CN106906215B - 一种与卵巢癌和子宫内膜癌相关的长链非编码RNA-TDRG1的siRNA和应用 - Google Patents
一种与卵巢癌和子宫内膜癌相关的长链非编码RNA-TDRG1的siRNA和应用 Download PDFInfo
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Abstract
本发明涉及生物医学领域，具体涉及长链非编码RNA‑TDRG1和siRNA‑TDRG1在卵巢癌和子宫内膜癌诊断和治疗的作用。所述的siRNA的sense和antisense的核苷酸序列分别为CCUUCCCAGGUCUAGGUUC；GAACCUAGACCUGGGAAGG。所述的长非编码RNA‑TDRG1可以作为诊断卵巢癌和子宫内膜癌的标志物。所述的siRNA‑TDRG1可以用于制备抗卵巢癌和子宫内膜癌药物。
Description
技术领域
本发明涉及生物医学领域，具体涉及长链非编码RNA-TDRG1和siRNA-TDRG1在卵巢癌和子宫内膜癌诊断和治疗的作用。
背景技术
卵巢癌是最致命的妇科恶性肿瘤之一。由于缺乏有效的筛选策略，约60%以上的上皮性卵巢癌患者被诊断为晚期疾病。到目前为止，卵巢癌治疗的基石涉及分期/减瘤手术和个别腹腔或静脉铂基辅助化疗。尽管这种模式的高级阶段呈现，五年存活率是46%。子宫内膜癌作为妇科最常诊断的恶性肿瘤之一，其发病率仅次于宫颈癌，且其发病率成上升趋势。子宫内膜癌的发生与高血压、糖尿病、肥胖、雌激素暴露等高危因素密切相关。尽管随着B超、分段诊刮、宫腔镜检查、细胞学检查等诊断方法以及手术、放化疗、激素等治疗技术的成熟，子宫内膜癌早期诊断5年生存率和预后较好，但其晚期死亡率仍处于一个较高的水平，生存率低于20%。然而，现有卵巢癌和子宫内膜癌诊断和治疗方法的存在诸多不足。越来越复杂的实验已经被用来研究靶向治疗，以提高卵巢癌和子宫内膜癌患者的五年存活率，这已经成为一个重要的问题。
作为妇女中最常诊断的癌症，上皮性卵巢癌和子宫内膜癌的发展和进展仍然是开放的研究领域。长非编码RNA（lncRNA）在妇科肿瘤中的作用是一个新兴的研究领域。长链非编码RNA（lncRNA）最近已经作为在各种生物学环境（例如肿瘤生长和发育，细胞凋亡，增殖，分化和自噬）中的基因表达的中心调节剂出现。因此，它们与致癌作用和其他疾病密切相关，拓宽了基因调节和细胞复杂性的范围。人睾丸发育相关基因1（TDRG1）是一种近期确定的基因，仅在睾丸中表达，并促进睾丸生殖细胞肿瘤的发展。然而，TDRG1在妇科肿瘤中的功能是未知的。
发明内容
针对上述问题，本发明提供一种与卵巢癌和子宫内膜癌相关的长链非编码RNA-TDRG1的siRNA-TDRG1及其应用，并研究设计TDRG1基因在制备卵巢癌和子宫内膜癌诊疗药物中的应用。
为了实现上述目的，本发明提供一种与卵巢癌和子宫内膜癌相关的长链非编码RNA TDRG1，其DNA序列如SEQ.ID. NO.1所示；针对该长链非编码RNA设计的siRNA- TDRG1，所述的siRNA的sense和antisense的核苷酸序列分别如SEQ.ID.NO.2和SEQ.ID.NO.3所示。
SEQ.ID.NO.2 ： CCUUCCCAGGUCUAGGUUC。
SEQ.ID.NO.3 ： GAACCUAGACCUGGGAAGG。
所述的长非编码RNA-TDRG1可以作为诊断卵巢癌和子宫内膜癌的标志物。
所述的siRNA-TDRG1可以用于制备抗卵巢癌和子宫内膜癌药物。
本发明的有益效果。
本发明发现TDRG1基因在正常组织中呈低水平表达状态，而在卵巢癌和子宫内膜癌组织中，TDRG1基因成高水平表达。TDRG1的上调诱导肿瘤细胞的增殖，抑制其凋亡。此外，TDRG1表达与分化成正相关。本发明首次发现了TDRG1在卵巢癌和子宫内膜癌的发生和发展中发挥重要作用。因此，通过检测TDRG1基因在受试者中的水平可以判断受试者是否患有卵巢癌和内膜癌，通过利用siRNA可以制备相应的抗肿瘤药物来进行相应的临床治疗，弥补现有诊断和治疗方法的不足，提高诊断的阳性率，完善治疗方案，最终提高卵巢癌和内膜癌的生存率，减少复发率和死亡率。因此，本发明发现TDRG1基因可以作为卵巢癌和子宫内膜癌的诊断的肿瘤标志物，其siRNA可以作为肿瘤的药物靶标。
