WO2023280243A1 - miR-339-增强子-靶基因网络激活模型及其应用 - Google Patents

miR-339-增强子-靶基因网络激活模型及其应用 Download PDF

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WO2023280243A1
WO2023280243A1 PCT/CN2022/104248 CN2022104248W WO2023280243A1 WO 2023280243 A1 WO2023280243 A1 WO 2023280243A1 CN 2022104248 W CN2022104248 W CN 2022104248W WO 2023280243 A1 WO2023280243 A1 WO 2023280243A1
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mirna
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breast cancer
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于文强
梁英
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Fudan University
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Definitions

  • the invention relates to the field of biotechnology, in particular to a miR-339-enhancer-target gene network activation model and its application.
  • non-coding RNA is a basic scientific research that has been gradually recognized by scientists in recent years and quickly invested in it, aiming to understand the mystery of life in essence.
  • miRNAs microRNAs
  • LncRNAs long non-coding RNAs
  • miRNAs are small non-coding RNAs with a length of 21-25 nucleotides that regulate gene expression at the post-transcriptional level.
  • the first miRNA lin-4 was accidentally discovered more than 30 years ago when studying the developmental regulation of nematodes, and was shown to inhibit protein translation or degrade mRNA by binding to the 3'UTR of lin-14 mRNA in the cytoplasm.
  • miRNAs Since then, the negative regulatory mechanism of miRNA has almost become the "golden rule" in the field of miRNA research. Although some miRNAs were later found to be able to promote gene expression or translation under special circumstances, they were basically case studies, limited to phenomenon descriptions and simple mechanism studies, which left follow-up researchers at a loss and lacked the significance of generalization. On the other hand, although miRNAs are involved in most life phenomena and processes, the regulation of miRNAs in vivo is usually considered as a fine-tune, and does not play a decisive "switch" role.
  • Enhancer has always been considered as an important DNA sequence cis-regulatory element, which can regulate gene expression in close or long distance.
  • H3K4me1 and H3K27ac are important epigenetic marks of enhancers, and H3K27ac is closely related to the activity of enhancers.
  • Super-enhancers can be formed when multiple transcriptionally active enhancers enrich transcription factors at high density. Enhancers, especially super-enhancers, can usually drive the expression of genes that control cell identity and cell fate, and play an important role in cell differentiation, tissue development, individual growth and disease pathogenesis.
  • Malignant tumors are a global public health problem that endangers human health. As a large developing country, my country has increasingly prominent problems of industrialization, urbanization, and population aging. Unhealthy lifestyles and environmental pollution directly lead to a decline in national health index. Malignant tumors urgently need to wait capture.
  • miRNA research has been proven to play an important regulatory role in the development and migration of breast cancer. Relevant research is usually based on the traditional negative regulatory mechanism of miRNA. So far, there has been no substantial breakthrough on how to use miRNA to overcome breast cancer, and there is still a lack of effective therapeutic drugs and treatment options.
  • miRNA is a classic nucleic acid molecule. Nucleic acid drugs are considered to be the next generation of drugs after antibody drugs and cellular immunotherapy. They can achieve effects that cannot be replaced by traditional drugs. For example, antibodies mostly function on the cell surface, while nucleic acid drugs can enter cells Internally play a therapeutic role; some nucleic acid drugs can even enter the nucleus to play a precise targeted therapeutic effect.
  • Onpattro patisiran
  • a new RNAi drug approved by the US FDA in 2018, is a nucleic acid drug.
  • the main treatment method is to encapsulate siRNA in lipid nanoparticles and deliver the drug directly to the liver, by targeting and silencing TTR mRNA, hindering the production of targeted TTR protein, thereby reducing the accumulation of amyloid deposits in peripheral nerves, and eventually
  • hATTR hereditary transthyretin
  • This invention applies the creative "miRNA-enhancer-target gene network activation model” theory for the first time to explain the occurrence and development of breast cancer, that is, miR-339 in the nucleus interacts with enhancers to positively activate target genes to affect tumor growth Proliferation and growth, this theory is completely different from the prior art "negative regulation mechanism of miRNA”.
  • This application screened multiple miRNAs located in the enhancer region, respectively overexpressed these miRNAs in different types of cells, and found that multiple genes adjacent to these miRNAs on the genome were transcriptionally activated, and these miRNAs regulated the transcriptional activation of genes Relies on the integrity of the miRNA and its targeting enhancer sequences. Based on this, this application uses the screened miRNAs to reveal the function of miRNAs in the nucleus, providing a new perspective for analyzing the involvement of miRNAs in the occurrence and development of diseases such as tumors.
  • miR-339 and the target gene GPER1 tend to be low-expressed in breast cancer clinical tissue samples, and further verification found that miR-339 positively regulates the expression of the GPER1 gene through an enhancer, thereby inhibiting the occurrence and development of breast cancer tumors. If new breast cancer drugs are developed from the perspective of nucleic acid drugs, then miR-339 may bring good news to patients with these diseases.
  • the present invention adopts the following technical solutions:
  • the first aspect of the present invention discloses a miRNA capable of activating breast cancer-related genes, the position of the gene locus of the miRNA coincides with the histone modification mark H3K4me1 or H3K27ac of the enhancer.
  • the breast cancer is Luminal breast cancer and triple-negative breast cancer.
  • miRNA means MicroRNAs, which refers to a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a size of about 20-25 nucleotides. Mature miRNAs are produced by a series of nuclease cleavage and processing of longer primary transcripts, and then assembled into RNA-induced silencing complexes, which recognize target mRNAs through complementary base pairing, and according to the degree of complementarity Differently direct the silencing complex to degrade the target mRNA or to repress the translation of the target mRNA.
  • nucleotide sequence of the miRNA is as shown in SEQ ID NO: 1, or
  • the sequence of the miRNA is 90% homologous and functionally identical to the SEQ ID NO: 1.
  • the SEQ ID NO: 1 sequence from 5' ⁇ 3' is: UCCCUGUCCUCCAGGAGCUCACG.
  • the breast cancer-related gene is GPER1 (G protein-coupled estrogen receptor 1) gene.
  • the second aspect of the present invention discloses a nucleic acid drug, which includes the above-mentioned miRNA or its artificial mimic; preferably, the nucleic acid drug further includes a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical dosage form of nucleic acid includes powder, tablet, granule, capsule, solution, aerosol, injection, emulsion or suspension.
