WO2018094955A1 - 一种长非编码rna及其在诊断/治疗子痫前期中的应用 - Google Patents

一种长非编码rna及其在诊断/治疗子痫前期中的应用 Download PDF

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WO2018094955A1
WO2018094955A1 PCT/CN2017/080715 CN2017080715W WO2018094955A1 WO 2018094955 A1 WO2018094955 A1 WO 2018094955A1 CN 2017080715 W CN2017080715 W CN 2017080715W WO 2018094955 A1 WO2018094955 A1 WO 2018094955A1
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pvt1
cells
htr
svneo
cell
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孙丽洲
许叶涛
左青
黄诗韵
吴丹
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江苏省人民医院
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Definitions

  • the invention belongs to the field of genetic engineering, and particularly relates to the application of a long non-coding RNA-PVT1 in the diagnosis and preparation of a medicament for treating pre-eclampsia.
  • Preeclampsia is one of the most common gestational complications in the world and is the leading cause of complication-related death in pregnancy. According to statistics from the World Health Organization, pre-eclampsia or eclampsia directly leads to about 14% (about 8,500) per year globally. Maternal death is one of the most common causes of preterm birth and fetal growth restriction and maternal mortality. Although the current medical treatment has been greatly developed, the overall incidence of patients is still high. With the rapid development of sequencing technology and molecular biology, genetic diagnosis and molecular targeted therapy have become a hot issue in the treatment of preeclampsia. Therefore, studying the molecular mechanisms involved in the development, progression, and metastasis of preeclampsia is critical to developing specific diagnostic methods and individualized treatment strategies.
  • lncRNAs are involved in the regulation of a variety of cellular processes through chromatin remodeling, including recombinant stem cell pluripotency, parental imprinting and tumor cell proliferation and metastasis, and epigenetic modification and adsorption of miRNAs.
  • chromatin remodeling including recombinant stem cell pluripotency, parental imprinting and tumor cell proliferation and metastasis, and epigenetic modification and adsorption of miRNAs.
  • lncRNA ROR can contribute to tumorigenesis by inhibiting methyltransferase G9A and promoting demethylation of the promoter region of TESC H3K9.
  • AOC4P inhibits epithelial-mesenchymal transition (EMT) and inhibits hepatocellular carcinoma metastasis by binding to vimentin and promoting its degradation.
  • EMT epithelial-mesenchymal transition
  • SPRY4-IT1 inhibits proliferative cell proliferation, migration and angiogenesis by pre-eclampsia by binding to HUR.
  • PVT1 is a 1957 nt lncRNA located on human chromosome 8q24.21. We found that PVT1 was down-regulated in the placenta of pregnant women in preeclampsia compared with normal pregnant women. After overexpressing or knocking out PVT1, the role of PVT1 in the development and progression of preeclampsia was studied and the function of PVT1 related target genes in trophoblasts of preeclampsia pregnant women was studied.
  • the present invention relates to a long-chain non-coding RNA having the nucleotide sequence of SEQ ID NO: 1; the sequence is: ctccgggcag agcgcgtgtg gcggccgagc acatgggccc gcgggccggg cgggctcggg gcggccggga cgaggagggg cgacgagcaa agatgtgccccgggaccccc ggcaccttcc agtggatttc cttgcggaaa ggatgttggc ggtccctgtg acctgtggag acacggccag atctgccctc cagctgatc tttggccag aaggagatta aaaagatgccctcaagatg
  • siRNA that interferes with PVT1 as set forth in SEQ ID NO: 2, SEQ ID NO: 3;
  • a kit comprising the primer
  • composition comprising the long chain non-coding RNA
  • the pharmaceutical composition further includes an adjuvant.
  • Excipients include: (lip2000, Opti-mem medium, PBS phosphate buffered saline)
  • the concentration in the primer was 10 mol/L, respectively.
  • PVT1 The differential expression of PVT1 in clinical tissues was screened by qPCR, and it was found that the expression level of PVT1 in placental tissues of pregnant women in preeclampsia was lower than that in normal pregnant women. Guess: Whether PVT1 is involved in the pathogenesis of pre-eclampsia.
  • the internationally recognized normal trophoblast cells were selected as experimental subjects, and the interference sequence of PVT1 was designed.
  • the lip2000 was used as the transfection vector to transfer the interference sequence into the cells to simulate the pathogenesis of pre-eclampsia diseases.
  • the PVT1 plasmid was constructed and positively verified the function of PVT1 in the nourishing cell HTR-8/SVneo.
  • the relevant downstream target genes of PVT1 may be involved in cell function (such as proliferation, apoptosis or migration), and then the preliminary study on the regulation mechanism of PVT1, through FISH and other means to find that PVT1 is more present in trophoblast cells.
  • PVT1 may regulate the corresponding target gene at the transcriptional level, and RIP and CHIP assays detect that PVT1 inhibits the expression of downstream target genes TP53INP1 and ANGPTL4 by binding EZH2 protein.
  • lip2000 a versatile liposome transfection reagent for DNA, RNA and oligonucleotide transfection, has high transfection efficiency for most eukaryotic cells. Its unique formulation allows it to be added directly to the culture medium. The presence of serum does not affect the efficiency of transfection, and the PVT1 interference sequence is transferred into the cell.
  • PBS phosphate buffer saline is generally used as a solvent to act as a dissolution protection reagent. It is the most widely used buffer in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. Due to the secondary dissociation of Na2HPO4 and KH2PO4, the buffer pH range is wide; while NaCl and KCl are mainly The role is to increase the salt ion concentration. Exclude its own impact on the subject.
  • HTR-8/SVneo The selection of trophoblast cells (HTR-8/SVneo) was provided by Queen's University of Canada. HTR-8/SVneo cells were cultured in RPMI 1640 medium; both contained 5% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Conventional culture was carried out in a 37 ° C incubator with 5% CO 2 . Replace fresh medium every 2-3 days, when the cell fusion degree reaches 80%-90% generation. All cell lines were verified by DNA analysis of short tandem repeats.
  • the reverse transcription reaction was carried out using TaKaRa Prime Script kit (TaKaRa, Dalian, China).
  • the reverse transcription kit was used to reverse transcribe 1 ⁇ g of total RNA to a final volume of 20 ⁇ l.
  • the specificity and amplification efficiency of the primers were analyzed, and the reaction specificity of the primers was judged according to the dissolution curve.
  • the Ct value was obtained according to the amplification curve, and the relative amount of the target gene was analyzed by the relative amount method and the internal reference GAPDH.
  • the human full-length PVT1 cDNA was synthesized and inserted into the eukaryotic expression vector pCDNA 3.1 to construct a PVT1 overexpression vector plasmid, and then the above plasmid was transformed into Escherichia coli, shaken, cultured, and selected for monoclonal culture.
  • Plasmid vectors (pCDNA3.1-PVT1, si-PVT1, pCDNA3.1-TP53INP1, and empty vector plasmid) for transfection were extracted with an endotoxin-removed plasmid extraction kit (DNA Midiprep kit, Qiagen).
  • the interference sequences of PVT1, namely: si-PVT 1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO: 2) and si-PVT 2#AACUCCUCAGCCUCCAAGCTT (SEQ ID NO: 3), and scrambled control (si-NC) were purchased from Invitrogen ( Invitrogen, CA, USA).
  • the cells HTR-8/SVneo were seeded in a 6-well culture plate at 2 ⁇ 105 cells per well. After the cells were attached, the original medium was discarded 6 h before transfection and replaced with a medium without a double antibody. 10 ⁇ L of lipid was taken.
