CN116875600A - hsa_circHIPK2在骨关节炎产品上的应用 - Google Patents
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Abstract
本发明属于生物技术领域，具体公开了hsa_circHIPK2在骨关节炎产品上的应用，核苷酸序列如SEQ ID NO:1所示。本发明公开的hsa_circHIPK2在骨关节炎软骨中低表达，与膝关节骨关节炎程度成负相关，可以作为临床诊断生物标记物的潜在价值；hsa_circHIPK2能够促进软骨基质的合成与分泌、抑制软骨退变，有望作为治疗骨关节炎的药物，对诊断、治疗骨关节炎具有重要意义。
Description
技术领域
本发明属于生物技术领域，尤其涉及hsa_circHIPK2在骨关节炎产品上的应用。
背景技术
骨关节炎(Osteoarthritis,OA)是一种以关节疼痛、僵硬、功能障碍及畸形为主要表现的慢性关节疾病，导致患者生活质量降低。目前，全世界有超过5亿OA患者，患病率在人群中占约7％；我国约有1.4亿，65岁以上人群OA发病率超过80％。据调查，长期罹患OA的患者容易出现睡眠障碍、抑郁甚至残疾，致残致畸率高达20％。OA的治疗目前主要是早期给予非甾体类抗炎药等药物消炎镇痛，终末期则往往需要关节置换术缓解疼痛和恢复功能，造成了严重的医疗负担。OA的病理发生发展涉及多个组织和过程，但主要病理改变为软骨退变及损伤。因此，如何有效地阻止软骨退变、维持软骨细胞稳态一直是关节外科的研究热点及重点。
环状RNA(Circular RNA,circRNA)是一类特殊的非编码RNAs，其由前体mRNA反向剪切成，在组织中广泛表达，具有结构稳定、高度保守、生物功能多样等特点。可通过海绵结合miRNA、翻译蛋白、结合蛋白等途径发挥重要功能，circRNA已成为当下癌症、炎症、退行性疾病等领域的研究热点。近年来，circRNAs在骨关节炎发生和发展中的作用被大量研究。因此，通过circRNAs早期诊断和干预骨关节炎具有重要意义。
发明内容
本发明的目的在于提供hsa_circHIPK2在骨关节炎产品上的应用，以解决上述技术问题之一。
本发明提供的技术方案为：hsa_circHIPK2在骨关节炎产品上的应用，核苷酸序列如SEQ ID NO:1所示；产品为辅助临床诊断骨关节炎的试剂或者能够提高hsa_circHIPK2表达量的药物。
优选的，相对于正常软骨细胞，hsa_circHIPK2在骨关节炎软骨中低表达。
优选的，hsa_circHIPK2能够促进软骨基质的合成与分泌、抑制软骨退变。
优选的，用于制备诊断或治疗骨关节炎产品。
本发明的有益效果在于：
1、本发明可以通过检测hsa_circHIPK2的表达量，辅助临床诊断骨关节炎(Osteoarthritis,OA)，还可以作为药物治疗OA。
2、通过收集骨关节炎软骨与正常软骨样本，提取细胞，利用高通量测序方法，比较成正常软骨与骨关节炎软骨中circRNAs的表达谱，发现相对于正常软骨细胞(NA)，hsa_circHIPK2在骨关节炎软骨中低表达，并且通过实时荧光定量PCR(RT-qPCR)实验发现hsa_circHIPK2表达与膝关节OA程度成负相关，揭示了hsa_circHIPK2具有作为临床诊断生物标记物的潜在价值。另外，本发明利用过表达和敲低hsa_circHIPK2，在软骨细胞中检测成与软骨基质合成相关指标，发现hsa_circHIPK2能够促进软骨基质的合成与分泌、抑制软骨退变，且hsa_circHIPK2表达的降低，是导致骨关节炎发生的重要因素，这表明hsa_circHIPK2有望作为治疗骨关节炎的药物，对治疗骨关节炎具有重要意义。
附图说明
图1为软骨标本的测序结果图；其中，A：circRNAs测序热图；B：在OA、NA细胞中hsa_circHIPK2表达量结果；C为在OA、NA细胞中hsa_circHIPK2表达量结果；