本发明提供的与卵巢癌和子宫内膜癌相关的LncRNA，是由上海SIGMA生物公司独家的Rosetta算法，确保基因设计时的高效特异性基础上设计siRNA序列；其质粒序列由苏州吉玛基因股份有限公司设计。分别将质粒载体和si-RNA干扰片段转染到卵巢癌细胞系A2780、OVCAR3及子宫内膜癌细胞系HEC-1B中，发现肿瘤细胞的增殖活性上升/下降，因此认为该基因的功能与癌细胞的增殖能力相关。本研究首次发现TDRG1在EOC组织和子宫内膜组织中的表达显着高于正常组织。TDRG1的异常表达与卵巢癌和子宫内膜癌的发生和发展密切相关，这为卵巢癌和子宫内膜癌的临床诊断和治疗提供了新的突破口。
附图说明
图1 qRT-PCR检测TDRG1在卵巢癌和内膜癌组织中的表达情况。
图2 qRT-PCR检测TDRG1或者siRNA-TDRG1转染后在卵巢癌和内膜癌细胞中的表达情况。
图3 MTT实验检测TDRG1对卵巢癌和内膜癌细胞的增殖的影响。
图4 凋亡实验检测TDRG1对卵巢癌和内膜癌细胞的凋亡的影响。
图5 MTT实验检测siRNA-TDRG1对卵巢癌和内膜癌细胞的增殖的影响。
图6 凋亡实验检测siRNA-TDRG1对卵巢癌和内膜癌细胞的凋亡的影响。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。以下实施例将有助于对本发明的了解，但这些实施例仅为了对本发明加以说明，本发明并不限于这些内容。在实施例中未作特殊说明的操作方法均为本技术领域常规操作方法。
实施例1。
TDRG1应用于卵巢癌和子宫内膜癌的诊断。
1、卵巢癌和内膜癌标本采集。
从中国医科大学第一附属医院妇科系（中国沈阳）手术切除的患者收集卵巢癌组织、子宫内膜癌组织和对应的正常组织标本；标本采集的患者没有接受术前化疗或放疗；在患者知情的情况下进行标本的采集，并且经过了中国医科大学伦理委员会批准。
2、引物设计。
PCR检测所用引物。
上游引物（SEQ.ID.NO.4）：TCTTCCCTGGCTTGGC。
下游引物（SEQ.ID.NO.5）：TGGGCTCTTTCGTGGC。
3、应用qRT-PCR方法分别检测TDRG1在卵巢癌、正常卵巢组织及子宫内膜癌组织、正常子宫内膜组织的表达量。
3.1收集的组织标本总RNA提取：取采集的标本组织，放到1.5mlEP管中，加入1mlTrizol，剪碎组织；来回震荡数次；加入200ul三氯甲烷，剧烈摇动30秒，静置5到10分钟；12000r离心20分钟；将上清移入另一个EP管中；加入等体积异丙醇，-20℃过夜；12000r离心20分钟；弃上清，加75%冰乙醇1ml，振荡；7500r离心20分钟；弃上清，用剪好的滤纸小心吸去沉淀周围残留液体，晾干5-10分钟；加入适量（10-20ul）的RNase-free水溶解沉淀后，用UV-2800A型紫外可见分光光度计测定RNA浓度和纯度（定量RNA浓度1ug/ul， OD260/OD2801.8-2.0之间表示RNA纯度较高）。
3.2逆转录合成cDNA。
采用 GoScript 反转录系统（A5000、A5001），按照如下操作步骤进行逆转录得到的 Cdna。
第一步：取一定量模板 RNA 加入引物。
RNA( 1µg/ul) 5 μl。
Random Primers (0.5 µg /ul) 1 μl。
Oligo(dT)15 Primer (0.5 µg/ul) 1 μl。
Nuclease-Free Water (加至 10 μl) 3 μl。
第二步：将模板 RNA 与反转录引物（ Random Primers和Oligo(dT)15 Primer）的混合物进行 70℃、5 min 预变性，完成后取出置于冰上。
第三步：配制 RT -Mix，向每个样品管加入10 μl。
组分 反转录混合液 终浓度。
Nuclease-Free Water 1.6 μl 。
GoScript™ 5X Reaction Buffer 4 μl 1X。
MgCl2 (25 mM) 2 μl 2.5mM。
PCR Nucleotide Mix 1 μl 0.5mM。