  • the plastids were diluted in 240 ⁇ L of OPTI-MEM, and gently incubated at room temperature for 5 min; 100 pmol siRNA, si-NC or 4 ug plasmid vector were diluted in 250 ⁇ L OPTI-MEM, and mixed by blowing and incubated for 5 min at room temperature; Mix well liposomes with siRNA or plasmid dilutions and mix gently by pipetting.
  • the treated cells were seeded in 96-well culture plates at 2000-3000 cells per well. After the cells were 80% adherent, the cells were synchronized for 12 h and the original medium was discarded. Six replicate wells were set per sample with a total reaction volume of 200 ⁇ l per well. 20 ⁇ l of MTT reaction solution (5 mg/ml in PBS) was added to each well, and incubated at 37 ° C for 4 h in the dark. The supernatant was discarded, 150 ⁇ l of dimethyl sulfoxide (DMSO) was added to each well, shaken for 10 min, and the absorbance at a wavelength of 490 nm was measured by a microplate reader.
  • DMSO dimethyl sulfoxide
  • HTR-8/SVneo cells were transfected for 48 hours after trypsinization, followed by Annexin V-FITC fluorescent probe and iodination according to FITC Annexin V Apoptosis Detection Kit (BD) and instructions for its use. Propylene (PI) staining. Flow cytometry detection and analysis.
  • a 8 ⁇ m pore size Transwell chamber was placed in a 24-well plate.
  • the upper chamber of the bottom membrane of the Transwell chamber was coated with a 50 mg/l BD Matrigel 1:6 dilution, and the well-coated chamber was placed in a 24-well plate and incubated for 6 h in the incubator.
  • the cells were digested, the digestion was terminated, the culture was discarded by centrifugation, washed 1-2 times with PBS, and resuspended in serum-free medium containing BSA. Adjust the cell density to 3x105. 300 ⁇ l of the cell suspension was added to the Transwell chamber.
  • 700 ⁇ l of medium containing 10% FBS was added to the lower chamber of the 24-well plate and placed in an incubator for 24 hours.
  • Cell migration experiments were performed to adjust cell density to 3x104.
  • 300 ⁇ l of the cell suspension was added to the Transwell chamber.
  • 700 ⁇ l of medium containing 10% FBS was added to the lower chamber of the 24-well plate and placed in an incubator for 24 hours.
  • the chamber was taken, the matrigel and the cells in the upper chamber were wiped with a cotton swab, and the cells on the bottom of the small outdoor surface were stained with 0.1% crystal violet.
  • the stained cells attached to the upper and lower chamber sides of the Transwell chamber basement membrane were photographed by an inverted microscope.
  • the nuclei and cytoplasm of HTR-8/SVneo cells were isolated using the PARIS kit (Life Technologies, USA) according to the manufacturer's instructions. The distribution of PVT1, GAPDH and U1 in the cytoplasm and nucleus was examined using the qPCR method. GAPDH is a cytoplasmic reference and U1 is a nuclear reference. The expression of PVT1, GAPDH and U1 in the cytoplasm and nucleus was expressed as a percentage of total RNA.
  • FISH In situ hybridization
  • the corresponding probes (synthesized by Shanghai Bogu Biotech Co., Ltd.) were designed, and the HTR-8/SVneo cells were planted in a 6-well plate containing 15 mm, until the cells were about 80%. After discarding the culture medium twice with PBS, add 2 ml of methanol for 30 min and then send the sample. The next treatment was carried out by Shanghai Bogu Biotechnology Co., Ltd., and the inverted silver microscope was used to photograph and qualitatively detect the sub-localization of PVT1 in the cells. Further verify the results of the nuclear separation experiment.
  • the cells were planted in a six-well plate. After the cells were grown to about 80%, they were treated with 10 ul of lip2000si-PVT1 and si-NC. After 48 hours, the cells were collected by Trizol treatment. The samples were sent by Beijing Genetic Testing Institute and selected for Illumina. Perform subsequent experiments to obtain and process the corresponding data.
  • the HTR-8/SVneo cells were lysed for use in an immunoblot assay of the endogenous PRC2 complex.
  • the cell supernatant was incubated with protein A/G agarose magnetic beads coated with EZH2, SNRNP70 and control IgG, respectively, for 6 hours at 4 °C. Subsequently, cleaning the magnetic The beads were incubated with 0.1% SDS/0.5 mg/ml proteinase K for 30 minutes at 55 °C to remove proteins.
  • RNA was extracted for qPCR analysis.
  • the 4% paraformaldehyde solid HTR-8/SVneo cells were incubated for 10 minutes to generate DNA-protein crosslinks.
  • the cells were sonicated to generate 200-300 bp chromosome fragments, followed by incubation of the pellet with EZH2 and H3K27me3 specific antibodies and control IgG antibodies. Chromatin DNA was restored and subsequently analyzed using qPCR.
  • the denatured cell protein lysate was added to a pre-made 10% denaturing polyacrylamide gel (SDS-PAGE) well and the protein in the sample was isolated. It was then transferred to an NC membrane and incubated with specific antibodies. Finally, the ECL illuminating hydraulic sheet is used for exposure.
  • the GAPDH antibody was a control
  • the TP53INP1 antibody was purchased from CST
  • the ANGPTL4 antibody was purchased from Proteintech.
  • the experimental data were analyzed by SPSS 17.0 software, expressed as the mean ⁇ standard error of three experiments, and the differences between groups were analyzed by two-tailed Student's T test, rank sum test and chi-square test. Multivariate analysis was performed using p ⁇ 0.05 in the univariate analysis.
  • 2e-f knockdown of PVT1 can attenuate the ability of HTR-8/SVneo cell clone formation; overexpression of PVT1 can enhance HTR-8/SVneo cell clone formation ability
  • 2g-h knockdown of PVT1 can reduce the proliferation of HTR-8/SVneo cells; overexpression of PVT1 can enhance HTR-8/SVneo cell proliferation
  • 3a knockdown of PVT1 can promote trophoblast cell HTR-8/SVneo apoptosis
  • FIG. 5 PVT1 inhibits transcription of the TP53INP1 and ANGPTL4 promoter regions by binding to EZH2 protein
  • 5a-b PVT1 localizes to the nucleus
  • downstream target gene TP53INP1 in 7a-b was significantly higher than that in normal pregnant women.
  • the solution was divided into three layers (aqueous phase - white precipitate - red organic), and the aqueous layer was transferred to a new 1.5 ml centrifuge tube. Try not to absorb a white precipitate. Add an equal volume of isopropanol, mix gently by inversion and leave for 5-10 min. Centrifuge at 12000 g for 4 min at 4 °C. The supernatant was aspirated and 1 ml of 75% ethanol (present) was added to wash the RNA pellet. Centrifuge at 7500g at 4 ° C for 5 min and discard the supernatant. Try to remove 75% of the alcohol and dry it at room temperature for about 15 minutes. The RNA pellet was dissolved in RNase-free water (20-25 ⁇ l).
  • RNA concentration was determined by ultraviolet absorption assay.
  • the RNA concentration and purity were determined using an ultraviolet spectrophotometer and zeroed with DEPC water for dissolving RNA prior to measurement.
  • a reading of 1 at 260 nm indicates 40 ng/ ⁇ l, and the ratio of A260/A280 of the RNA solution was used for the detection of RNA purity, with a ratio ranging from 1.8 to 2.1 indicating compliance.
  • the integrity of the RNA was identified by agarose gel electrophoresis. Prepare 1% agarose gel. The agarose was dissolved by heating, cooled, and 1 ⁇ l of ethidium bromide (EB, 10 mg/ml) was added. After shaking, the gel was poured.