  • the fourth aspect of the present invention is a biological material related to the above-mentioned nucleic acid drug, the biological material is selected from one of the following A), B) or C):
  • the expression vector includes at least one of pCDH vector, pCMVp-NEO-BAN vector, pEGFP vector, pEGFT-Actin, pSV2 vector, pCDNA vector, pLVX vector, pAAV vector, pET vector and pDsRed vector.
  • the step of constructing the recombinant vector comprises: 1) designing primers, PCR amplifying the miRNA sequence; 2) digesting the amplified sequence fragment and the expression vector, and connecting the target sequence fragment and the expression vector; 3) connecting the sequence fragment and the expression vector; The product is transformed into Escherichia coli and cultivated; 4) After identification, the recombinant plasmid is extracted and packaged to obtain the recombinant vector.
  • the fifth aspect of the present invention discloses the application of the above-mentioned miRNA or the above-mentioned nucleic acid drug in the diagnosis, prevention and/or treatment of breast cancer.
  • the sixth aspect of the present invention discloses a method for screening the aforementioned miRNAs that can activate disease-related genes, comprising the following steps:
  • step S2 verifying the relationship between the miRNA screened in step S1 and the target gene within 200kb around it;
  • the method for verifying the relationship between the miRNA and its surrounding target genes within 200kb includes: detecting the expression level of the target gene after the miRNA is overexpressed by real-time fluorescent quantitative PCR.
  • the seventh aspect of the present invention discloses a model for studying the mechanism of breast cancer, the model is a miRNA-enhancer-target gene network activation model.
  • the miRNA interacts with the enhancer to positively activate the target gene to affect the proliferation and growth of the tumor, and the miRNA is miR-339;
  • the method for constructing the model includes: screening multiple miRNAs located in the enhancer region, respectively overexpressing the miRNAs in different types of cells, and finding that multiple genes adjacent to the miRNAs on the genome are transcribed Activation; get miRNA-enhancer-target gene network activation model.
  • the eighth aspect of the present invention discloses the application of the above model in the field of breast cancer; preferably, the application of the model in the preparation of drugs for treating breast cancer.
  • the inventors conducted a systematic bioinformatics analysis on 1594 miRNA precursors in different tissue cells in the early stage, and found that the positions of more than 300 miRNA precursors in the genome highly overlapped with the histone modification marks H3K4me1 or H3K27ac of enhancers . It was also found that most of these miRNAs can be localized in the nucleus, and further studies have found that these miRNAs can bind to enhancers, thereby activating gene expression at the genome-wide level. In view of the fact that both enhancer and miRNA are involved in processes such as individual development and disease occurrence, the inventors linked the two to explore their important roles in the occurrence of tumors and other diseases.
  • the present invention firstly overexpresses the miRNA miR-339 gene sequence, activates the expression level of surrounding genes, and affects the biological function of the breast cancer cell line.
  • the above screening and verification have good application value in the diagnosis and detection of breast cancer and drug development.
  • the miR-339 involved in the present invention can activate the expression of genes through enhancers, and is closely related to breast cancer, and its artificially synthesized analogs or inhibitors can be used in the development of nucleic acid drugs.
  • the present invention Compared with the prior art, the present invention has the following advantages: the present invention provides that miR-339, which can activate disease-related genes, is related to breast cancer, and provides new strategies and potential drug targets for the diagnosis and treatment of breast cancer and other diseases. Moreover, the present invention applies the theory of "miRNA-enhancer-target gene network activation model" to explain the occurrence and development of breast cancer for the first time, that is, miR-339 in the nucleus interacts with enhancers to positively activate target genes, thereby affecting tumor growth and development. Proliferation and growth, this theory is completely different from the prior art "negative regulation mechanism of miRNA", and overcomes the prejudice of the prior art.
  • Figure 2 shows the expression levels of miR-339 and target gene GPER1 in the breast cancer cell line 4175 in Example 2 of the present invention
  • Fig. 3 is the dual fluorescein reporter gene experiment detection enhancer sequence activity in the embodiment 3 of the present invention.
  • Figure 4 illustrates the effect of the expression of miR-339 on enhancer activity in the double fluorescein reporter gene experiment in Example 3 of the present invention
  • FIG. 5 shows that after the ChIP-seq analysis in Example 3 of the present invention overexpressed miR-339, the enrichment of H3K27ac near the position of miR-339 DNA locus was significantly higher than that of the control group;
  • Fig. 6 shows that the overexpression of miR-339 in Example 3 of the present invention can activate enhancer activity.
  • Figure 7 shows that miR-339 was overexpressed after knocking out the enhancer sequence in Example 3 of the present invention, and the target gene GPER1 could not be reactivated;
  • FIG. 8 is a graph showing the proliferation ability of breast cancer cell line 4175 with miR-339 overexpression in Example 3 of the present invention.
  • FIG. 9 is a graph showing the proliferation ability of breast cancer cell line T47D with miR-339 overexpression in Example 3 of the present invention.
  • Fig. 10 is a diagram of the proliferation ability of breast cancer cell line 4175 after interfering with the expression of GPER1 in Example 3 of the present invention.
  • Fig. 11 is a diagram of the proliferation ability of the breast cancer cell line T47D after interfering with the expression of GPER1 in Example 3 of the present invention.
  • Figure 12 is a clone formation diagram of breast cancer cell line 4175 overexpressing miR-339 in Example 3 of the present invention.
  • Figure 13 is a clone formation diagram of the breast cancer cell line T47D overexpressing miR-339 in Example 3 of the present invention.
  • Figure 14 is a diagram of the clone formation of the breast cancer cell line 4175 after interfering with the expression of GPER1 in Example 3 of the present invention.
  • Figure 15 is a diagram of the clone formation of the breast cancer cell line T47D after interfering with the expression of GPER1 in Example 3 of the present invention.
  • Figure 16 is a diagram of the tumors of three groups of mice in Example 3 of the present invention.
  • FIG 17 is a statistical chart of the tumor weights of the three groups of mice in Example 3 of the present invention ("Tumor weight (g)" in the figure refers to "tumor weight”).
  • the term "protein” is a general term for different peptides consisting of 25 natural amino acids in different compositions and arrangements, ranging from dipeptides to complex linear and circular structures, and is a multifunctional compound derived from proteins.
