CN117467770A - 一种结肠癌诊断生物标志物及其应用 - Google Patents
一种结肠癌诊断生物标志物及其应用 Download PDFInfo
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- CN117467770A CN117467770A CN202311700000.8A CN202311700000A CN117467770A CN 117467770 A CN117467770 A CN 117467770A CN 202311700000 A CN202311700000 A CN 202311700000A CN 117467770 A CN117467770 A CN 117467770A
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Classifications
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
本发明公开了一种结肠癌诊断生物标志物及其应用,所述生物标志物为基因LINC00963,核苷酸序列如SEQ ID NO.1所示,位于第9号染色体,长度为2123bp。本发明揭示了基因LINC00963在结肠癌细胞中的重要角色,其表达水平在结肠癌细胞中显著升高,因此可以作为结肠癌检测的有效生物标志物。同时本发明提出了一种以基因LINC00963为靶点,制备功能性表达抑制剂来预防或治疗结肠癌的方案。这一发现为结肠癌的治疗提供了新的策略和方向,具有极高的临床应用价值,有望为结肠癌患者带来更好的治疗效果。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种结肠癌诊断生物标志物及其应用。
背景技术
结直肠癌,简称CRC,是全球病患数量最庞大的恶性肿瘤之一。由于早期症状难以察觉,近八成的病患在确诊时已发展到晚期。然而,这种疾病的发病机制和治疗方案仍有待深入探索。
近年来,科学家们在肿瘤的分子生物学、细胞遗传学、免疫学等领域取得了显著进展,为CRC的治疗也带来了新的希望。长非编码RNA(lncRNA)是一类不编码蛋白质、长度超过200个碱基的RNA分子。它们在细胞的生命活动中起到关键作用,而它们在肿瘤的发生和进展中也逐渐受到科学家们的关注。
LINC00963就是这样一种lncRNA,它位于第9号染色体上,产生一个长度为2123个碱基的lncRNA。这个基因在转录的表观遗传调控中发挥重要作用,并已被证实存在差异表达,与多种人类肿瘤如骨肉瘤、黑色素瘤、前列腺癌和肾癌的发生有关。然而,LINC00963在CRC中的角色和功能仍然是一个谜。这个新近被发现的在结肠癌中异常表达的lncRNA,其调控机制及对病理的作用尚未被详细报道。
发明内容
针对现有技术的不足,本发明提供一种结肠癌诊断生物标志物及其应用,为结肠癌的治疗提供了新的策略和方向,具有极高的临床应用价值。
本发明是通过以下技术方案实现的:
一种结肠癌诊断生物标志物,所述生物标志物为基因LINC00963,核苷酸序列如SEQ ID NO.1所示,位于第9号染色体,长度为2123bp。
用于检测上述的生物标志物的产品在制备结肠癌诊断试剂盒中的应用。
优选地,所述产品为检测试剂,所述检测试剂包括引物对,所述引物对的核苷酸序列如SEQ ID NO.2-3所示。
上述的生物标志物的功能性表达抑制剂在制备治疗结肠癌的药物和/或辅助药物中的应用。
优选地,所述功能性表达抑制剂为shRNA慢病毒、RNAi、sgRNA、ZFN、TALEN元件或同源重组载体。
优选地,所述shRNA慢病毒的核苷酸序列如SEQ ID NO.5所示。
一种治疗结肠癌的药物组合物和/或辅助药物组合物,包括上述的功能性表达抑制剂,以及药学上可接受的辅料。
优选地,所述辅料包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂中的一种或多种。
优选地,所述药物组合物为口服剂型或非口服剂型。
优选地,所述药物组合物为片剂、胶囊剂、散剂、丸剂、颗粒剂、溶液剂、混悬剂、糖浆剂、注射剂、栓剂、吸入剂或喷雾剂。
本发明的有益效果如下:
(1)揭示了基因LINC00963在结肠癌细胞中的重要角色,其表达水平在结肠癌细胞中显著升高,因此可以作为结肠癌检测的有效生物标志物。
(2)提出了一种以基因LINC00963为靶点,制备功能性表达抑制剂来预防或治疗结肠癌的方案。