  • EB ethidium bromide
  • the gel was condensed, it was placed in an electrophoresis tank, immersed in 1 ⁇ TAE buffer for 10 min, and used. Spotting.
  • the 5 ⁇ nucleic acid electrophoresis loading buffer was mixed with the sample at 1:4 (v/v), and each sample contained 1 ⁇ g of RNA in the gel well. 80V constant pressure electrophoresis for 50min. After the end of the electrophoresis, the results were observed on a gel imager.
  • TAE Tris-Acetic Acid
  • the reverse transcription reaction conditions were as follows: 37 ° C for 15 min (reverse transcription reaction); 85 ° C for 5 sec (reverse reaction of reverse transcriptase). Design primer sequences based on the gene sequences provided by Genebank,
  • the primers for PVT1 are as follows:
  • the normal trophoblast cell line HTR-8/SVneo was selected as the study object, and the PHT1 interference sequence si-PVT 1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO: 2) and si-PVT2#AACUCCUCAGCCUCCAAGCTT were transfected using lip2000 as a vector.
  • SEQ ID NO: 3 mimics the expression of PVT1 to mimic the pathogenesis of pre-eclampsia.
  • MTT proliferation assay revealed that the interference sequence of PVT1 was transfected into HTR-8/SVneo cells, and PVT1 expression was knocked down to inhibit cell growth.
  • PVT1 plasmid was transfected into normal trophoblast cells to further validate PVT1 function and it was found that overexpression of PVT1 promoted HTR-8/SVneo cell proliferation (Fig. 2A-D).
  • the results of colony formation assay showed that the ability of HTR-8/SVneo cell clone formation was decreased after down-regulation of PVT1, and the overexpression of PVT1 enhanced the ability of HTR-8/SVneo cell clone formation (Fig. 2E and Fig. 2F).
  • EDU staining demonstrated that proliferation of HTR-8/SVneo cells was reduced after knockdown of PVT1, whereas proliferation of HTR-8/SVneo cells was enhanced after overexpression of PVT1 (Fig. 2G and Fig. 2H).
  • PVT1 is involved in HTR-8/SVneo cell migration and invasion
  • the HTR-8/SVneo cell line was used as a research object, and the PHT1 interference sequence si-PVT 1#GCUUGGAGGCUGAGGAGUUTT (SEQ ID NO: 2) and si-PVT 2#AACUCCUCAGCCUCCAAGCTT (SEQ ID NO: 3) were transfected with lip2000 as a vector.
  • the expression of low PVT1 mimics the pathogenesis of pre-eclampsia.
  • the effects of PVT1 knockdown on HTR-8/SVneo cell migration and invasion were studied by transwells experiments.
  • lncRNAs Abnormal expression of lncRNA in preeclampsia in a variety of human diseases.
  • the abnormal expression of lncRNA MEG3 in preeclampsia affects the proliferation and apoptosis of trophoblasts.
  • some studies have shown that lncRNAs are more tissue-specific than protein-coding genes, suggesting that lncRNAs are more likely to be more sensitive biomarkers for different tumors. Therefore, the discovery of preeclampsia-related lncRNAs, their clinical significance and biological function, can promote the diagnosis and treatment of lncRNA-guided preeclampsia.
  • lncRNAs regulate the expression of target genes through various mechanisms, such as recruiting chromatin-regulating enzymes to target genes and cis- or trans-regulated transcription, as scaffolding to bind related molecular elements or to adsorb miRNAs.
  • PHT1 binds to EZH2 in HTR-8/SVneo cells, recruiting them to the TP53INP1 and ANGPTL4 promoter regions to inhibit their transcription.
  • the core unit of EZH2-PRC2 is a histone methyltransferase that catalyzes the H3K27 trimethylation of target genes.
  • TP53INP1 and ANGPTL4 are co-transcribed in trophoblastic cell HTR-8/SVneo. Inhibited by EZH2 recruited by PVT1.
  • TP53INP1 is an important member of a large family of cytostatic factors and has been identified as a new cell balance regulator.
  • Our results also indicate that up-regulation of its expression levels can inhibit the growth of trophoblastic cells HTR-8/SVneo and induce apoptosis, attenuating cell migration and invasion.