Recombinant RNasin® Ribonuclease Inhibitor 0.4 μl 20units。
GoScript™ Reverse Transcriptase 1 μl。
第四步：设置反转录程序，包括退火、延伸、逆转录酶失活三步（退火25℃ 5min，延伸42℃ 60 min，失活70℃ 15 min，4℃ + ∞。程序完成后得到 cDNA。
3.3 Real-time PCR。
（1）按下列配置PCR反应混合液（反应液配置可在室温进行），并分至各反应管，然后加入2ul模板。
组分 体积（20ul反应体系） 终浓度。
Nuclease-Free Water 7 μl 。
上游引物（10 uM) 0.4ul 0.2uM。
下游引物（10 uM) 0.4ul 0.2uM。
GoTaq®qPCR Master Mix,2X 10ul 1X。
CXR 100X 0.2ul 1X。
（2）采用ABI PRISM®7500 Real-Time PCR System两步法进行PCR标准扩增程序。
（3）导出数据，Realtime PCR结果用2-△△CT法进行分析。利用Graphad Prism软件分别绘制出卵巢癌和正常卵巢组织、子宫内膜癌及正常子宫内膜组织表达水平的图表，结果如图1-1至图1-4和图2；其中图1-1表示lncRNA TDRG1表达在上皮性卵巢癌中比在正常卵巢组织中显着升高；1-2表示 lncRNA TDRG1在分化程度高组较在低/中度分化组低，*P <0.05；1-3表示lncRNA TDRG1在浆液性腺癌中比其它类型的癌症中的表达低；1-4表示lncRNA TDRG1表达在子宫内膜癌中比在正常子宫内膜组织中显着增高；图2中2-1，2-2，2-5表示转染lncRNA TDRG1后的卵巢癌及内膜癌细胞中的lncRNA TDRG1的表达显着增高；图2中2-3，2-4，2-6表示转染lncRNA si-TDRG1后的卵巢癌及内膜癌细胞中的lncRNA TDRG1的表达显着下降，说明lncRNA TDRG1与卵巢癌、子宫内膜癌发生有关。
3、细胞培养和转染。
卵巢癌细胞株A2780、OVCAR3购自中科院细胞库，子宫内膜癌细胞株HEC-1B、Ishikawa购自中科院细胞库，细胞培养基DMEM/RPMI 1640和胎牛血清均购自HyClone公司，TDRG1质粒表达载体购自苏州吉玛技术有限公司，siRNA干扰片段序列由上海SIGMA生物公司合成，RNA抽提试剂RNAiso Plus购自宝生物工程有限公司，逆转录试剂盒High CapacitycDNA Reverse Transcription Kits购自Invitrogen公司，定量PCR试剂盒Power SYBRGreen PCR Master Mix购自Invitrogen公司，TDRG1基因和管家基因18S引物由上海生工生物工程技术服务有限公司合成。
将A2780和OVCAR3人卵巢癌细胞系和HEC-1B人类子宫内膜癌细胞系用补充有青霉素/链霉素（100U / mL）和10％胎牛血清的Dulbecco's改良的Eagle's培养基（A2780细胞和HEC-1B； HyClone，Logan，UT，USA）或RPMI 1640（OVCAR3细胞，HyClone）在5％CO 2中在37℃下进行培养。根据制造商的说明书使用Lipofectamine 2000进行TDR1质粒转染。
4、MTT测定。
将细胞以3000个细胞/孔的密度接种在96孔板中；在瞬时转染后0小时，24小时，48小时和72小时，将细胞与20μL MTT（5mg/mL）在37℃下孵育4小时；然后除去培养基，将沉淀溶于150μl二甲基亚砜中；使用微孔板分光光度计（BioTek Instruments，Winooski，VT，USA）在490nm处检测吸光度，结果见图3。图3-1、3-2表示转染lncRNA TDRG1后促进卵巢癌细胞的增殖；图3-3表示转染lncRNA TDRG1后促进子宫内膜癌细胞的增殖。
5、凋亡测定。