  • Active peptides have a variety of human metabolism and physiological regulation functions, are easy to digest and absorb, and have the functions of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, and lowering blood fat. They are extremely safe to eat and are currently the most popular in the international food industry. Research topics and highly promising functional factors.
  • the term "cloning” refers to duplication, copying and doubling, that is, to produce the same copy from the prototype, whose appearance and genetic genes are exactly the same as the prototype.
  • sequencing refers to a method for determining the sequence of a gene.
  • This embodiment is the construction of miR-339 overexpression vector, and its steps mainly include:
  • the DNA sequence information of pri-miR-339 was obtained from the UCSC database and the miRbase database, and the relevant primer sequences were designed using Primer 5.0 software, and then the applicability of the primers was tested by BLAST.
  • the primers were synthesized by Shanghai Sonny Biotechnology Co., Ltd.
  • the final designed primers are: PCDH339-EcoRI-F and PCDH339-BamHI-R.
  • PCDH339-EcoRI-F GAAGATTCTAGAGCTAGCGAATTCGGCCACAGGCAGGTGCCACC (SEQ ID NO: 2);
  • PCDH339-BamHI-R GCAGATCCTTCGCGGCCGCGGATCCACACCAGTGGAACATCCCATGC (SEQ ID NO: 3).
  • KODFX High Fidelity Enzyme was purchased from TOYOBO Company, product number KFX-101.
  • PCR products were detected by 1% agarose gel electrophoresis, and the target fragment was recovered using a common agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.).
  • Enzyme digestion and purification refers to the enzyme digestion system on the NEB website, which is digested overnight at 37°C, and then purified and recovered using a PCR product recovery kit (Tiangen Biochemical Technology Co., Ltd.).
  • the digested PCR product was ligated with the digested pCDH vector, and ligated overnight at 16°C.
  • 2 ⁇ Taq Master Mix was purchased from lifetech company.
  • the miR-339 sequence was overexpressed in breast cancer cells T47D and 4175, and the expression levels of surrounding genes were detected.
  • Encapsulation of lentivirus by liposome method According to molecular cloning, the plasmid pCDH-miR339 obtained in Example 1, the virus packaging plasmid psPAX2 and the capsid plasmid pMD2.G-VSVG were transferred into 293T cells. The supernatant was collected once after 48h and 72h respectively, and the collected supernatant was filtered through a 0.45 ⁇ m filter to obtain the lentivirus stock solution.
  • Infected cells spread the cells to be infected in 6cm culture dishes at a rate of ⁇ 200,000 per dish in advance, and perform the first infection after the cells adhere to the wall on the second day, and repeat the infection once on the third day; on the fourth day The cells were allowed to recover for one day without any stimulation; on the fifth day, drug screening was carried out on the corresponding drug-killing markers carried by the plasmids.
  • Takara (D2680A) reverse transcription PCR kit was used for reverse transcription.
  • Reverse transcription PCR conditions 42°C for 10 minutes, 95°C for 2 minutes.
  • Takara real-time fluorescent quantitative PCR kit was used to detect the expression of the target gene at the transcriptional level.
  • the miR-339 expression plasmid (PSR-339) and the pGL 3-p-339 vector inserted into the miR-339 locus were simultaneously transfected into 293T cells for a double-fluorescein reporter gene experiment.
  • the results showed that after cotransfection of miR-339 expression plasmid (PSR-339) and pGL3-p-339 vector inserted into miR-339 locus, the reporter gene activity was significantly higher than that of the control group transfected only with pGL3-p-339 vector The activity was higher, suggesting that the enhancer activity was enhanced by miR-339 (Fig. 4-a,b).
  • H3K27ac ChIP-purified DNA from the experimental group and control group that overexpressed miR-339 in 293T cells, and the qualified library was analyzed by high-throughput sequencing to determine whether the enhancer near miR-339 was overexpressed by miR -339 activated.
  • H3K27ac ChIP-seq data analysis results ( Figure 5) and QPCR results ( Figure 6) showed that, compared with the control cells, a DNA sequence located about 60 kb upstream of the GPER1 gene was significantly enriched in H3K27ac after miR-339 overexpression set, suggesting that this region is an enhancer activated by miR-339, and this location coincides with the miR-339 locus.
  • Example 3 miR-339 inhibits the proliferation of breast cancer cells by targeting and activating the target gene GPER1
  • Example 2 On the basis of Example 2, further detect and evaluate the proliferation ability of breast cancer cell lines 4175 and T47D stably expressing miR-339 constructed by lentivirus through the proliferation of CCK8.
  • each group was set up as a control group: breast cancer cell lines 4175 and T47D transfected with empty virus fluid.
  • Example 2 further detect and evaluate the proliferation ability of breast cancer cell lines 4175 and T47D stably expressing miR-339 constructed by lentivirus through the proliferation of clone formation.
  • each group was set up as a control group: breast cancer cell lines 4175 and T47D transfected with empty virus fluid.
  • Figure 16 is a graph of tumors in each group
  • Figure 17 is a statistical graph of tumor weight.
  • the nude mice tumors in the experimental group overexpressing miR-339 were significantly smaller than those in the control group, suggesting that miR-339 has the potential to inhibit the occurrence and development of breast cancer tumors; after overexpressing miR-339 and re-knocking down GPER1, the nude mice formed tumors The size is larger than that of nude mice in the experimental group overexpressing miR-339, indicating that GPER1 can inhibit tumor growth in nude mice.
  • miR-339 is located on chromosome 7 of the human genome.
  • Current research focuses on the involvement of miR-339 in tumor development and metastasis through negative regulation of target genes.
  • the present application first finds that miR-339 is a key factor in the occurrence and development of breast cancer through bioinformatics analysis.
  • exogenous miR-339 was overexpressed in Luminal breast cancer and triple-negative breast cancer cell lines T47D and 4175, respectively, and the results showed that after overexpression of miR-339, the expression of tumor suppressor gene GPER1 in these two breast cancer cells Both of them were up-regulated, and their proliferation and clonogenic ability were significantly inhibited.
  • GPER1 is a G protein-coupled receptor, which has been reported many times as a tumor suppressor gene to be down-regulated or inactivated in estrogen-related cancers, and is associated with poor prognosis of tumors.
  • the research results of this application suggest that miR-339 analogs or mimics can be used as a potential strategy for the treatment of triple-negative breast cancer.