  • our results suggest that reduced expression of lncRNA PVT1 may result in shallow implantation of the placenta and induce the development and progression of preeclampsia.

Abstract

提供了一种长非编码RNA，PVT1，其核苷酸序列如SEQ ID NO:1所示，包含该PVT1的药物组合物，检测该PVT1的引物和包含所述引物的试剂盒，干扰该PVT1的siRNA，以及所述PVT1、引物、药物组合物在制备诊断/治疗子痫前期试剂/药物中的应用。

Description

一种长非编码RNA及其在诊断/治疗子痫前期中的应用 技术领域
本发明属于基因工程领域，特别涉及长非编码RNA--PVT1在诊断及制备治疗子痫前期药物的应用。
背景技术
子痫前期是全球最常见妊娠期合并症之一，是妊娠期并发症相关死亡的主要原因,世界卫生组织统计报道表明，子痫前期或者子痫直接导致全球每年约14％(约8500)的孕产妇死亡，其是胎儿早产和胎儿生长受限以及孕产妇死亡最常见的原因之一。尽管目前内科治疗得到了长足的发展,但病人总体发生率依然较高。随着测序技术和分子生物学的快速发展，基因诊断和分子靶向治疗成为子痫前期治疗中一个热点问题。因此,研究参与子痫前期的发生发展和转移的的分子机制，对制定特定的诊断方法和个性化的治疗策略至关重要。
在过去的十年里,基于快速出现的高通量测序的基因表达分析技术和生物信息学推动了大规模的人类基因组学的研究,进而发现了非编码RNAs。人类基因组中只有2％的编码转录成蛋白,而绝大多数转录成非编码RNAs，包括小分子核糖核酸、长非编码rna(lncRNAs)和假基因。最近,miRNA在细胞进程的各个方面的作用已经得到证实，然而,lncRNAs的功能研究不是很透彻。ENCODE计划中GENECODE研究组新数据显示有成千上万的lncRNAs,但只有其中的一些有生物学功能。有趣的是,这些lncRNAs通过染色质重塑参与调控多种细胞进程,包括重组干细胞多能性,父母的印记和肿瘤细胞的扩散和转移以及表观遗传修饰和吸附miRNAs。最近,大量的研究显示，异常lncRNAs表达与多样的人类疾病相关。例如,lncRNA ROR可通过抑制甲基转移酶G9A,促进TESC的启动子区H3K9去甲基化参与肿瘤发生。同时,AOC4P通过与波形蛋白绑定和促进其降解，抑制上皮间质转化(EMT)从而抑制肝细胞癌转移。此外,上调的SPRY4-IT1通过与HUR绑定，抑制子痫前期滋养细胞增殖，迁移和血管形成能力。这些发现表明lncRNAs在人类疾病尤其是子痫前期的发生发展过程中扮演至关重要的角色。因此,发现更多的子痫前期相关的lncRNAs并研究他们的生物功能和机制，对更好地理解子痫前期发生发展的的分子生物学有重要的意义。
PVT1是一条长度为1957nt的lncRNA,其位于人类染色体8q24.21。我们发现PVT1在子痫前期孕妇胎盘组织中较正常孕妇胎盘中表达量下调。在过表达或敲除PVT1后，研究了PVT1在子痫前期发生和发展的作用并研究了PVT1在子痫前期孕妇滋养细胞中的相关靶基因的功能。
发明内容
技术目的
本发明的目的是提供PVT1在诊断子痫前期及在制备治疗子痫前期药物中的应用。
本发明涉及一种长链非编码RNA，其核苷酸序列为SEQ ID NO:1；其序列为：即：ctccgggcag agcgcgtgtg gcggccgagc acatgggccc gcgggccggg cgggctcggg gcggccggga cgaggagggg  cgacgacgag ctgcgagcaa agatgtgccc cgggaccccc ggcaccttcc agtggatttc cttgcggaaa ggatgttggc ggtccctgtg acctgtggag acacggccag atctgccctc cagcctgatc ttttggccag aaggagatta aaaagatgcc cctcaagatg gctgtgcctg tcagctgcat ggagcttcgt tcaagtattt tctgagcctg atggatttac agtgatcttc agtggtctgg ggaataacgc tggtggaacc atgcactgga atgacacacg cccggcacat ttcaggatac taaaagtggt tttaagggag gctgtggctg aatgcctcat ggattcttac agcttggatg tccatggggg acgaaggact gcagctggct gagagggttg agatctctgt ttacttagat ctctgccaac ttcctttggg tctccctatg gaatgtaaga ccccgactct tcctggtgaa gcatctgatg cacgttccat ccggcgctca gctgggcttg agctgaccat actccctgga gccttctccc gaggtgcgcg ggtgaccttg gcacatacag ccatcatgat ggtactttaa gtggaggctg aatcatctcc cctttgagct gcttggcacg tggctccctt ggtgttcccc ttttactgcc aggacactga gatttggaga gagtctcact ctgtggtcca ggctgaagta cagtggcatg atcccaggtc actgcaaccc ccacctcccg ggttcaagtg atcctcctgc ctcagcctcc cgagtagctg gtattacagg cgtgtgccac aaagcctggc taagttttgt atttttagta gagacggggt ttcaccatgt tggccaggtt ggtctcgaac tcctgacctc aagtgatcca ctcactttgg cctttcaacg tgctgggatt acaggcgaga gtcaccgcac ccggacgact ctgacatttt tgaagagtcc agaatcctgt tacacctggg atttaggcac tttcaatctg aaaaaataca tatcctttca gcactctgga cggacttgag aactgtcctt acgtgaccta aagctggagt attttgagat tggagaatta agagccagtc ttggtgctct gtgttcacct ggttcatctg aggagctgca tctaccctgc ccatgccata gatcctgccc tgtttgcttc tcctgttgct gctagtggac atgagaagga cagaataacg ggctcccaga ttcacaagcc ccaccaagag gatcacccca ggaacgcttg gaggctgagg agttcactga ggctactgca tcttgagact caggatgaag acccagcttg gggctgtcaa agaggcctga agaggcagaa caccccagag gagcctgggg ccaccaccca gcatcactgt gggaaaacgg cagcaggaaa tgtcctctcg cctgcgtgct ccacctcggt ccacgccttc cctccttctg gaagccttgc ctgaccactg gcctgcccct tctatgggaa tcactactga ccttgcagct tattatagac ttatatgttt tttgcatgtc tgacacccat gactccacct ggaccttatg gctccaccca gaagcaattc agcccaacag gaggacagct tcaacccatt acgatttcat ctctgcccca accactcagc agcaagcacc tgttacctgt ccacccccac cccttccccc aaactgcctt tgaaaaatcc ctaacctatg agctttgaat aagatgagta cgaacttcat cgcccacgtg gcgtggccgg cctcgtgtct attaaattct ttttctacta aaaaaaaaaa aaaaaaa
一种长链非编码RNA在制备治疗子痫前期药物中的应用；
一种长非编码RNA在制备诊断子痫前期试剂中的应用；
一种检测PVT1的引物，如SEQ ID NO：4、SEQ ID NO：5所示；
一种干扰PVT1的siRNA,如SEQ ID NO：2、SEQ ID NO：3所示；
一种试剂盒，包括所述引物；
一种包括所述长链非编码RNA的药物组合物；
所述引物在制备诊断子痫前期试剂中的应用；