(3)为结肠癌的治疗提供了新的策略和方向,具有极高的临床应用价值,有望为结肠癌患者带来更好的治疗效果。
附图说明
图1为实施例1中荧光定量PCR检测基因LINC00963在结肠癌细胞LoVo及SW480中的表达情况;
图2为实施例2中体外实验的细胞划痕图(A)和细胞增殖图(B);
图3为实施例2中体外实验的细胞侵袭图(A)及统计图(B);
图4为实施例2中体内实验的肿瘤图(A)及肿瘤体积统计图(B)。
具体实施方式
下面结合附图与具体实施例对本发明做进一步详细说明。
若无特殊说明,以下实施例中所用的技术手段,均为本领域技术人员所熟知的常规手段,未注明具体条件的实验方法,均为本领域常规方法。
以下实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中所用实验动物是6周龄的BALB/c裸鼠购自Gem pharma(中国南京)。
以下实施例中所用到的结直肠癌细胞株SW480、LoVo来自American Type CultureCollection(ATCC,USA)。
实施例1
本实施例选用SW480、LoVo细胞作为结肠癌细胞的代表进行实验,通过荧光定量PCR检测了结肠癌细胞SW480和LoVo中基因LINC00963的表达情况,具体如下:
1、应用Trizol试剂(Invitrogen,Carlsbad,USA)从细胞和组织样品中取总RNA。
(1)吸尽6孔板中结肠癌细胞SW480、LoVo的培养液,每孔加入1mL Trizol,使其覆盖细胞,再用吸管或加样器吹打3次,细胞应完全裂解,然后转移至离心管中。
(2)在装有裂解物的离心管中加入0.2mL的氯仿(1mL Trizol加入0.2mL氯仿),振荡器上充分振荡混匀20s,室温放置5min。12000rpm 4℃离心10min,然后吸取含总RNA的上层水相至一新的离心管中,每毫升Trizol约可吸取0.6mL上层水相。有机相和中间层含有DNA和蛋白质,应避免触及。
(3)加入和上层水相等体积的异丙醇,颠倒数次混匀,室温沉淀5min。12000rpm 4℃离心15min,在管底可见RNA沉淀。弃上清,按每毫升Trizol加入1mL75%乙醇,轻轻颠倒混匀,以清洗RNA沉淀。12000rpm 4℃离心2min,弃去液体,小心勿丢弃RNA沉淀。室温倒置晾干5~10min。
(4)溶解:加入适量DEPC处理水使RNA沉淀溶解。存放于-80℃。
2、应用M-MLV逆转录酶合成第一链cDNA。
3、在总体积为20μL的SYBR Green PCR Master Mix(Roche,Germany)中进行实时定量PCR(qPCR)。所有反应均一式两份进行。使用2-ΔΔCT法方法计算基因LINC00963的相对表达量。用于磷酸化AKT的PCR引物在Invitrogen合成。以GAPDH作为内参照。基因LINC00963的引物对序列具体如下:
上游引物(SEQ ID NO.2):5’-GTCAGGCCACTCTGCTACTG-3’;
下游引物(SEQ ID NO.3):5’-CCACTGCGATGGTTGTGCTC-3’。
基因LINC00963,核苷酸序列如SEQ ID NO.1所示。
4、实验结果
如图1所示,基因LINC00963在结肠癌细胞中的表达明显上调,且在高侵袭性细胞LoVo中的表达量相对较高,而在低侵袭性细胞SW480中的表达量相对较低(**P<0.01)。
本实施例的实验结果表明,基因LINC00963在结肠癌细胞中的表达明显上调,因此可以作为检测结肠癌的一个有效指标(如生物标志物)。
实施例2
为了深入研究基因LINC00963在结肠癌预防和治疗中的作用,本实施例构建了体外LINC00963敲除结肠癌细胞模型和体内LINC00963敲除结肠癌原位移植瘤模型,以验证基因LINC00963的敲除是否能抑制结肠肿瘤的生长并促进癌细胞凋亡。具体如下:
1、体外实验
(1)将对数生长期的结直肠癌细胞SW480和LoVo置于6孔板中,分别转染LINC00963干扰慢病毒(LINC00963-shRNA)或NC-shRNA(均购自上海和元生物技术股份有限公司),LINC00963-shRNA的感染复数(MOI)为20,转染后24h更换培养基。转染48h后,加入1μg/mL嘌呤霉素进行筛选。qPCR检测LINC00963的表达以验证转染成功。
NC-shRNA(SEQ ID NO.4):5’-UUCUCCGAACGUGUCACGUTT-3’;