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Abstract

提供了可激活乳腺癌相关基因的miR-339、miR-339-增强子-靶基因网络激活模型及其应用。该miR-339的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合，与乳腺癌相关，为乳腺癌等疾病的诊断、治疗提供新的策略和潜在的药物靶点。并将"miRNA-增强子-靶基因网络激活模型"理论应用于解释乳腺癌的发生发展现象。

Description

miR-339-增强子-靶基因网络激活模型及其应用 技术领域
本发明涉及生物技术领域，具体涉及miR-339-增强子-靶基因网络激活模型及其应用。
背景技术
非编码RNA的研究是近年来逐渐被科学家认识并迅速投入其中的基础科学研究，旨在从本质上认识生命的奥秘。非编码RNA种类繁多，目前研究较多的是microRNA(miRNA)和长非编码RNA(LncRNA)。miRNA是一些长度为21-25个核苷酸，在转录后水平调控基因表达的非编码小RNA。第一个miRNA lin-4是30多年前在研究线虫的发育调控时被意外发现的，并被证明通过结合细胞浆中lin-14 mRNA的3’UTR发挥抑制蛋白翻译或降解mRNA的作用。此后，miRNA的负向调控作用机制几乎成为miRNA研究领域“金科玉律”。尽管后来也发现部分miRNA在特殊情况下能够促进基因表达或翻译，但基本都是个例研究，仅限于现象描述和简单的机制研究，致使后续研究者不得要领，缺乏推而广之的意义。另一方面，虽然miRNA参与大多数生命现象和过程，但是miRNA在体内的调控通常被认为是微调控(fine-tune)，并没有起到决定性的“开关”作用。
增强子(Enhancer)一直被认为是重要的DNA序列顺式调控元件，它可以近距离或远距离调控基因的表达。H3K4me1以及H3K27ac是增强子重要的表观遗传学标记，其中H3K27ac与增强子的活性密切相关。当多个具有转录活性的增强子高密度富集转录因子后，可以形成超级增强子。增强子尤其是超级增强子通常可以驱动控制细胞身份和细胞命运的基因表达，在细胞分化、组织发育、个体生长和疾病致病过程中发挥很重要的作用。
恶性肿瘤是危害人类健康的全球公共卫生问题，我国作为一个发展中大国，工业化、城镇化和人口老龄化问题日益突出，不良生活方式及环境污染等直接导致国民身体健康指数 下降，恶性肿瘤急需等待攻克。
miRNA研究已经被证明在乳腺癌发生发展与迁移中发挥重要的调控作用。相关研究通常是基于传统的miRNA负向调控机制，到目前为止，如何利用miRNA攻克乳腺癌尚未有实质性的突破，尚缺乏有效的治疗药物和治疗方案。miRNA是经典的核酸分子，核酸药物被认为是继抗体药物及细胞免疫疗法之后的下一代药物，其可以达到传统药物无法替代的效果，比如抗体多在细胞表面发挥作用，而核酸药物可以进入细胞内部发挥治疗作用；有的核酸药物甚至可以进入到细胞核发挥精确的靶向治疗作用。比如，2018年美国FDA批准上市的RNAi新药Onpattro(patisiran)是一款核酸药物。主要的治疗方法是把siRNA包裹在脂质纳米颗粒中并将药物直接递送至肝脏，通过靶向和沉默TTR mRNA，阻碍靶向TTR蛋白生成，从而降低周围神经中淀粉样沉积物的积累，最终达到治疗hATTR(遗传性转甲状腺素蛋白)淀粉样变性疾病的目的。目前核酸药物开发日益成熟，在癌症和遗传性疾病以及流感和病毒感染方面取得一些重要成果，但是有待开发的潜力巨大。
发明内容
本发明首次将创造性的“miRNA-增强子-靶基因网络激活模型”理论应用于解释乳腺癌的发生发展现象，即核内miR-339与增强子互作进而正向激活靶基因从而影响肿瘤的增殖生长，这一理论与现有技术“miRNA的负向调控机制”完全不同。
本申请筛选了位于增强子区域的多个miRNA，分别将这些miRNA在不同种类的细胞中过表达，发现在基因组上与这些miRNA邻近的多个基因被转录激活，这些miRNA对基因的转录激活调控依赖于miRNA及其靶向增强子序列的完整性。基于此，本申请将筛选出来的miRNA用于揭示细胞核内miRNA功能，为解析miRNA参与肿瘤等疾病发生发展提供新的视角。
具体地，本申请发现miR-339和靶基因GPER1倾向于在乳腺癌临床组织样本中低表 达，进一步验证发现miR-339通过增强子正向调控GPER1基因的表达，进而抑制乳腺癌肿瘤发生发展。如果从核酸药物的角度去开发新型乳腺癌的药物，那么miR-339有可能会为这些疾病患者带来福音。
为实现上述目的，本发明采用如下技术方案：
本发明第一个方面公开了一种可激活乳腺癌相关基因的miRNA，所述miRNA的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。
优选的，所述乳腺癌为Luminal型乳腺癌和三阴性乳腺癌。
术语“miRNA”即MicroRNAs，其是指在真核生物中发现的一类内源性的具有调控功能的非编码RNA，其大小长约20～25个核苷酸。成熟的miRNAs是由较长的初级转录物经过一系列核酸酶的剪切加工而产生的，随后组装进RNA诱导的沉默复合体，通过碱基互补配对的方式识别靶mRNA，并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译。
优选的，所述miRNA的核苷酸序列如SEQ ID NO：1所示，或
所述miRNA的序列为与所述SEQ ID NO：1具有90％同源性且功能相同的序列。
所述SEQ ID NO：1序列从5’→3’为：UCCCUGUCCUCCAGGAGCUCACG。
优选的，所述乳腺癌相关基因为GPER1(G蛋白偶联雌激素受体1)基因。
本发明第二个方面公开了一种核酸药物，所述核酸药物包括上述的miRNA或其人工模拟物；优选的，所述核酸药物还包括药学上可接受的载体或赋形剂。
更优选的，所述核酸药物剂型包括散剂、片剂、颗粒剂、胶囊剂、溶液剂、气雾剂、注射剂、乳剂或混悬剂。
本发明第四个方面一种与是上述核酸药物相关的生物材料，所述生物材料选自下述A)、B)或C)中的一种：
A)与上述的miRNA的核苷酸序列互补配对的干扰RNA分子或者抑制剂；
B)包含上述miRNA的核苷酸序列的表达载体或重组载体；
C)包含有B)中所述的表达载体或重组载体的细胞或微生物。
优选的，所述表达载体包括pCDH载体、pCMVp-NEO-BAN载体、pEGFP载体、pEGFT-Actin、pSV2载体、pCDNA载体、pLVX载体、pAAV载体、pET载体和pDsRed载体中的至少一种。