所述药物组合物在制备治疗子痫前期药物中的应用。
所述药物组合物，其中还包括辅料。辅料包括：(lip2000,Opti-mem培养液,PBS磷酸缓冲盐溶液)
所述试剂盒，所述引物中的浓度分别为10mol/L。
技术方案
通过qPCR筛查临床组织中PVT1的差异表达，发现在子痫前期孕妇胎盘组织中PVT1的表达量较正常孕妇胎盘中表达量低。猜想：PVT1是否参与了子痫前期疾病的发病过程。
随后选用国际认可的正常滋养细胞作为实验研究对象，设计PVT1的干扰序列，以lip2000为转染载体将干扰序列转入细胞后模拟子痫前期疾病疾病的发病过程。通过检测在干扰序列转入细胞后细胞的功能如增殖，凋亡，迁移和能力等。从而证明在正常的滋养细胞HTR-8/SVneo中敲低PVT1的表达，影响了细胞的功能，诱导或加速子痫前期疾病的发病进程。相反，构建PVT1质粒，正反验证PVT1在滋养细胞HTR-8/SVneo中的功能。
通过转录组测序，检测PVT1的可能参与细胞功能(如增殖，凋亡或迁移)的相关下游靶基因，随后对PVT1调控机制的初步探讨，通过FISH等手段发现PVT1更多的存在于滋养细胞的细胞核中，考虑PVT1可能在转录水平调控相应的靶基因，通过RIP和CHIP实验检测PVT1通过绑定EZH2蛋白抑制下游靶基因TP53INP1和ANGPTL4的表达。
转染过程所需的各种试剂，
(1)lip2000，一种多用途的脂质体转染试剂，适用于DNA、RNA和寡核苷酸的转染，对大多数真核细胞具有很高的转染效率。其独特的配方使其可直接加入培养基中，血清的存在不会影响转染效率，进而将PVT1干扰序列转进到细胞内。
(2)Opti-mem培养液，含HEPES，2400mg/l碳酸氢钠，次黄嘌呤，胸腺嘧啶，丙酮酸钠，L-谷氨酰胺，微量元素，生长因子，以及减量至1.1mg/l的酚红，作为转染试剂的辅料，其本身对细胞无任何害处，而更好更有效的转进细胞中，以获得预期目的。
(3)PBS磷酸缓冲盐溶液(phosphate buffer saline)一般作为溶剂，起溶解保护试剂的作用。它是生物化学研究中使用最为广泛的一种缓冲液，主要成分为Na2HPO4、KH2PO4、NaCl和KCl，由于Na2HPO4和KH2PO4它们有二级解离，缓冲的pH值范围很广；而NaCl和KCl主要作用为增加盐离子浓度。排除其本身对实验对象的影响。
组织收集
我们收集了52对2014年至2015年在江苏省人民医院，江苏省妇幼保健院接受剖宫产手术，诊断患有子痫前期的孕妇胎盘组织和不含有任何基础疾病的正常孕妇胎盘组织。并记录临床的特点：包括孕妇年龄，有无吸烟史，孕周数，收缩压，舒张压以，蛋白尿以及胎儿体重。组织样本收集的均为第一时间液氮或储存在-80℃,直至RNA提取。该研究经过南京医科大学伦理委员会批准。获得所有病人的书面知情同意。
细胞系
选取滋养细胞(HTR-8/SVneo)来自加拿大女皇大学提供。HTR-8/SVneo细胞用RPMI 1640培养基培养；培养基中均含有5％的胎牛血清、100U/ml的青霉素和100mg/ml的链霉素。5％CO2的37℃恒温培养箱中常规培养。每2-3天更换新鲜培养基，当细胞融合度达到80％-90％时传 代。所有细胞系被短串联重复序列的DNA分析验证。
RNA提取和定量PCR分析
根据试剂的使用说明，用Trizol试剂分离总RNA。逆转录反应应用TaKaRa Prime Script试剂盒(TaKaRa,大连,中国)。逆转录试剂盒对1μg总RNA进行逆转录，最终体积为20μl。结果分析：分析引物的特异性及扩增效率，根据溶解曲线判断引物的反应特异性。根据扩增曲线得到Ct值，采用相对量法与内参GAPDH进行目的基因相对表达量的分析。PVT1的引物如下：Primer F 5’-TGAGAACTGTCCTTACGTGACC-3’(SEQ ID NO：4)，Primer R 5’-AAGGGCATTCACCAACTCC-3’(SEQ ID NO：5)计算公式为：2^(-△Ct)，△Ct＝Ct gene-Ct control。
质粒构建
合成人全长PVT1 cDNA，并将其插入真核表达载体pCDNA 3.1中，构建PVT1过表达载体质粒，随后将上述质粒转化大肠杆菌，摇菌、培养、挑选单克隆菌培养扩增。
细胞转染
用于转染的质粒载体(pCDNA3.1-PVT1、si-PVT1、pCDNA3.1-TP53INP1、和空载体质粒)，均用去除内毒素的质粒提取试剂盒(DNA Midiprep试剂盒，Qiagen)提取。PVT1的干扰序列,即：si-PVT 1#GCUUGGAGGCUGAGGAGUUTT(SEQ ID NO：2)和si-PVT 2#AACUCCUCAGCCUCCAAGCTT(SEQ ID NO：3)，及乱序对照(si-NC)均购自Invitrogen公司(Invitrogen公司，CA，USA)。将细胞HTR-8/SVneo按每孔2×105个细胞种于6孔培养板，待细胞贴壁后，于转染前6h吸弃原有培养基，换成无双抗培养基；取10μL脂质体稀释于240μL的OPTI-MEM中，温和吹打混匀室温下孵育5min；取100pmol siRNA,si-NC或4ug质粒载体分别稀释于250μL OPTI-MEM中，吹打混匀室温下孵育5min；将孵育好的脂质体与siRNA或质粒稀释液混合，温和吹打混匀。于室温下继续孵育20min；将上述混合物均匀滴入事先加好1.5mL OPTI-MEM的6孔培养板中，轻轻混匀。37℃,5％CO2培养箱中继续培养6h后，换完全培养基。转染后36h，收集细胞提取RNA或蛋白进行实时定量RT-PCR或免疫印迹分析。
细胞增殖活性检测
MTT实验，将处理后的细胞按每孔2000-3000个细胞接种于96孔培养板。待细胞80％贴壁后，细胞同步化12h，弃去原有培养基。每个样本设置6个复孔，每孔总反应体积为200μl。每孔加入20μl的MTT反应液(5mg/ml，溶于PBS)，37℃避光孵育4h。弃去上清液，每孔加入150μl二甲亚砜(DMSO)，震荡10min，酶标仪测定490nm波长处的吸光度。
EdU实验，将适量处理后的细胞中在24孔板中，每孔加入10μM EdU试剂。2h后，用4％多聚甲醛固定30分钟。清洗，使用Click-iTR Edu试剂盒染色30分钟，随后用DAPI染色5分钟，随后使用荧光显微镜拍摄(奥林巴斯，日本)。最后，使用Image-Pro Plus软件分析。
流式细胞术
凋亡检测，用胰酶消化收集转染48小时后的HTR-8/SVneo细胞，随后根据FITC Annexin V凋亡检测试剂盒(BD)及其使用说明予以Annexin V-FITC荧光探针和碘化丙锭(PI)染色。流式细胞仪检测和分析。
细胞周期检测，根据说明书使用CycleTESTTM PLUS DNA试剂盒(BD)予以PI染色，随后用FACScan分析。
细胞迁移和侵袭实验
24孔板中放置8μm孔径大小的Transwell小室。细胞侵袭实验，用50mg/l BD Matrigel1:6稀释液包被Transwell小室底部膜的上室面，包被好的小室放入24孔板中，孵箱中孵育6h。消化细胞，终止消化后离心弃去培养液，用PBS洗1-2遍，用含BSA的无血清培养基重悬。调整细胞密度至3x105。取细胞悬液300μl加入Transwell小室。24孔板下室加入700μl含10％FBS的培养基，放入孵箱中常规培养24h。细胞迁移实验，调整细胞密度至3x104。取细胞悬液300μl加入Transwell小室。24孔板下室加入700μl含10％FBS的培养基，放入孵箱中常规培养24h。取小室，用棉签擦去基质胶和上室内的细胞，用0.1％结晶紫将小室外底面的细胞染色利用倒置显微镜对Transwell小室底膜上下室侧附着的染色的细胞拍照计数。
亚细胞结构定位
根据使用说明书使用PARIS试剂盒(Life Technologies，USA)分离HTR-8/SVneo细胞的细胞核和细胞质。使用qPCR方法检测PVT1、GAPDH和U1在细胞质和细胞核中的分布。GAPDH为细胞质参照，U1为细胞核参照。以总RNA百分比呈现PVT1、GAPDH和U1在细胞质和细胞核中的表达情况。
原位杂交技术(FISH)
根据PVT1基因转录本的特点，设计相对应的探针(由上海博谷生物科技公司合成)，将HTR-8/SVneo细胞种植于含有15mm爬片6孔板里，待细胞至80％左右时，弃培养基用PBS清洗两遍后，加2ml的甲醇固定30min后送样，由上海博谷生物科技公司进行接下来的处理，选用倒置银光显微镜拍照，定性检测PVT1在细胞中的亚定位，进一步验证核质分离实验结果。
RNA测序
将细胞种植于六孔板中，待细胞长至80％左右后给于10ul的lip2000si-PVT1和si-NC处理，48h后用Trizol处理收集细胞，送样，由北京基因检测机构实施，选用Illumina进行随后的实验，得到并处理相应的数据。
RNA免疫印迹(RIP)
裂解HTR-8/SVneo细胞供内源性PRC2复合物免疫印迹实验使用。将细胞上清与包被分别识别EZH2、SNRNP70和对照IgG的蛋白A/G琼脂糖磁珠在4℃孵育6个小时。随后，清洗磁 珠，用0.1％SDS/0.5mg/ml蛋白酶K在55℃孵育30分钟以去除蛋白。提取RNA供qPCR分析。