对于TDRG1质粒转染，在根据制造商的方案用7AAD和PE标记的膜联蛋白V（BDBiosciences）染色后使用流式细胞术定量凋亡；转染后48小时收集细胞，用冷PBS洗涤两次，以1×10 6细胞/ mL重悬，与100μL1×缓冲液和5μL膜联蛋白V-PE和7AAD混合，在黑暗中孵育15分钟，加入400μL1×缓冲液，并在1小时内对细胞进行流式细胞术分析，结果见图4。图4-1表示转染lncRNA TDRG1后抑制卵巢癌细胞的凋亡；图4-2表示转染lncRNA TDRG1后抑制子宫内膜癌细胞的凋亡。
结果发现，TDRG1基因在肿瘤组织和细胞内高表达而在正常组织和细胞中低表达；转染TDRG1入细胞后，肿瘤的增殖能力增强；TDRG1基因导致肿瘤细胞的凋亡减少；因此，TDRG1基因的表达水平可以作为肿瘤是否存在的特异性指标，也可以为肿瘤的发生发展提供判断依据。故TDRG1基因可以成为一种新的诊断卵巢癌和子宫内膜癌的方法。
实施例2。
siRNA-TDRG1应用于卵巢癌和子宫内膜癌的治疗。
1、细胞培养和转染。
细胞培养方法和条件如前所述，后根据制造商的说明书使用Lipofectamine 2000进行SiRNA- TDRG1转染。
2、MTT测定。
实验步骤如实施例1所述，最终测定SiRNA- TDRG1转染后对肿瘤细胞增殖的影响。
3、凋亡测定。
siRNA转染后，根据制造商的方案在PI和FITC标记的膜联蛋白V（BD Biosciences）染色后进行流式细胞术，以检测磷脂酰丝氨酸外化作为早期凋亡的终点指标；在48小时孵育后，将细胞用冰冷的PBS洗涤两次，以1×106个细胞/ mL重悬于100μL1×结合缓冲液中，并用5μLFITC-膜联蛋白V和PI孵育。 轻轻涡旋样品，在室温下在黑暗中孵育15分钟，然后向每个管中加入400μL1×结合缓冲液，并在1小时内对细胞进行流式细胞术分析。
结果发现，如图5-1、5-2,siRNA-TDRG1转染入卵巢癌细胞后，可以降低卵巢癌细胞的增殖能力；如图5-3，siRNA-TDRG1转染入子宫内膜癌细胞后，可以降低子宫内膜癌细胞的增殖能力；如图6-1和6-2，转染siRNA- TDRG1的卵巢癌细胞，其凋亡增加；如图6-3，转染siRNA- TDRG1的子宫内膜癌细胞，其凋亡增加。由此可见，siRNA-TDRG1可以抑制肿瘤细胞的增长，也可以促进肿瘤细胞的凋亡。鉴于此，siRNA- TDRG1可以作为一种抗肿瘤药物的形式存在，为卵巢癌和子宫内膜的的治疗提供了一种新的靶向治疗方法。
序列表
＜110＞中国医科大学附属第一医院
＜120＞一种与卵巢癌和子宫内膜癌相关的长链非编码RNA-TDRG1的siRNA和应用
＜160＞5
＜210＞1
＜211＞1097
＜212＞DNA
＜213＞长链非编码RNA TDRG1的核苷酸序列
ctcgagaaga tcaggactgc tgaaagaatg aagaagaact tacttggcct aggatgtggc 60
tagagaacgg agcgcacttt cacttacctg gcaaggtggt tttggaagtt tccacggaag 120
ccttcccagg gccgctgctg cccgtcctcg cctaggtgtg gagcttcccg accggctggg 180
gagggaatgt cctgcgggag ccgccgagga ccctctttag cctcttccct ggcttggctg 240
cagctgcagt ctggtactcg cccattctgc tcttcttcac ctccgtattt tctctctcac 300
ggaagaagaa ttccattctc ccatcccagg aaggagagtg ccctgcgtgc cacgaaagag 360
cccaggaccc agaggacaaa tacgtcactg gcagacctgc cttcgccagc gccagcccat 420
ctctgaaacc tccagcattt gtgccccggt ccggcccctc ccaggcctga ctctttccgt 480
gaacggtcac tgcgcaggat caagctacaa tgaagaggag ggaggcagtc tgcgcgcacc 540
gccattttct aggaactggg aagccccccc accccttagg aagatccatc cctgtggaac 600
