WO2023155857A1 - 一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用 - Google Patents

一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用 Download PDF

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WO2023155857A1
WO2023155857A1 PCT/CN2023/076595 CN2023076595W WO2023155857A1 WO 2023155857 A1 WO2023155857 A1 WO 2023155857A1 CN 2023076595 W CN2023076595 W CN 2023076595W WO 2023155857 A1 WO2023155857 A1 WO 2023155857A1
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circpolk
lung cancer
tumor
cells
polk
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French (fr)
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张翀
李杨玲
曾玲晖
刘也涵
凌世生
董文坤
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浙大城市学院
杭州安旭生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
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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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    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present application relates to the technical field of tumor diagnosis, in particular to a circPOLK for tumor treatment targets and diagnostic biomarkers and its application.
  • DNA polymerase kappa is a special DNA polymerase involved in translesion DNA synthesis (TLS), which belongs to the Y family DNA polymerase.
  • the POLK gene is located in subband 3, zone 1, long arm of chromosome 5 (5q13.3), with a total length of 87.5 kb, encoding a protein Polk with 870 amino acids.
  • POLK enables DNA to replicate in the event of damage through a series of regulatory mechanisms to maintain the integrity of the genome, and at the same time affects oxidative stress in vivo through the repair of single and double bond breaks. Human cell lines lacking a functional copy of the POLK gene have compromised genome integrity and affected the oxidative damage process, so it plays an important role in the DNA repair process and oxidative damage process.
  • the POLK gene is a key gene in the trans-damage DNA synthesis-related pathway. When this gene is dysregulated, a series of events such as DNA breakage and chromosomal abnormality will occur to destabilize the genome, and it is related to tumor progression. Studies have found that genetic variation of the POLK gene is associated with the efficacy, adverse reactions and prognosis of platinum-based chemotherapy in patients with non-small cell lung cancer. However, the current exploration of POLK cannot fully reveal the diversity of the gene involved in tumorigenesis and development. There are few reports on non-coding RNA produced by POLK gene.
  • CircRNA is a new type of single-stranded closed non-coding RNA molecule formed by back-splicing of pre-mRNA without 5'cap and 3'poly(A) tail.
  • circRNA plays a role in eukaryotic cells
  • Lung cancer and liver cancer are currently the two malignant tumors with the highest mortality rates in my country, and they are also the two most common metastatic cancers with tumor metastasis. Smoking is still the main cause of lung cancer. In recent years, second-hand and third-hand smoke have gradually become the inducing factors of lung cancer. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the main risk factors for liver cancer, while excessive drinking and aflatoxin are also important reasons for the increased risk of liver cancer. However, the specific pathological mechanism of lung cancer and liver cancer is still unclear. The diagnosis of lung cancer and liver cancer mainly relies on imaging diagnosis, serum tumor marker examination and tissue biopsy.
  • circRNAs have been widely reported to be involved in the occurrence and development of non-small cell lung cancer.
  • circRNA mainly promotes the process of non-small cell lung cancer by adsorbing miRNA or encoding protein.
  • Botai Li et al. found that circNDUFB2 suppresses the progression of non-small cell lung cancer by destabilizing IGF2BPs and activating antitumor immunity.
  • Daishi Chen et al found that hsa_circ_100395 regulates the miR-1228/TCF21 pathway to inhibit lung cancer progression.
  • the purpose of the present invention is to provide a circPOLK for tumor treatment target and diagnostic biomarker and its application.
  • a circPOLK used as a tumor treatment target and a diagnostic biomarker the nucleotide sequence of the circPOLK is shown in SEQ ID NO:1.
  • kits for diagnosing tumorigenesis including a molecular probe that is specifically complementary to circPOLK described in the first aspect or used to amplify the circPOLK described in the first aspect. Primer pairs for circPOLK.
  • the application of the circPOLK described in the first aspect in the preparation of a drug for preventing and/or treating tumors is provided.
  • the tumor includes liver cancer and lung cancer.
  • the tumor comprises non-small cell lung cancer.
  • the tumor includes primary tumor and metastatic tumor.
  • a drug for preventing and/or treating tumors the drug using the circPOLK described in the first aspect as an active ingredient.
  • the tumor includes liver cancer and lung cancer.
  • the tumor includes primary tumor and metastatic tumor.
  • this application discloses for the first time the circular RNA (circPOLK) formed by back splicing of exons 1-3 of POLK mRNA. Characterization of R digestion and actinomycin D treatment.