染色质免疫共沉淀(CHIP)
使用4％多聚甲醛固HTR-8/SVneo细胞，孵育10分钟以产生DNA-蛋白交联。超声裂解细胞以产生200-300bp的染色体碎片，随后用EZH2和H3K27me3特异性抗体和对照IgG抗体孵育沉淀。恢复染色质DNA，随后使用qPCR检测分析。
蛋白质免疫印迹(Westblotting)
将变性好的细胞蛋白裂解产物加入至预先制好的10％变性聚丙烯酰氨凝胶(SDS-PAGE)的加样孔中，分离样本中蛋白。随后转至NC膜，并以特异性抗体孵育。最后用ECL发光液压片曝光。GAPDH抗体为对照，TP53INP1抗体购自CST公司，ANGPTL4抗体购自Proteintech公司。
数据处理
实验数据皆用SPSS17.0软件分析，以三次实验的平均值±标准误表示，组间差异用双尾Student’s T检验、秩和检验和卡方检验。单因素分析中p<0.05的随后再使用多因素分析。
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附图说明
图1、PVT1在子痫前期孕妇胎盘组织中表达下调
1a PVT1在子痫前期孕妇胎盘组织(n＝52)表达较正常组织下调
1b PVT1在子痫前期孕妇胎盘组织(n＝52)表达较正常组织下调
图2、PVT1对滋养细胞HTR-8/SVneo细胞增殖能力的影响
2a-b 在HTR-8/SVneo细胞中敲低PVT1和过表达PVT1
2c-d 敲低PVT1表达抑制HTR-8/SVneo细胞的增殖能力；过表达PVT1促进HTR-8/SVneo细胞增殖
2e-f 敲低PVT1能够使HTR-8/SVneo细胞克隆形成能力减弱；过表达PVT1能够增强HTR-8/SVneo细胞克隆形成能力
2g-h 敲低PVT1能够减少HTR-8/SVneo细胞的增殖；过表达PVT1能够增强HTR-8/SVneo细胞增殖
图3 PVT1对HTR-8/SVneo细胞周期、凋亡、迁移和侵袭的影响
3a 敲低PVT1能够促进滋养细胞HTR-8/SVneo凋亡
3b 敲低PVT1诱导细胞周期阻滞在G0/G1期；过表达PVT1促进G0/G1期向S期转换。
3c-d 过表达PVT1能够促进HTR-8/SVneo细胞的迁移能力
3e-f 过表达PVT1能够促进HTR-8/SVneo细胞的侵袭能力
图4 通过测序检测参与PVT1介导滋养细胞生长的潜在下游靶基因
4a-b 细胞测序的相关性分析以及GO分析
4c-d 敲低PVT1和过表达PVT1验证TP53INP1和ANGPTL4是其潜在的靶基因
图5 PVT1通过结合EZH2蛋白,抑制TP53INP1和ANGPTL4启动子区域转录
5a-b PVT1定位于细胞核
5c RIP实验验证PVT1与EZH2结合
5d-f 蛋白免疫印记实验(CHIP)，验证得到PVT1通过与EZH2蛋白的绑定抑制TP53INP1和ANGPTL4的表达
图6 过表达TP53INP1对滋养细胞生长增殖的影响
6a-c 过表达TP53INP1抑制细胞增殖能力，同时也减弱细胞克隆形成能力
6d 过表达TP53INP1抑制细胞侵袭能力
6e-f 补救实验验证TP53INP1参与了PVT1调控滋养细胞的增殖过程
图7 临床标本中检测下游靶基因TP53INP1的表达
7a-b 检测下游靶基因TP53INP1在子线前期孕妇胎盘样本中表达较正常孕妇胎盘表达量明显增高
7c 运用免疫组化检测TP53INP1在胎盘中的表达，得出TP53INP1在子线前期孕妇胎盘样本中表达较正常孕妇胎盘表达量明显增高。
具体实施方式
以下通过实施例对本发明作进一步的阐述，但不限制本发明。
实施例中末注明具体条件的的实验方法，基本上都按照Sambrook，J等人编著的《分子克隆实验指南(第3版)》(MolecularCloning:ALaboratoryManual,3rded.黄培堂等译，科学出版社.2002.8)中所述的条件及方法或按照材料提供商所建议的条件及方法进行，其它没有详细描述的技术相应于本领域人员来说是熟知的标准方法。
本发明的材料：本申请中提及的细胞株以及培养基均有商品供应或以别的途径能为公众所得，它们仅作举例，对本发明不是唯一的，可分别用其它适合的工具和生物材料来代替。
实施例1
检测PVT1在组织和细胞中的表达情况
取0.1g组织，液氮研磨充分(成粉末状)或1-5×107细胞弃培养基，预冷的PBS润洗2次。加入1ml的Trizol裂解液，以无酶枪头吹打混匀，静置5min，将裂解液移入预先标 记好的无酶1.5ml的离心管中。4℃7500g离心5分钟，取上清加入1/5体积的氯仿，颠倒混匀30s，静置2min。4℃，12000g离心，15min。溶液分三层(水相-白色沉淀-红色有机物)，转移水相层至新的1.5ml离心管中，尽量不要吸到白色沉淀。加入等体积异丙醇，轻轻颠倒混匀，放置5-10min。4℃，12000g离心，10min。吸弃上清，加入1ml 75％的乙醇(现配)，洗涤RNA沉淀。4℃，7500g离心，5min，弃上清。尽量去除75％的酒精，于室温中晾干，约15min。用无RNA酶水(20-25μl)溶解RNA沉淀。
紫外吸收测定法测定RNA的浓度。使用紫外分光光度计测定RNA浓度和纯度，测量前先用溶解RNA用的DEPC水调零。在260nm处读值1为表示40ng/μl，RNA溶液的A260/A280的比值用于RNA纯度的检测，比值范围在1.8到2.1表明符合要求。琼脂糖凝胶电泳鉴定RNA的完整性。配制1％的琼脂糖胶。加热溶解琼脂糖，冷却，加入1μl溴化乙锭(EB,10mg/ml)。摇匀后倒胶，待胶冷凝后，置于电泳槽中，浸于1×TAE缓冲液中平衡10min，待用。点样。按1:4(v/v)将5×核酸电泳上样缓冲液与样本混合，准确将各样本含有1μg的RNA加入凝胶孔中。80V恒压电泳50min。电泳结束后，在凝胶成像仪上观察结果。
Tris-乙酸(TAE)缓冲液配方(1L)50×：
Figure PCTCN2017080715-appb-000001
子痫前期孕妇胎盘组织及正常孕妇胎盘组织标本，HTR-8/SVneo细胞的总RNA，逆转录反应应用TaKaRa PrimeScript试剂盒(大连宝生物工程有限公司)。逆转录反应体系如下：
Figure PCTCN2017080715-appb-000002
反转录反应条件如下：37℃ 15min(反转录反应)；85℃ 5sec(反转录酶的失活反应)。根据Genebank提供的基因序列，设计引物序列，
QPCR应用7300PCR系统(Applied Biosystems，Warrington，UK)。cDNA样品采用三部法PCR扩增标准程序。反应体系：
Figure PCTCN2017080715-appb-000003
反应条件：
Figure PCTCN2017080715-appb-000004
结果分析：分析引物的特异性及扩增效率，根据溶解曲线判断引物的反应特异性。根据扩增曲线得到Ct值，采用相对量法与内参GAPDH进行目的基因相对表达量的分析。计算公式为：2^(-△Ct)，△Ct＝Ct gene-Ct control。
PVT1的引物如下：
Primer F 5’-TGAGAACTGTCCTTACGTGACC-3’，见SEQ ID NO：4，
Primer R 5’-AAGGGCATTCACCAACTCC-3’，见SEQ ID NO：5。
结果表明,lncRNA PVT1在子痫前期孕妇胎盘组织表达较正常组织下调(图1A)。我们利用实时定量PCR检测了52对子痫前期孕妇胎盘组织表达较正常组织中PVT1的表达水平。结果显示与正常孕妇胎盘组织相比，PVT1的表达在78.8％(41/52)的子痫前期孕妇胎盘组织中较正常组织表达降低(倍数>1.5,P<0.05)(图1B)。提示PVT1可能在子痫前期疾病的诊断，发生发展及治疗中扮演着重要的作用。
Table 1子痫前期孕妇和正常妊娠孕妇的临床数据
子痫前期(N＝52)  正常妊娠(N＝52) P值
母体年龄(年)  29.96±5.639 34.69±3.226 p>0.05
母体体重  74.75±10.885 72.28±9.185 p>0.05
吸烟  0 0 p>0.05
收缩压(mmHg)  162.51±15.472 116.73±7.728 p<0.01
舒张压(mmHg)  106.71±11.155 74.59±7.57 p<0.01
蛋白尿(g/day)  >0.3g <0.3g p<0.05
新生儿体重(g)  2365.57±1013.032 3389.42±387.72 p<0.05
实施例2。
为了研究PVT1对正常滋养细胞HTR-8/SVneo细胞表型的影响。