cttgcccagg cttaccagcc tttgctgagg ttgatctatt gtccctcctt gtccccatca 660
aaatatccag cactccacct tcagggagta gacttgaccc tcaaatagca agttcagcct 720
tcccaggtct aggttccctg ggaggtcaag attcgtctgg ttccttagta cagagggcta 780
gctgtgagtt ggaatccccc tatgagcttt agaatcagtc aagaggaatt gggccccttc 840
ccttcatccc tcttcttttc cctttttgtc ccagagctca gctctgactc aaaagttttt 900
ccatttacca tcaacatgga aacttggctc ctcacgtagg tatattatcc cccttttgta 960
cgtggtcttg ttgatccaaa ctccctttct gtgaaagagg cctgtggggc tcaagaagcc1020
tggtcagcca gccaggctag tcccacatac ctcagaacca gtttaataaa ggctctatgt1080
cattcttttt tggtacc 1097
＜210＞2
＜211＞19
＜212＞RNA
＜213＞外引物sense
＜400＞2
ccuucccagg ucuagguuc 19
＜210＞3
＜211＞19
＜212＞RNA
＜213＞外引物antisense
＜400＞3
gaaccuagac cugggaagg 19
＜210＞4
＜211＞16
＜212＞DNA
＜213＞人工序列
＜400＞4
tcttccctgg cttggc 16
＜210＞5
＜211＞16
＜212＞DNA
＜213＞人工序列
＜400＞5
tgggctcttt cgtggc 16
Claims (1)
1.一种与卵巢癌和子宫内膜癌相关的长链非编码RNA-TDRG1的siRNA用于制备抗卵巢癌和子宫内膜癌药物，其特征在于，所述的siRNA的sense和antisense的核苷酸序列分别如SEQ.ID.NO.2和SEQ.ID.NO.3所示：
SEQ.ID.NO.2 ： CCUUCCCAGGUCUAGGUUC；
SEQ.ID.NO.3 ： GAACCUAGACCUGGGAAGG。
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| LncRNA TDRG1 enhances tumorigenicity in endometrial carcinoma by binding and targeting VEGF-A protein;Chen S等;《BBA - Molecular Basis of Disease》;20180618;第1864卷(第9期);第3013-3021页 * |
| Novel insights into a treatment for aplastic anemia based on the advanced proliferation of bone marrow‑derived mesenchymal stem cells induced by fibroblast growth factor 1;Jiang S等;《Mol Med Rep》;20151009;第12卷(第6期);第7877-7782页 * |
| The role of the long non-coding RNA TDRG1 in epithelial ovarian carcinoma tumorigenesis and progression through miR-93/RhoC pathway;Chen S等;《Mol Carcinog》;20171106;第57卷(第2期);第225-234页 * |
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