LINC00963-shRNA(SEQ ID NO.5):5’-GGGTCAAAGGAGAGCACAA-3’。
(2)CCK-8实验
96孔板分别接种经转染LINC00963干扰慢病毒(LINC00963-shRNA)或NC-shRNA处理的LoVo细胞,密度分别为1.5×103细胞。在含10% FBS的DMEM培养基中,于37℃、5% CO2培养箱中培养24h、48h、72h。随后,在96孔板中加入10μL CCK-8(道进岛,日本)并培养1h。最后,使用酶标仪(BioTek,美国)在450nm下计数和分析细胞(BioTek,美国)。
(3)细胞划痕实验
将转染LINC00963干扰慢病毒(LINC00963-shRNA)或NC-shRNA处理的LoVo细胞(2×105细胞/孔)接种于6孔板,在37℃、5% CO2培养箱中培养。200μL移液管尖端从细胞单层产生浆液直到细胞融合达到90%。将脱落的细胞用PBS轻轻洗涤3次。用新鲜无血清培养基孵育24h后,倒置显微镜下观察创面形态;迁移率=[0~24h时创面宽度(WW)/0h时创面宽度(WW)]。
(4)Transwell实验
在细胞侵袭实验中,将上述稳定转染细胞加入Transwell迁移小室上层(孔径8μm),在含有1%胎牛血清的培养基中覆盖Matrigel(356234,Biocoat,美国)。下室加入含10%胎牛血清的培养基,平板置于含5% CO2的37℃培养箱中培养24h。转染LINC00963干扰慢病毒(LINC00963-shRNA)或NC-shRNA处理的LoVo细胞培养12h,用棉签擦拭上室底部。用甲醇固定30min,结晶紫染色30min,显微镜下计数。
2、体内实验
6周龄的BALB/c裸鼠饲养在标准的12h光/暗循环条件下。将5×106感染了shRNA慢病毒的LoVo细胞(LINC00963-shRNA,LINC00963干扰组)与空载干扰组(NC-shRNA)以及正常对照组(Control),然后将其注射到BALB/c裸鼠背部皮下。于种瘤后7、14、28d测量肿瘤体积。肿瘤体积(mm3)通过长度(mm)×宽度(mm)×高度(mm)×1/2计算。所有三组裸鼠都成功成瘤,成瘤时间平均为10天。在成瘤后20天,对小鼠进行安乐处死,并取瘤称重。
3、实验结果
体外实验如图2中A所示,与空载干扰组(NC-shRNA)相比,感染了shRNA慢病毒的LoVo细胞(LINC00963-shRNA)其增殖能力明显受到抑制,细胞增殖活性显著减弱(**P<0.01)。如图2中B所示,LINC00963的敲除(LINC00963-shRNA)也明显抑制了结肠癌细胞的迁移能力(**P<0.01)。同时,如图3所示,与对照组(NC-shRNA)相比,LINC00963的敲除(LINC00963-shRNA)也明显抑制了结肠癌细胞的侵袭能力(**P<0.01)。
体内实验如图4所示,LINC00963干扰的转染病毒组(LINC00963干扰组)的肿瘤体积明显低于空载干扰组和空白对照组(**P<0.01),这表明敲除LINC00963能显著抑制结肠癌细胞的增殖能力。
本实施例的实验结果表明,敲除LINC00963能显著抑制结肠癌细胞的增殖能力,同时敲除LINC00963还能明显促进结肠癌细胞的凋亡。因此开发基因LINC00963的功能性表达抑制剂(如本实施例的shRNA慢病毒)可以用于制备治疗结肠癌的药物和/辅助药物。
据文献记载,除了shRNA慢病毒,还有多种技术手段可以实现抑制LINC00963的功能性表达,或者抑制LINC00963下游物质的功能性表达,从而起到抑制LINC00963用于预防或治疗结肠癌方面的作用。这些技术手段包括但不限于其他RNAi技术,例如通过其他siRNA抑制LINC00963的功能性表达;成簇规律间隔短回文重复(CRISPR/Cas9技术为代表)技术,使用一段序列特异性向导RNA分子(sgRNA)引导核酸内切酶到靶点处,从而完成基因组编辑;锌指核酸酶(ZFN)技术,通过加工改造ZFN的锌指DNA结合域,靶向定位于不同的DNA序列;转录激活样效应因子核酸酶(TALEN)技术,通过DNA识别模块将TALEN元件靶向特异性的DNA位点并结合,然后在FokI核酸酶的作用下完成特定位点的剪切,并借助于细胞内固有的同源定向修复(HDR)或非同源末端连接途径(NHEJ)修复过程完成特定序列的插入(或倒置)、删失及基因融合;以及利用同源重组载体敲除基因的技术,例如Cre/LoxP系统或来自酵母的FLP-frt系统,利用基因同源重组原理,用设计的同源片段替代靶基因片段,从而达到基因敲除的目的。