图2为hsa_circHIPK2在软骨中的作用研究结果图；其中，A、B：在软骨细胞中敲低或过表达hsa_circHIPK2；C、D：在软骨细胞中敲低hsa_circHIPK2，qPCR和WB验证软骨细胞外基质合成指标COL2A1、SOX9、MMP13、ADAMTS4的表达变化；E、F：在软骨细胞中过表达hsa_circHIPK2，qPCR，WB验证软骨细胞外基质分解指标COL2A1、SOX9、MMP13、ADAMTS5的表达变化。
图3为鼠关节腔过表达hsa_circHIPK2对DMM小鼠模型OA进展的影响研究结果图。
具体实施方式
下面通过具体实施方式进一步详细说明：
以下实验所用到的软骨组织获取于广东省广州市中山大学附属第一医院就诊，并进行膝关节置换手术的患者，手术时获取成人患者的股骨髁和胫骨平台，后续用于提取软骨细胞。成人患者的年龄在40岁以上，临床诊断患有膝骨关节炎，Kellgren&Lawrence影像分级Ⅲ至Ⅳ级，Thompson大体形态学分级Ⅲ至Ⅳ级。
根据国际软骨修复学会(ICRS)分级系统，膝骨关节炎软骨可分成完整区(ICRS＝0)和磨损区(ICRS＝1-4)，以下实验按完整区及磨损区分开收集。将非磨损的完整区软骨细胞定义为相对正常软骨细胞(NA)。
将膝关节表面关节软骨切成1mm3大小并进行称重，加入4mg/mL蛋白酶(Roche，Inc)后放入37℃恒温无菌摇床，90转/分钟，消化90分钟，PBS清洗后加入胶原酶P(Roche)0.25mg/mL，0转/分钟，消化8-10小时，所获得细胞悬液过滤、离心后获得人膝关节软骨细胞，然后进行常规平面培养，具体培养方法如下：将离心后软骨细胞以配制10％FBS和1％青霉素/链霉素的DMEM F-12培养基(Gibico)重悬，种入6孔板，每孔细胞数约2×106-4×106，每3天换液一次。
1、通过高通量测序确定hsa_circHIPK2
采用 mini试剂盒(购买自Qiagen)分别提取正常软骨(NA软骨)细胞与骨关节炎软骨(OA)细胞中总RNA。利用高通量测序获得两组细胞circRNAs的表达谱，找出在NA软骨细胞核骨关节炎软骨细胞中差异表达的环状RNA hsa_circHIPK2。
图1为软骨标本的测序结果图，图1A为circRNAs测序热图；如图1A所示，相对于NA，hsa_circHIPK2在骨关节炎软骨中表达较NA软骨低。
2、采用实时荧光定量PCR(RT-qPCR)验证上述测序结果
分别收集10例不同患者来源的NA软骨及骨关节炎软骨，从组织中提取出正常软骨细胞和骨关节炎软骨细胞，提取外泌体总RNA并进行逆转录，随后通过RT-qPCR对hsa_circHIPK2进一步验证。
逆转录反应体系及各成分剂量见表1。准备新无酶200μl EP管，将各组分与总RNA混合，随后置于RT-PCR仪器中，按照以下条件进行逆转录反应：37℃孵育15分钟启动扩增，85℃孵育5秒以终止反应，随后将产物置于4℃保存备用。
表1 mRNA的逆转录反应混合液的配方
| 试剂组分 | 体积(μL) |
| 5x EVo M-MLVRTmAster Mix | 4 |
| 总RNA | 1μg/RNA浓度 |
| dd H2O(RNase/DNase free) | 与RNA体积补足16μL |
| 总体积 | 20 |
RT-qPCR检测采用2X Green Pro Taq HS Premix II(ROX plus)试剂(购买自AG生物)并根据其说明书进行实验，随后使用ABI Quant StudioTMReal-Time PCR仪器和Quant StudioTM Real-Time PCR软件进行分析。hsa_circHIPK2引物如下：
hsa_circHIPK2F1(SEQ ID NO:2)：5'-AGGTCTTATCCACGCTGACC-3'；
hsa_circHIPK2R1(SEQ ID NO:3)：5'-GAAGGGTGTGAGGGGAGAAA-3'。
内参引物如下：
hsa_GAPDH F1(SEQ ID NO:4)：5'-GGAGCGAGATCCCTCCAAAAT-3'；
hsa_GAPDH R1(SEQ ID NO:5)：5'-GGCTGTTGTCATACTTCTCATGG-3'。