优选的，所述重组载体构建步骤包括：1)设计引物，PCR扩增所述miRNA序列；2)将扩增的序列片段和表达载体酶切，连接目的序列片段和表达载体；3)将连接产物转化大肠杆菌，培养；4)鉴定后提取重组质粒并进行包装即得到重组载体。
本发明第五个方面公开了上述的miRNA或上述的核酸药物在诊断、预防和/或治疗乳腺癌中的应用。
本发明第六个方面公开了一种筛选上述的可激活疾病相关基因的miRNA的方法，包括以下步骤：
S1、通过生物信息学分析，筛选基因座位置与H3K4me1或H3K27ac重合的miRNA；
S2、验证步骤S1筛选的miRNA与其周围200kb范围内的靶基因的关系；
S3、获取S2中与所述靶基因具有相关性的miRNA，即为可激活疾病相关基因的miRNA。
优选的，步骤S2中，验证miRNA与其周围200kb范围内的靶基因的关系的方法包括：实时荧光定量PCR检测过表达所述miRNA后所述靶基因的表达量。
本发明第七个方面公开了一种用于研究乳腺癌机制的模型，所述模型为miRNA-增强子-靶基因网络激活模型。
优选的，miRNA与增强子互相作用进而正向激活靶基因从而影响肿瘤的增殖生长，所述miRNA为miR-339；
优选的，所述模型的构建方法包括：筛选位于增强子区域的多个miRNA，分别将所述miRNA在不同种类的细胞中过表达，发现在基因组上与所述miRNA邻近的多个基因被转 录激活；得到miRNA-增强子-靶基因网络激活模型。
本发明第八个方面公开了上述的模型在乳腺癌领域中的应用；优选的，所述模型在制备治疗乳腺癌药物中的应用。
发明人前期在不同的组织细胞中，对1594条miRNA前体进行了系统地生物信息学分析，发现300多条miRNA前体在基因组中的位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac高度重叠。并且发现，这些miRNA大多能够定位于细胞核内，进一步研究发现这些miRNA能够与增强子结合，进而在全基因组的水平上激活基因表达。鉴于增强子和miRNA都参与个体发育和疾病发生等过程，发明人将二者联系起来，探究其在肿瘤等疾病发生中的重要作用。
本发明首先通过将miRNA miR-339基因序列过表达，激活周围基因表达水平，影响乳腺癌细胞系的生物学功能。上述筛选及验证对乳腺癌的诊断及检测以及药物开发方面均具有很好的应用价值。本发明所涉及miR-339可以通过增强子激活基因的表达，与乳腺癌密切相关，其人工合成的类似物或者抑制剂可用于核酸药物的开发。
在符合本领域常识的基础上，上述各优选条件，可任意组合，而不超出本发明的构思与保护范围。
本发明相对于现有技术具有如下优点：本发明提供了可激活疾病相关基因的miR-339与乳腺癌相关，为乳腺癌等疾病的诊断、治疗提供新的策略和潜在的药物靶点。并且，本发明首次将“miRNA-增强子-靶基因网络激活模型”理论应用于解释乳腺癌的发生发展现象，即核内miR-339与增强子互作进而正向激活靶基因从而影响肿瘤的增殖生长，这一理论与现有技术“miRNA的负向调控机制”完全不同，克服了现有技术的偏见。
附图说明
图1为本发明实施例2中的乳腺癌细胞系T47D中miR-339和靶基因GPER1的表达量 (图中“Relative expression levels”指“相对表达水平”)；
图2为本发明实施例2中的乳腺癌细胞系4175中miR-339和靶基因GPER1的表达量；
图3为本发明实施例3中的双荧光素报告基因实验检测增强子序列活性；
图4为本发明实施例3中的双荧光素报告基因实验阐述miR-339的表达可以对增强子活性的影响；
图5为本发明实施例3中的ChIP-seq分析过表达miR-339后，miR-339 DNA locus位置附近H3K27ac富集明显高于对照组；
图6为本发明实施例3中的过表达miR-339可以激活增强子活性。ChIP-QPCR检测过表达miR-339后，miR-339 DNA locus位置附近存在显著的H3K27ac富集(图中“input”指“阳性对照”)；
图7为本发明实施例3中的敲除增强子序列后过表达miR-339，无法重激活靶基因GPER1；
图8为本发明实施例3中的miR-339过表达的乳腺癌细胞株4175的增殖能力图；
图9为本发明实施例3中的miR-339过表达的乳腺癌细胞株T47D的增殖能力图；
图10为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株4175的增殖能力图；
图11为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株T47D的增殖能力图；
图12为本发明实施例3中的miR-339过表达的乳腺癌细胞株4175的克隆形成图；
图13为本发明实施例3中的miR-339过表达的乳腺癌细胞株T47D的克隆形成图；
图14为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株4175的克隆形成图；
图15为本发明实施例3中的干扰GPER1表达后的乳腺癌细胞株T47D的克隆形成图；
图16为本发明实施例3中的3组小鼠肿瘤图；
图17为本发明实施例3中的3组小鼠肿瘤重量统计图(图中“Tumor weight(g)”指“肿瘤重量”)。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述，但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明所用试剂和原料均市售可得。
本申请早期在研究miRNA自身的表观遗传学调控机制时，在7种不同的组织细胞中，对1594条miRNA前体进行了系统分析，结果发现有300多条miRNA前体在基因组中的位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac高度重叠。进一步应用18种组织细胞的增强子数据库进行比对分析，发现人类的1881条miRNA的60％以上与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。基于前期的工作，本申请提出miRNA—增强子—靶基因网络激活模型理论，这些miRNA大多能够定位于细胞核内，进一步研究发现这些miRNA能够与增强子结合，进而在全基因组的水平上激活基因表达。进一步地，本申请通过生物信息学数据整合分析，把基因座位置与H3K4me1或H3K27ac重合的miRNA所在位置的200kb范围内的靶基因联系起来，发现更多的miRNA—增强子—靶基因网络可用于解释生命个体的不同生理现象。