本发明利用在结直肠癌细胞中显著高表达的LINC00963,有效地用于结直肠癌的检测。通过构建体外LINC00963敲除的结直肠癌细胞模型和体内LINC00963敲除的结直肠癌皮下肿瘤模型,我们进行了系统的实验研究。这些研究明确展示了LINC00963能显著抑制结直肠癌细胞的增殖能力,并显著促进其凋亡。因此,敲除LINC00963具有预防或治疗结直肠癌的潜力,使我们得以开发以LINC00963为功能性表达抑制剂的新型治疗方法,对抗结直肠癌。
因此,本发明的目的在于研发一种LINC00963功能性表达抑制剂用于制备治疗结肠癌的药物组合物和/或辅助药物组合物。为实现这一目标,本发明提出使用针对LINC00963的RNAi、针对LINC00963的shRNA慢病毒、针对LINC00963的sgRNA(需配合CRRSPR/Cas9使用)、针对LINC00963的ZFN(需配合ZFN技术使用)、针对LINC00963的TALEN元件(需配合TALEN技术使用)以及针对LINC00963的同源重组载体(利用Cre/LoxP系统或来自酵母的FLP-frt系统)。此外,本发明建议在药物组合物中加入其他药类以及药学上可接受的载体,包括:稀释剂如乳糖、氯化钠、葡萄糖、尿素、淀粉和水等,填充剂如淀粉和蔗糖等,粘合剂如单糖浆、葡萄糖溶液、淀粉溶液、纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮,湿润剂如甘油,崩解剂如干淀粉、海藻酸钠、海带多糖粉末、琼脂粉末、碳酸钙和碳酸氢钠,吸收促进剂如季铵化合物和十二烷基硫酸钠等,表面活性剂如聚氧化乙烯山梨聚糖脂肪酸酯、十二烷基硫酸钠、硬脂酸单甘油酯和十六烷醇等,致湿剂如甘油和淀粉等,吸附载体如淀粉、乳糖、斑脱土、硅胶和高岭土等,以及润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇和硼酸粉末等。
综上所述,本发明的研究结果表明,基因LINC00963在结肠癌的诊断和治疗中具有重要作用。敲除LINC00963可以显著抑制结肠癌细胞的增殖能力,并促进其凋亡。此外,本发明的研究还为开发LINC00963功能性表达抑制剂用于制备治疗结肠癌的药物和/或辅助药物提供了重要依据。
以上所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。本发明的保护范围以权利要求书要求的范围为准,基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
Claims (10)
1.一种结肠癌诊断生物标志物,其特征在于,所述生物标志物为基因LINC00963,核苷酸序列如SEQ ID NO.1所示,位于第9号染色体,长度为2123bp。
2.用于检测权利要求1所述的生物标志物的产品在制备结肠癌诊断试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述产品为检测试剂,所述检测试剂包括引物对,所述引物对的核苷酸序列如SEQ ID NO.2-3所示。
4.权利要求1所述的生物标志物的功能性表达抑制剂在制备治疗结肠癌的药物和/或辅助药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述功能性表达抑制剂为shRNA慢病毒、RNAi、sgRNA、ZFN、TALEN元件或同源重组载体。
6.根据权利要求5所述的应用,其特征在于,所述shRNA慢病毒的核苷酸序列如SEQ IDNO.5所示。
7.一种治疗结肠癌的药物组合物和/或辅助药物组合物,其特征在于,包括权利要求5所述的功能性表达抑制剂,以及药学上可接受的辅料。
8.根据权利要求7所述的药物组合物,其特征在于,所述辅料包括稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体和润滑剂中的一种或多种。
9.根据权利要求7所述的药物组合物,其特征在于,所述药物组合物为口服剂型或非口服剂型。
10.根据权利要求7所述的药物组合物,其特征在于,所述药物组合物为片剂、胶囊剂、散剂、丸剂、颗粒剂、溶液剂、混悬剂、糖浆剂、注射剂、栓剂、吸入剂或喷雾剂。
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