对RT-qPCR测定得到的基因表达量进一步采用ΔΔCT法进行统计学分析，基因表达量的展示为2^(-ΔΔCT)，结果如图1B所示，hsa_circHIPK2在骨关节炎软骨中表达较NA软骨低。图1B和图1C为在OA细胞、NA细胞中hsa_circHIPK2表达量结果。
参考Kellgren-Lawrence Grade(0-4)分级系统，对不同退变程度的软骨进行分级，结合hsa_circHIPK2表达趋势进行相关性分析。图1C显示，软骨退变程度分级越高，hsa_circHIPK2表达越低，膝关节OA程度和hsa_circHIPK2表达趋势显著正相关，因此hsa_circHIPK2可以很好的作为早期OA的诊断分子。
3、利用过表达hsa_circHIPK2与敲低hsa_circHIPK2技术在软骨细胞中检测细胞外基质与成软骨分化相关指标，发现hsa_circHIPK2在软骨细胞退变进程中的抑制作用。
(1)构建过表达与敲低hsa_circHIPK2的质粒
选用pcDNA3.1作为载体构建过hsa_circHIPK2表达及hsa_circHIPK2敲低质粒，hsa_circHIPK2敲低序列(SEQ ID NO:6)：GTCCAGATATTACAGGTAT。
(2)稳定株构建成功后对软骨细胞进行总蛋白与总RNA提取
本实验使用3000转染试剂盒进行转染。按试剂说明书的比例，分别配置A液：125μl Opti-MEM、5μl />3000；B液：125μl Opti-MEM、5μl/>2μg待转染质粒。
(3)将A、B液轻柔且充分混合，室温下静置15分钟后进行细胞转染。利用RT-qPCR技术检测软骨退变相关指标，具体步骤与采用RT-qPCR验证测序结果的步骤相同，所用引物如下：
COL2A1F1(SEQ ID NO:7)：5'-CCAGATGACCTTCCTACGCC-3'；
COL2A1R1(SEQ ID NO:8)：5'-TTCAGGGCAGTGTACGTGAAC-3'；
SOX9F1(SEQ ID NO:9)：5'-AGCGAACGCACATCAAGAC-3'；
SOX9R1(SEQ ID NO:10)：5'-CTGTAGGCGATCTGTTGGGG-3'；
Aggrecan F1(SEQ ID NO:11)：5'-GATGTTCCCTGCAATTACCACCTC-3'；
Aggrecan R1(SEQ ID NO:12)：5'-TGATCTCATACCGGTCCTTCTTCTG-3'；
MMP13F1(SEQ ID NO:13)：5'-CCAGACTTCACGATGGCATTG-3'；
MMP13R1(SEQ ID NO:14)：5'-GGCATCTCCTCCATAATTTGGC-3'；
ADAMTS4F1(SEQ ID NO:15)：5'-GAGGAGGAGATCGTGTTTCCA-3'；
ADAMTS4R1(SEQ ID NO:16)：5'-CCAGCTCTAGTAGCAGCGTC-3'；
RUNX2F1(SEQ ID NO:17)：5'-CACTGGCGCTGCAACAAGA-3'；
RUNX2R1(SEQ ID NO:18)：5'-CATTCCGGAGCTCAGCAGAATAA-3'；
图2为hsa_circHIPK2在软骨中的作用研究结果图；图2A为在软骨细胞中敲低或过表达hsa_circHIPK2。结果如图2A所示，RT-qPCR结果显示在软骨细胞中过表达hsa_circHIPK2，可显著增加hsa_circHIPK2的表达水平，并且软骨保护性分子Aggrecan、COL2A1、SOX9的表达也较oe-NC组(过表达对照组)显著增加，而软骨破坏性分子MMP13、ADAMTS4、RUNX2的表达水平较oe-NC组显著减少。在软骨细胞中敲低hsa_circHIPK2，可显著减少hsa_circHIPK2的表达水平，并且软骨保护性分子Aggrecan、COL2A1、SOX9的表达也较sh-NC组(敲低对照组)显著减少，而软骨破坏性分子MMP13、ADAMTS4、RUNX2的表达水平较sh-NC组显著增加。