目前研究主要集中在miR-339通过负性调控靶基因，参与肿瘤发展和转移过程。发明人首先通过生物信息学分析发现NamiRNA miR-339是乳腺癌发生发展的关键因子。接着，在Luminal型乳腺癌和三阴性乳腺癌细胞系T47D和4175中分别过表达外源性miR-339，结果表明过表达miR-339后，这两个乳腺癌细胞中抑癌基因GPER1的表达均上调，它们的增殖和克隆形成能力均受到明显抑制。最后本研究结果在动物实验中进一步得到验证，提示过表达miR-339可以再激活抑癌基因GPER1从而抑制乳腺癌细胞的生长与增殖。
术语说明
如本发明中所述，术语“蛋白”是25个天然氨基酸以不同组成和排列方式构成的从二肽到复杂的线性、环形结构的不同肽类的总称，是源于蛋白质的多功能化合物。活性肽具有多种人体代谢和生理调节功能，易消化吸收，有促进免疫、激素调节、抗菌、抗病毒、降血压、降血脂等作用，食用安全性极高，是当前国际食品界最热门的研究课题和极具发展前景的功能因子。
如本发明中所述，术语“克隆”是指复制、拷贝和翻倍，就是从原型中产生出同样的复制品，它的外表及遗传基因与原型完全相同。
如本发明中所述，术语“测序”指的是对基因的序列进行判断的一种方法。
实施例1 miRNA过表达载体构建
本实施例为miR-339过表达载体的构建，其步骤主要包括：
1.1、序列获取和引物设计
首先从UCSC数据库和miRbase数据库上获取pri-miR-339的DNA序列信息，利用Primer 5.0软件设计相关引物序列，再用BLAST检测引物适用性。引物委托上海桑尼生物科技有限公司进行合成。最终设计的引物为：PCDH339-EcoRI-F和PCDH339-BamHI-R。
PCDH339-EcoRI-F：GAAGATTCTAGAGCTAGCGAATTCGGCCACAGGCAGGTGCCACC(SEQ ID NO：2)；
PCDH339-BamHI-R：GCAGATCCTTCGCGGCCGCGGATCCACACCAGTGGAACATCCCATGC(SEQ ID NO：3)。
1.2、PCR扩增获取pri-miR-339 DNA序列
使用293T细胞的基因组作为模板以及步骤1.1设计的引物进行PCR扩增。其中，
PCR反应体系：
Figure PCTCN2022104248-appb-000001
Figure PCTCN2022104248-appb-000002
其中，KODFX高保真酶购于TOYOBO公司，货号KFX-101。
PCR反应条件：
Figure PCTCN2022104248-appb-000003
1.3、PCR产物的回收、酶切及纯化
PCR产物的回收：将PCR产物进行1％琼脂糖凝胶电泳检测，并使用普通琼脂糖凝胶DNA回收试剂盒(天根生化科技公司)回收目的片段。
酶切及纯化：酶切过程参照NEB网站酶切体系37℃酶切过夜，并采用PCR产物回收试剂盒(天根生化科技公司)纯化回收。
1.4、连接
利用T4连接酶，将酶切后的PCR产物和酶切后的pCDH载体连接，16℃连接过夜。
连接体系：
Figure PCTCN2022104248-appb-000004
Figure PCTCN2022104248-appb-000005
1.5、转化、挑取单克隆
(1)将10μl步骤1.4得到的连接产物加入50μl的DH5α感受态细胞中，冰上孵育30min。
(2)42℃热激感受态细胞90s后，立即放到冰上5min。
(3)在超净工作台中加入300μl不含抗生素的LB液体培养基，37℃恒温摇床中摇30min。
(4)菌液1000g离心5min后弃上清，剩50μl菌液，将菌液均匀涂到氨苄抗性的LB固体平板上，37℃恒温培养箱培养过夜。
(5)从过夜培养的平板上挑取适量单克隆菌落，分别放入加有200μl氨苄抗性LB液体培养基的EP管中，37℃恒温摇床中摇菌2h后通过PCR克隆鉴定。
(6)PCR克隆鉴定，使用293T细胞的基因组作为模板进行PCR扩增。
PCR反应体系：
Figure PCTCN2022104248-appb-000006
其中，2×Taq Master Mix购于lifetech公司。
PCR反应条件：
Figure PCTCN2022104248-appb-000007
取少量菌液接入到LB培养基中，加入相应的抗生素(氨苄或者卡那)，以250-300rpm/min的转速，37℃振荡培养过夜。抽提质粒获得过表达miR-339的质粒PCDH-miR339。
实施例2 miR-339激活靶基因GPER1
本实施例在乳腺癌细胞T47D和4175中过表达miR-339序列，对周围基因表达水平进行检测。
2.1、脂质体法包制慢病毒：依照分子克隆，将实施例1得到的质粒PCDH-miR339及病毒包装质粒psPAX2和衣壳质粒pMD2.G-VSVG转入293T细胞。分别于48h和72h后各收取一次上清，将收取的上清于0.45μm滤器过滤细胞碎片后得到慢病毒原液。
2.2、感染细胞：提前将需要被感染的细胞按照～20万每皿铺于6cm培养皿中，待第二天细胞贴壁之后进行第一次感染，第三天再重复感染一次；第四天让细胞恢复一天不加入任何刺激；第五天开始对质粒所带的对应杀药标记进行药物筛选。
2.3、实时荧光定量PCR
(1)总RNA提取
准备约10 6-10 7被感染的细胞，用PBS重悬后离心去除上清，加入1ml Trizol室温裂解5min，然后加入0.2ml氯仿，涡旋震荡仪震荡15s，室温静置2min。4℃离心机离心15min，13300rpm。转移上层无色水相于另一EP管中。加入等体积异丙醇，涡旋震荡仪充分混合， 入4℃离心机离心10min，13300rpm。倒掉上清，加入DEPC水配制的75％乙醇1ml，上下颠倒至沉淀悬浮起来，入4℃离心机离心5min，13,300rpm。用移液器吸尽上清，室温干燥5-20min，期间观察沉淀形态，待其刚刚变透明时，根据沉淀的量将其溶解于40-100μl DEPC水中。取1μl在Nanodrop上测定其浓度以及OD260/OD280。提取的RNA于-80℃冰箱保存。
(2)逆转录合成cDNA
逆转录使用Takara(D2680A)的反转录PCR试剂盒。
反应体系：
Figure PCTCN2022104248-appb-000008
反转录PCR条件：42℃10min，95℃2min。
(3)RT-qPCR
使用Takara实时荧光定量PCR试剂盒检测目的基因转录水平上的表达情况。
