑制其表达。
实施例4.LncRNA MSTRG.24062.2在高脂血症大鼠中表达受到抑制
通过对4周龄成年雄性Wistar大鼠给予普通饲料和高脂饲料喂养8w构建正常及高脂血症大鼠血症模型。在大鼠双侧股骨干骺端分别植入种植体,2w后获取种植体周1mm骨组织。Micro-CT结果显示,高脂大鼠组种植体周围新骨形成较正常组减少,BV/TV为正常组0.73倍。(如图7所示)RT-PCR结果分析显示,高脂组大鼠种植体周围骨组织中LncRNAMSTRG.24062.2、Runx2、ALP、FZD9的RNA水平降低,miRNA-125a-5p和miRNA-125b-5p的表达量升高。(如图8所示)
实施例5.LncRNA MSTRG.24062.2促进高脂血症大鼠种植体骨结合
发明人为了进一步研究未表征功能的LncRNA MSTRG.24062.2在种植体骨结合的作用。在高脂血症大鼠模型建成后,种植术前3天,通过在双侧股骨干骺端邻近部位以1×108个病毒数目滴度分别肌肉注LncRNA MSTRG.24062.2和LV5-nc慢病毒载体,实现LncRNAMSTRG.24062.2的过表达。
Micro-CT结果显示:与LV5-nc组相比,LncRNA MSTRG.24062.2-overexpressed组可看到较多的新骨形成,BV/TV升高1.29倍。(如图9所示)
在证实LncRNA MSTRG.24062.2能通过慢病毒载体被高表达后,RT-PCR及Western--blot检测种植体周骨组织结果显示,与lv5-nc组相比,LncRNA MSTRG.24062.2-enhancer组FZD9、Runx2的表达升高,而miRNA-125a-5p、miRNA-125b-5p的表达均降低。(如图10所示)
LncRNA MSTRG.24062.2过表达通过负调控miRNA-125a-5p和miRNA-125b-5p促进高脂血症大鼠的种植体骨结合,新骨形成增加,BV/TV、BIC%升高,提示高脂环境下种植体骨结合率降低与LncRNA MSTRG.24062.2表达降低与有着密切联系,揭示LncRNA作为骨生物标志物的潜在价值。
综合上述各种验证实验可知,本申请所提供的LncRNA MSTRG.24062.2可以通过结合miRNA-125a-5p和miRNA-125b-5p调控FZD9基因表达,从根本上解释了LncRNAMSTRG.24062.2的作用机理,为进一步临床诊疗和新型的靶向药物的研发提供理论和实验依据,同时该LncRNA MSTRG.24062.2还可以作为诊断早期骨质疏松的一个可行生物标志物,提高检测的准确性和速度。
<110>山东大学
<120>一种调控FZD9的非编码RNA及其应用
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>441
<212>DNA/RNA
<213>人工序列(人工序列)
<400>1
agggatccac cccaggcttt ctgcatgctt agcagatatt gtaccactta gggatcagac 60
atagacagtg tacactcagg gatcagacat agacagtgta ccagggatca gacatagaca 120
gtgtacactc agggatcaga cacagacagt gtacactcag ggatcagaca cagacacttg 180
tacactcagg gatcagacac agacactgta ctacttaggg atcagacaca ggcactgtac 240
tacttaggga tcagacatta tgccttcagc aacctgccat gcctatggat gaaatgaggt 300
caagaatatt gtaattatat cctaattata gtttgaggaa ttctttcagg aatttcctga 360
gtgaaataaa taaggaaact tagtttactg tttaatcata gtttttcctt tgcgtggcgc 420
tgactgacaa ctatttcccc t 441
<210>2
<211>453
<212>DNA
<213>人工序列(人工序列)
<400>2
gagctcaggg atccacccca ggctttctgc atgcttagca gatattgtac cacttaggga 60
tcagacatag acagtgtaca ctcagggatc agacatagac agtgtaccag ggatcagaca 120
tagacagtgt acactcaggg atcagacaca gacagtgtac actcagggat cagacacaga 180
cacttgtaca ctcagggatc agacacagac actgtactac ttagggatca gacacaggca 240
ctgtactact tagggatcag acattatgcc ttcagcaacc tgccatgcct atggatgaaa 300
tgaggtcaag aatattgtaa ttatatccta attatagttt gaggaattct ttcaggaatt 360
tcctgagtga aataaataag gaaacttagt ttactgttta atcatagttt ttcctttgcg 420
tggcgctgac tgacaactat ttcccctgtc gac 453
<210>3
<211>453
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<213>人工序列(人工序列)
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gagctcaggg atccacccca ggctttctgc atgcttagca gatattgtac cacttaggga 60
tcagacatag acagtgtaca tctgaaagtc agacatagac agtgtaccag ggatcagaca 120
tagacagtgt acatctgaaa gtcagacaca gacagtgtac atctgaaagt cagacacaga 180
cacttgtaca tctgaaagtc agacacagac actgtactac ttagggatca gacacaggca 240
ctgtactact tagggatcag acattatgcc ttcagcaacc tgccatgcct atggatgaaa 300
tgaggtcaag aatattgtaa ttatatccta attatagttt gaggaattct ttcaggaatt 360
tcctgagtga aataaataag gaaacttagt ttactgttta atcatagttt ttcctttgcg 420
tggcgctgac tgacaactat ttcccctgtc gac 453
Claims (5)
1.一种调控FZD9的非编码RNA,其特征在于:所述非编码RNA为LncRNA MSTRG.24062.2,其核苷酸序列如SEQ ID NO.1所示。
2.一种调控FZD9的非编码RNA的应用,其特征在于:所述的应用为过表达LncRNAMSTRG.24062.2基因。
3.根据权利要求2所述调控FZD9的非编码RNA的应用,其特征在于:过表达LncRNAMSTRG.24062.2载体在制备治疗骨质疏松药物中的应用。
4.根据权利要求3所述调控FZD9的非编码RNA的应用,其特征在于:过表达LncRNAMSTRG.24062.2载体为LncRNA MSTRG.24062.2和LV5-nc慢病毒载体病毒数目滴度分别为1×108的混合物。
5.一种调控FZD9的非编码RNA的应用,其特征在于:LncRNA MSTRG.24062.2作为早期骨质疏松生物标志物的应用。
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