4、利用Western Blot技术检测软骨退变相关指标
使用PAGE凝胶快速制备试剂盒(购买自雅酶)制备凝胶。凝胶中每孔蛋白上样量为25μg，恒压80V电泳30分钟，更换恒压120V电泳60分钟。随后恒流250mA转膜90分钟，根据蛋白分子量适当增减转膜时间。在室温下用快速封闭液封闭10分钟(购买自雅酶)，一抗4℃孵育过夜，清洗后孵育相应种属二抗室温孵育1h。
所使用抗体如下：Aggrecan抗体，ADAMTS4抗体，SOX9抗体，GAPDH抗体，MMP13抗体，COL2A1抗体，RUNX2抗体(均购买自Proteintech)。
配制超敏ECL发光液(购买自Merck millipore)并使用成像仪(ChemiDoc Touch，BIO-RAD)获取条带图像。
图2为hsa_circHIPK2在软骨中的作用研究结果图；图2B为在软骨细胞中敲低或过表达hsa_circHIPK2。实验结果如图2B所示，在软骨细胞中过表达hsa_circHIPK2，软骨保护性分子Aggrecan、COL2A1、SOX9的蛋白表达水平较oe-NC组显著增加，而软骨破坏性分子MMP13、ADAMTS4、RUNX2的蛋白表达水平较oe-NC组显著减少。在软骨细胞中敲低hsa_circHIPK2，软骨保护性分子Aggrecan、COL2A1、SOX9的蛋白表达水平较sh-NC组显著减少，而软骨破坏性分子MMP13、ADAMTS4、RUNX2的蛋白表达水平较sh-NC组显著增加。
上述实验结果表明hsa_circHIPK2对软骨细胞有保护作用。
5、采用动物实验验证hsa_circHIPK2能够抑制关节软骨退变
(1)小鼠同源hsa_circHIPK2为mmu_circ_0001468，根据该circRNA序列信息构建过表达腺相关病毒(hsa_circHIPK2过表达腺相关病毒)。
(2)选取24只12周龄C57BL/6小鼠进行实验，并随机将小鼠分为四种：
假手术组(Sham)、DMM组、DMM+空载腺相关病毒组(vector)、DMM+hsa_circHIPK2过表达腺相关病毒组(oe hsa_circHIPK2)。
除假手术组(Sham)外，其余三组于小鼠左膝进行内侧半月板失稳(DMM)手术。两周后于Sham组、DMM组注射同等剂量生理盐水，DMM+vector组注射空载腺相关病毒，DMM+oehsa_circHIPK2组注射hsa_circHIPK2过表达腺相关病毒，连续注射4周，术后第10周处死小鼠收取标本。
(3)标本使用micro-CT、HE染色、番红O固绿染色、阿利新蓝染色和免疫组织化学(IHC)染色，并按照国际关节炎研究协会(OARSI)系统分析骨性关节炎情况及骨赘情况。
图3为鼠关节腔过表达hsa_circHIPK2对DMM小鼠模型OA进展的影响研究结果图。实验结果如图3所示，小鼠关节腔内注射过表达hsa_circHIPK2后，关节标本关节炎程度低于DMM+vector组，说明过表达hsa_circHIPK2能够抑制软骨退变，促进软骨基质的合成与分泌。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
Claims (4)
1.hsa_circHIPK2在骨关节炎产品上的应用，其特征在于，核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的hsa_circHIPK2在骨关节炎产品上的应用，其特征在于，相对于正常软骨细胞，hsa_circHIPK2在骨关节炎软骨中低表达。
3.根据权利要求1所述的hsa_circHIPK2在骨关节炎产品上的应用，其特征在于，hsa_circHIPK2能够促进软骨基质的合成与分泌、抑制软骨退变。
4.根据权利要求1所述的hsa_circHIPK2在骨关节炎产品上的应用，其特征在于，用于制备诊断或治疗骨关节炎产品。
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