首先，选取正常滋养细胞HTR-8/SVneo细胞系作为本实验的研究对象，利用lip2000作为载体，转染PVT1干扰序列si-PVT 1#GCUUGGAGGCUGAGGAGUUTT(SEQ ID NO：2)和si-PVT2#AACUCCUCAGCCUCCAAGCTT(SEQ ID NO：3)以敲低PVT1的表达模拟子痫前期的发病过程，MTT增殖实验检测发现,在HTR-8/SVneo细胞中转染PVT1的干扰序列，敲低PVT1表达后抑制细胞生长。相比之下,在正常滋养细胞中转染PVT1质粒，进一步验证PVT1功能作用，发现PVT1的过表达促进HTR-8/SVneo细胞增殖(图2A-D)。此外,克隆形成试验结果表明,下调PVT1后HTR-8/SVneo细胞克隆形成能力减弱,而PVT1过表达后增强了HTR-8/SVneo细胞克隆形成能力(图2E和图2F)。此外,EDU染色证明,敲低PVT1后减少HTR-8/SVneo细胞的增殖,而过表达PVT1后HTR-8/SVneo细胞增殖增强(图2G和图2H)。由此可知，这些数据表明,PVT1可促进HTR-8/SVneo细胞的增殖能力。
实施例3
PVT1对胎盘滋养细胞HTR-8/SVneo周期和凋亡的影响
为了研究是否PVT1对HTR-8/SVneo细胞的增殖影响了细胞周期转换,以正常滋养细胞HTR-8/SVneo细胞系作为研究对象，利用lip2000作为载体，转染PVT1干扰序列si-PVT 1#GCUUGGAGGCUGAGGAGUUTT(SEQ ID NO：2)和si-PVT 2#AACUCCUCAGCCUCCAAGCTT(SEQ ID NO：3)以敲低PVT1的表达模拟子痫前期的发病过程。利用流式细胞术分析细胞周期。结果表明,HTR-8/SVneo细胞转染PVT1干扰序列后，发现敲低PVT1的表达后细胞周期阻滞在G1/G0期，相反，在正常滋养细胞中转染PVT1质粒，进一步验证PVT1功能作用，发现过表达PVT1后细胞周期阻滞在G1/G0期所占比率较对照组减少(图3B，P<0.05)。此外,我们进行了流式细胞仪分析细胞凋亡是否参与了PVT1敲低后诱导的细胞生长受抑。如图3A所示,早期凋亡(UR)和晚期凋亡率(LR)在PVT1敲低的HTR-8/SVneo细胞中高于对照组细胞。PVT1影响了滋养细胞的周期和凋亡
实施例4
PVT1参与HTR-8/SVneo细胞迁移和侵袭
滋养细胞浸润和转移子痫前期发病机制中的一个重要方面。HTR-8/SVneo细胞系作为研究对象，利用lip2000作为载体，转染PVT1干扰序列si-PVT 1#GCUUGGAGGCUGAGGAGUUTT(SEQ ID NO：2)和si-PVT 2#AACUCCUCAGCCUCCAAGCTT(SEQ ID NO：3)以敲低PVT1的表达模拟子痫前期的发病过程。利用transwells实验研究了PVT1敲低以后对HTR-8/SVneo细胞迁移和侵袭能力影响。结果表明,敲低PVT1后与对照组细胞相比，抑制了滋养细胞HTR-8/SVneo迁移能力,抑制侵袭能力(图3C和D)。这些结果表明,敲低PVT1表达后抑制滋养细胞的表型,阻碍了滋养细胞HTR-8/SVneo的迁移和侵袭。可见，PVT1的低表达表达影响着正常滋养细胞HTR-8/SVneo的细胞迁移和侵袭能力，进一步影响着胎盘的浅着床，诱导子痫前期疾病的发生，给药PVT1可以治疗子痫前期疾病。
讨论
在过去的十年里,随着测序技术和生物信息学的快速发展,越来越多的lncRNAs被人们认知。在多种人类疾病中包括子痫前期中lncRNA呈异常表达。例如,在子痫前期中lncRNA MEG3的表达异常影响滋养细胞的增殖及凋亡能力。此外,一些研究显示,lncRNAs比蛋白质编码基因更具组织特异性,这说明lncRNA更有可能作为不同肿瘤更加敏感的生物标志物。因此,发现子痫前期相关的lncRNAs,研究他们的临床意义和生物功能，可促进lncRNA指导的子痫前期的诊断和治疗。
本研究中,我们发现了一个新的lncRNA PVT1并证明其在子痫前期组织中表达下调。下调的PVT1表达与子痫前期发病机制有着密切的关系。此外，敲低PVT1后抑制细胞增殖,迁移和侵袭,促进滋养细胞凋亡。此外,我们认为敲低PVT1介导的滋养细胞生长抑制在一定程度上依赖于TP53INP1和ANGPTL4的表达。在这里,我们首次证实PVT1在人类滋养细胞中行使相关功能，是通过抑制的滋养细胞抑制因子TP53INP1和ANGPTL4的表达。lncRNAs通过各种机制调节靶基因的表达,如招募染色质调节酶到靶基因和顺式或反式调节转录,作为脚手架结合相关分子原件或吸附miRNA。在这项研究中,我们发现在HTR-8/SVneo细胞中PVT1可以结合EZH2,招募他们到TP53INP1和ANGPTL4启动子区域抑制其转录。
EZH2-PRC2的核心单元,是一个组蛋白甲基转移酶，催化靶基因的H3K27三甲基化；本实验中，我们进一步证明，在滋养细胞HTR-8/SVneo中TP53INP1与ANGPTL4共同转录，可被PVT1募集的EZH2抑制。TP53INP1是一个细胞抑制因子大家庭的重要成员之一,已被确定为一个新的细胞平衡调节器。我们的结果也表明了,上调其表达水平可以抑制滋养细胞HTR-8/SVneo生长和诱导细胞凋亡，减弱细胞的迁移和侵袭能力。综上所述,我们的研究结果表明,lncRNA PVT1表达减少可能导致胎盘的浅着床，诱发子痫前期的发生和发展。
我们的研究首次发现,在子痫前期孕妇胎盘组织和细胞PVT1表达下调。在子痫前期孕妇胎盘滋养细胞HT-8/SVneo敲低PVT1的表达，表现出细胞抑制功能，表现为细胞增殖、迁移减少以及细胞凋亡增加。此外。我们的发现会进一步丰富子痫前期发病机制,并促进lncRNA指导的诊断和治疗。

Claims (11)

  1. 一种长非编码RNA，PVT1，其核苷酸序列为SEQ ID NO：1，即：ctccgggcag agcgcgtgtg gcggccgagc acatgggccc gcgggccggg cgggctcggg gcggccggga cgaggagggg cgacgacgag ctgcgagcaa agatgtgccc cgggaccccc ggcaccttcc agtggatttc cttgcggaaa ggatgttggc ggtccctgtg acctgtggag acacggccag atctgccctc cagcctgatc ttttggccag aaggagatta aaaagatgcc cctcaagatg gctgtgcctg tcagctgcat ggagcttcgt tcaagtattt tctgagcctg atggatttac agtgatcttc agtggtctgg ggaataacgc tggtggaacc atgcactgga atgacacacg cccggcacat ttcaggatac taaaagtggt tttaagggag gctgtggctg aatgcctcat ggattcttac agcttggatg tccatggggg acgaaggact gcagctggct gagagggttg agatctctgt ttacttagat ctctgccaac ttcctttggg tctccctatg gaatgtaaga ccccgactct tcctggtgaa gcatctgatg cacgttccat ccggcgctca gctgggcttg agctgaccat actccctgga gccttctccc gaggtgcgcg ggtgaccttg gcacatacag ccatcatgat ggtactttaa gtggaggctg aatcatctcc cctttgagct gcttggcacg tggctccctt ggtgttcccc ttttactgcc aggacactga gatttggaga gagtctcact ctgtggtcca ggctgaagta cagtggcatg atcccaggtc actgcaaccc ccacctcccg ggttcaagtg atcctcctgc ctcagcctcc cgagtagctg gtattacagg cgtgtgccac aaagcctggc taagttttgt atttttagta gagacggggt ttcaccatgt tggccaggtt ggtctcgaac tcctgacctc aagtgatcca ctcactttgg cctttcaacg tgctgggatt acaggcgaga gtcaccgcac ccggacgact ctgacatttt tgaagagtcc agaatcctgt tacacctggg atttaggcac tttcaatctg aaaaaataca tatcctttca gcactctgga cggacttgag aactgtcctt acgtgaccta aagctggagt attttgagat tggagaatta agagccagtc ttggtgctct gtgttcacct ggttcatctg aggagctgca tctaccctgc ccatgccata gatcctgccc tgtttgcttc tcctgttgct gctagtggac atgagaagga cagaataacg ggctcccaga ttcacaagcc ccaccaagag gatcacccca ggaacgcttg gaggctgagg agttcactga ggctactgca tcttgagact caggatgaag acccagcttg gggctgtcaa agaggcctga agaggcagaa caccccagag gagcctgggg ccaccaccca gcatcactgt gggaaaacgg cagcaggaaa tgtcctctcg cctgcgtgct ccacctcggt ccacgccttc cctccttctg gaagccttgc ctgaccactg gcctgcccct tctatgggaa tcactactga ccttgcagct tattatagac ttatatgttt tttgcatgtc tgacacccat gactccacct ggaccttatg gctccaccca gaagcaattc agcccaacag gaggacagct tcaacccatt acgatttcat ctctgcccca accactcagc agcaagcacc tgttacctgt ccacccccac cccttccccc aaactgcctt tgaaaaatcc ctaacctatg agctttgaat aagatgagta cgaacttcat cgcccacgtg gcgtggccgg cctcgtgtct attaaattct ttttctacta aaaaaaaaaa aaaaaaa。