反应体系：
Figure PCTCN2022104248-appb-000009
Figure PCTCN2022104248-appb-000010
通过分析试验结果发现，通过克隆入miR-339序列，乳腺癌细胞T47D和4175中过表达miR-339分别为10.56倍和3.79倍。且乳腺癌细胞T47D和4175中的GPER1均被激活。结果如图1和图2所示。图1中，乳腺癌细胞系4175中miR-339过表达3.79倍，靶基因GPER1被激活2.80倍；图2中，乳腺癌细胞系T47D中miR-339过表达10.56倍，靶基因GPER1被激活3.54倍。上述结果表明，乳腺癌细胞系中miRNA-339可以激活抑癌基因GPER1 mRNA转录水平。
2.3、双荧光素报告基因实验
(1)将miR-339 DNA locus位置的上下游588bp DNA序列扩增并插入到报告基因载体PGL3-promoter上，验证miR-339 DNA locus的这一段序列是否具有增强子活性。我们在293T细胞中转染插入miR-339 DNA locus序列的报告基因载体(pGL3-miR-339)，和空载的对照组相比较(pGL3)，24小时后收集细胞检测荧光强度，结果显示实验组的活性更高(图3)，提示插入的miR-339 locus序列具有增强子活性，可以增强报告基因的活性。
(2)接下来，本发明想进一步探讨miR-339是否可以促进增强子的活性。将miR-339的表达质粒(PSR-339)和插入miR-339 locus的pGL 3-p-339载体同时转染到293T细胞中进行双荧光素报告基因实验。结果表示，在共转入miR-339的表达质粒(PSR-339)和插入miR-339 locus的pGL3-p-339载体后，报告基因活性明显比只转染pGL3-p-339载体的对照组活性更高，提示增强子活性被miR-339提高(图4-a，b)。
(3)突变结合位点。通过构建miR-339表达质粒的突变质粒(mut-PSR-339)以及增强子序列质粒pGL3-p-339的突变质粒(mut-pGL3-p-339)，结合位点突变后的报告基因活性不能继续被激活(图4-a，b，c，d)；如果结合位点按照界碱基互补配对的方式突变，报告基因的活性能继续被激活(图4-a，c，d，e)。提示miR-339激活增强子依赖于miR-339和 增强子结合序列的完整性，也就是种子序列结合位点的互补性。
2.4、染色质免疫共沉淀实验
对293T细胞过表达miR-339的实验组和对照组H3K27ac ChIP纯化的DNA进行文库构建，质检合格的文库进行高通量测序分析，以确定miR-339附近的增强子是否被过表达的miR-339激活。H3K27ac ChIP-seq数据分析结果(图5)以及QPCR结果(图6)显示，与对照组细胞相比，位于GPER1基因上游大约60kb位置的一段DNA序列在miR-339过表达后出现明显的H3K27ac富集，提示该区域为一个被miR-339所激活的增强子，而这个位置正好是和miR-339 locus重合。
2.5、CRISPR敲除实验
(1)首先设计增强子区域miR-339结合位点的gRNA，构建了CRISPR敲除质粒(CRISPR-339/GFP)并转染到293T细胞中；
(2)通过96孔板稀释法，结合快速克隆验证和Sanger测序等方法我们筛选获得了miR-339结合位点缺失的单克隆细胞株；
(3)对敲除增强子序列后的细胞再进行过表达miR-339，利用QPCR实验进一步研究增强子序列的完整性对miR-339激活作用的影响。敲除增强子序列后过表达miR-339，24小时后收集细胞进行QPCR，实验结果表明，如图7所示，miR-339成功过表达，但是GPER1的表达却明显下调，提示即使在敲除增强子序列中的细胞中再过表达miR-339也不能激活靶基因GPER1。
实施例3 miR-339通过靶向激活靶基因GPER1抑制乳腺癌细胞增殖
3.1、在实施例2的基础上进一步通过CCK8的增殖检测并评估慢病毒构建的稳定表达miR-339的乳腺癌稳转细胞株4175和T47D的增殖能力。并且，每组设立对照组为：转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图8和9所示。与对照组相比，miR-339过表达的乳腺癌细胞株4175和T47D的增殖能力均下降。
3.2、进一步研究GPER1对乳腺癌细胞系增殖能力的影响。通过转染shGPER1干扰乳腺癌细胞系4175和T47D中GPER1的表达，通过CCK8的增殖检测GPER1对两个乳腺癌细胞系的增殖影响。并且，每组设立对照组为：转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图10和11所示，两组乳腺癌细胞4175和T47D中，干扰GPER1表达后的实验组比对照组细胞数目明显多，提示GPER1对乳腺癌细胞系的增殖有抑制作用。
3.3、在实施例2的基础上进一步通过克隆形成的增殖检测并评估慢病毒构建的稳定表达miR-339的乳腺癌稳转细胞株4175和T47D的增殖能力。并且，每组设立对照组为：转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图12和13所示。与对照组相比，miR-339过表达的乳腺癌细胞株4175和T47D的增殖能力均下降。
3.4、进一步研究GPER1对乳腺癌细胞系增殖能力的影响。通过转染shGPER1干扰乳腺癌细胞系4175和T47D中GPER1的表达，通过克隆形成实验的增殖检测GPER1对两个乳腺癌细胞系的增殖影响。并且，每组设立对照组为：转染空载病毒液的乳腺癌细胞株4175和T47D。
结果如图14和15所示，两组乳腺癌细胞4175和T47D中，干扰GPER1表达后的实验组比对照组形成克隆数目明显多，提示GPER1对乳腺癌细胞系的增殖有抑制作用。
3.5、将稳定表达miR-339和相应空载的对照组稳转细胞株及过表达miR-339后敲低GPER1的乳腺癌细胞株，3组细胞株分别注射到裸鼠的后背，饲养并观察每组裸鼠后背成瘤状况。注射细胞4周后裸鼠背上开始成瘤，记录为d0天，在d0后的第28天，部分成瘤裸鼠出现死亡，立即将所有裸鼠引颈处死，收集瘤子，统计肿瘤大小和重量。
收集的肿瘤如图16和17所示，图16为各组肿瘤图，图17为肿瘤重量统计图。过表达miR-339的实验组裸鼠瘤子明显比对照组要小，提示miR-339有抑制乳腺癌肿瘤的发生发展的潜力；而在过表达miR-339后重新敲低GPER1的裸鼠成瘤大小要比过表达miR-339的实验组裸鼠瘤子大，说明GPER1在裸鼠中可以抑制肿瘤生长。