  2. 如权利要求1所述一种长非编码RNA在制备诊断子痫前期试剂中的应用。
  3. 如权利要求1所述一种长非编码RNA在制备治疗子痫前期药物中的应用。
  4. 一种检测PVT1的引物，如SEQ ID NO：4、SEQ ID NO：5所示。
  5. 一种干扰PVT1的s iRNA,如SEQ ID NO：2、SEQ ID NO：3所示。
  6. 一种试剂盒，包括权利要求4所述引物。
  7. 一种包括权利要求1所述RNA的药物组合物。
  8. 如权利要求4所述引物在制备诊断子痫前期试剂中的应用。
  9. 如权利要求7所述组合物在制备治疗子痫前期药物中的应用。
  10. 根据权利要求7所述药物组合物，其中还包括辅料。
  11. 根据权利要求6所述试剂盒，其特征在于，所述引物中的浓度分别为10mol/L。
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Publication number Priority date Publication date Assignee Title
WO2018167790A1 (en) * 2017-03-16 2018-09-20 Ramot At Tel-Aviv University Ltd. Methods for identifying and monitoring pregnant women at risk of preeclampsia
CN108220434A (zh) * 2017-12-13 2018-06-29 南京医科大学第二附属医院 一种长链非编码rna及其组合物在诊断/治疗胆管癌中的应用
CN108186665B (zh) * 2018-01-02 2020-03-20 深圳市第二人民医院 干扰长链非编码rna pvt1表达的试剂及其应用
US11225666B2 (en) * 2018-03-16 2022-01-18 Research Foundation Of The City University Of New York Plasmid vector for expressing a PVT1 exon and method for constructing standard curve therefor
CN109750038B (zh) * 2018-12-29 2021-08-31 烟台毓璜顶医院 一种长非编码rna及在制备诊断子痫前期及靶点药物治疗中的应用
CN111321148A (zh) * 2020-01-09 2020-06-23 宁夏医科大学总医院 一种用于子痫前期临床风险评估的标志基因及其应用
CN111172161B (zh) * 2020-01-15 2023-04-21 江苏省人民医院（南京医科大学第一附属医院） 一种长非编码rna及其在诊断/治疗子痫前期中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105648075A (zh) * 2016-02-29 2016-06-08 山东省立医院 一种肝癌诊断组合及含有该组合的试剂盒

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6336755B2 (ja) * 2010-11-12 2018-06-06 ザ ジェネラル ホスピタル コーポレイション ポリコームに関連する非コードｒｎａ
CN106047880B (zh) * 2016-08-18 2019-02-12 暨南大学 抑制血液肿瘤细胞增殖的PVT1 siRNA-1055及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105648075A (zh) * 2016-02-29 2016-06-08 山东省立医院 一种肝癌诊断组合及含有该组合的试剂盒

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
" "Homo sapiens Pvt1 oncogene (non-protein coding) (PVT1), long non-coding RNA"", GENBANK NCBI REFERENCE SEQUENCE: NR_003367.3, 17 July 2013 (2013-07-17) *
TAKAHASHI, Y. ET AL.: "Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers", BRITISH JOURNAL OF CANCER, vol. 110, no. 1, 5 November 2013 (2013-11-05), pages 164 - 171, XP055629631, ISSN: 0007-0920, DOI: 10.1038/bjc.2013.698 *
WAN, LI ET AL.: "Progession of long non-coding RNA PVT1 in tumor", CHINESE CLINICAL ONCOLOGY, vol. 21, no. 6, 30 June 2016 (2016-06-30), pages 569 - 572, ISSN: 1009-0460 *
ZHANG, X.W. ET AL.: "Overexpression of long non-coding RNA PVT1 in gastric cancer cells promotes the development of multidrug resistance.", BIOCHEM BIOPHYS RES COMMUN., vol. 462, no. 3, 3 July 2015 (2015-07-03), pages 227 - 232, XP029141467, DOI: 10.1016/j.bbrc.2015.04.121 *
ZHENG, CHENGWU ET AL.: "Overexpression of the long non-coding RNA PVT1 is correlated with leukemic cell proliferation in acute promyelocytic leukemia", J HEMATOL ONCOL., vol. 8, no. 126, 6 November 2015 (2015-11-06), pages 1 - 6, XP055629639, DOI: 10.1186/s13045-015-0223-4 *
ZHI, XIAOSONG ET AL.: "Progerssion of the role of long non-coding RNA PVT1 in tumor (non-official translation)", JOURNAL OF XINXIANG MEDICAL UNIVERSITY , vol. 33, no. 3, 31 March 2016 (2016-03-31), pages 243 - 245, ISSN: 1004-7239 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023215761A1 (en) * 2022-05-03 2023-11-09 Tacit Therapeutics, Inc. Localization of trans-splicing nucleic acid molecules to and within the cellular nucleus

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