综合实施例1-3，miR-339位于人类基因组7号染色体，目前研究主要集中在miR-339通过负性调控靶基因，参与肿瘤发展和转移过程。本申请首先通过生物信息学分析发现miR-339是乳腺癌发生发展的关键因子。并且，在Luminal型乳腺癌和三阴性乳腺癌细胞系T47D和4175中分别过表达外源性miR-339，结果表明过表达miR-339后，这两个乳腺癌细胞中抑癌基因GPER1的表达均上调，它们的增殖和克隆形成能力均受到明显抑制。最后申请的实验结果在动物实验中进一步得到验证，提示过表达miR-339可以再激活抑癌基因GPER1从而抑制乳腺癌细胞的生长与增殖。GPER1是G蛋白偶联受体，多次被报道作为抑癌基因在与雌激素相关的癌症中表达下调或者失活，并与肿瘤的不良预后相关。本申请的研究结果提示miR-339的类似物或者模拟物可以作为三阴性乳腺癌治疗的潜在策略。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (11)

  1. 一种可激活乳腺癌相关基因的miRNA，其特征在于，所述miRNA的基因座位置与增强子的组蛋白修饰标志H3K4me1或H3K27ac重合。
  2. 根据权利要求1所述的miRNA，其特征在于，所述miRNA的核苷酸序列如SEQ ID NO：1所示，或
    所述miRNA的序列为与所述SEQ ID NO：1具有90％同源性且功能相同的序列。
  3. 根据权利要求1所述的miRNA，其特征在于，所述乳腺癌相关基因为GPER1基因。
  4. 一种核酸药物，其特征在于，所述核酸药物包括权利要求1-3任一项所述的miRNA或其人工模拟物；优选的，所述核酸药物还包括药学上可接受的载体或赋形剂。
  5. 一种与权利要求4所述的核酸药物相关的生物材料，其特征在于，所述生物材料选自下述A)、B)或C)中的一种：
    A)与权利要求1-3任一项所述的miRNA的核苷酸序列互补配对的干扰RNA分子或者抑制剂；
    B)包含有权利要求1-3任一项所述miRNA的核苷酸序列的表达载体或重组载体；
    C)包含有B)中所述的表达载体或重组载体的细胞或微生物。
    优选的，所述表达载体包括pCDH载体、pCMVp-NEO-BAN载体、pEGFP载体、pEGFT-Actin、pSV2载体、pCDNA载体、pLVX载体、pAAV载体、pET载体和pDsRed载体中的至少一种。
    优选的，所述重组载体构建步骤包括：1)设计引物，PCR扩增所述miRNA序列；2)将扩增的序列片段和表达载体酶切，连接目的序列片段和表达载体；3)将连接产物转化大肠杆菌，培养；4)鉴定后提取重组质粒并进行包装即得到重组载体。
  6. 权利要求1-3任一项所述的miRNA或权利要求4所述的核酸药物在诊断、预防和/或治疗乳腺癌中的应用。
  7. 一种筛选权利要求1-3任一项所述的可激活疾病相关基因的miRNA的方法，其特征 在于，包括以下步骤：
    S1、通过生物信息学分析，筛选基因座位置与H3K4me1或H3K27ac重合的miRNA；
    S2、验证步骤S1筛选的miRNA与其周围200kb范围内的靶基因的关系；
    S3、获取S2中与所述靶基因具有相关性的miRNA，即为可激活疾病相关基因的miRNA。
  8. 根据权利要求7所述的方法，其特征在于，步骤S2中，验证miRNA与其周围200kb范围内的靶基因的关系的方法包括：实时荧光定量PCR检测过表达所述miRNA后所述靶基因的表达量。
  9. 一种用于研究乳腺癌机制的模型，其特征在于，所述模型为miRNA-增强子-靶基因网络激活模型。
  10. 根据权利要求9所述的模型，其特征在于，miRNA与增强子互相作用进而正向激活靶基因从而影响肿瘤的增殖生长，所述miRNA为miR-339；
    优选的，所述模型的构建方法包括：筛选位于增强子区域的多个miRNA，分别将所述miRNA在不同种类的细胞中过表达，发现在基因组上与所述miRNA邻近的多个基因被转录激活；得到miRNA-增强子-靶基因网络激活模型。
  11. 根据权利要求9-10所述的模型在乳腺癌领域中的应用；优选的，所述模型在制备治疗乳腺癌药物中的应用。
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WANG YAN: "The Screening of Invasion-related miRNAs in Breast Cancer Cell and the Preliminary Study on Biological Function of hsa-miR-339-5p", MEDICINE AND HEALTH SCIENCES, CHINA MASTER'S THESES/DOCTORAL DISSERTATIONS FULL-TEXT DATABASE (MASTER), no. 1, 15 January 2011 (2011-01-15), XP093021144 *
WU YAN, WENYU GAO, SUPING GAO, ZHIHONG LI: "Application value of serum miR-196a-5p and miR-339-5p expression levels in diagnosis of breast cancer", ZHONGGUO PUTONG WAIKE ZAZHI = CHINESE JOURNAL OF GENERAL SURGERY, ZHONGNAN DAXUE, CENTRAL SOUTH UNIVERSITY, CN, vol. 30, no. 5, 31 May 2021 (2021-05-31), CN , pages 551 - 557, XP093021146, ISSN: 1005-6947, DOI: 10.7659/j.issn.1005-6947.2021.05.007 *

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CN119799702A (zh) * 2025-01-06 2025-04-11 河南农业大学 miR-31-5p模拟物在增强金龟子绿僵菌对烟粉虱杀伤效果中的应用

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