  • the expression of circPOLK is up-regulated in tumor tissues and cells, and the prognosis of tumor patients with high expression of circPOLK is poor, and the expression level of circPOLK is positively correlated with lymph node metastasis and distant metastasis in patients with non-small cell lung cancer;
  • Both tumor cell lines expressing circPOLK and endogenous circPOLK knockout cell lines can prove that high expression of circPOLK significantly promotes the migration and invasion of non-small cell lung cancer cells, but has no significant effect on cell apoptosis, proliferation and clone formation ability.
  • circPOLK circular RNA
  • Figure 1 is a Sanger sequencing map of the circPOLK reverse splicing site sequence provided by the embodiment of the present invention
  • Figure 2 is a polyacrylamide gel image of the amplification product of circPOLK reverse splicing provided by the embodiment of the present invention
  • Figure 3 is a diagram of the results of the RNase R digestion experiment to verify the stability of circPOLK provided by the embodiments of the present invention
  • Figure 4 is a diagram of the results of actinomycin D treatment for detecting the half-life of circPOLK provided by the embodiment of the present invention
  • Figure 5 is a FISH result diagram for detecting the distribution of circPOLK cytoplasm and nucleus provided by the embodiment of the present invention
  • Fig. 6 is a result diagram of analyzing the correlation between the expression level of circPOLK and cancer patients provided by the embodiment of the present invention.
  • Figure 7 is a graph of RT-PCR results for detecting the basal expression level of circPOLK in lung cancer cells provided by the embodiment of the present invention.
  • Figure 8 is a graph of RT-PCR results for detecting the knockout efficiency of circPOLK in lung cancer cells provided by the embodiment of the present invention.
  • Figure 9 is a Transwell result diagram for detecting the effect of knocking down circPOLK on the migration ability of lung cancer provided by the embodiment of the present invention.
  • Fig. 10 is a Transwell result diagram for detecting the effect of knocking down circPOLK on the invasion ability of lung cancer provided by the embodiment of the present invention.
  • Figure 11 is a graph of the wound healing results of detecting the effect of knocking down circPOLK on the metastatic ability of lung cancer provided by the embodiment of the present invention.
  • Fig. 12 is a western blot result graph analyzing the effect of knocking down circPOLK on the metastatic ability of lung cancer provided by the embodiment of the present invention.
  • Figure 13 is a RT-PCR result diagram for detecting the overexpression efficiency of circPOLK in lung cancer cells provided by the embodiment of the present invention.
  • Figure 14 is a Transwell result diagram of detecting the effect of overexpression of circPOLK on the migration and invasion ability of lung cancer provided by the embodiment of the present invention.
  • Figure 15 is a diagram of the wound healing results of detecting the effect of overexpression of circPOLK on the metastatic ability of lung cancer provided by the embodiment of the present invention.
  • Fig. 16 is a graph showing the results of in vivo experiments in mice to detect the effects of circPOLK on lung metastasis and liver metastasis of lung cancer provided by the embodiment of the present invention
  • Figure 17 is a schematic diagram of the effect of circPOLK secreted by exosomes of lung cancer cells on the angiogenesis ability provided by the embodiment of the present invention.
  • Figure 18 is a graph of the wound healing results of detecting the effect of circPOLK secreted by lung cancer cell exosomes on the angiogenesis ability after knocking down circPOLK provided by the embodiment of the present invention
  • Figure 19 is a Transwell result diagram for detecting the effect of circPOLK secreted by exosomes of lung cancer cells on the angiogenesis ability provided by the embodiment of the present invention.
  • Figure 20 is a graph of the wound healing results of detecting the effect of circPOLK secreted by exosomes of lung cancer cells on the angiogenesis ability provided by the embodiment of the present invention.
  • Fig. 21 is a Transwell result diagram for detecting the effect of circPOLK secreted by exosomes of lung cancer cells on the angiogenesis ability provided by the embodiment of the present invention.
  • the present invention provides a kind of circPOLK that is used for tumor treatment target and diagnostic biomarker, the nucleotide sequence of described circPOLK is as SEQ ID NO: 10 (ataagtttataccatggatagcacaaaggagaagtgtgacagttacaaagatgatcttctgcttaggatgggacttaatgataataaagcaggaatggaaggattagataaagagaaaattaacaaaattataatggaagcc acgaaggggtccagattttatggaaatgagctcaagaaagaaaagcaagtcaaccaacgaattgaaaatatgatgcaacaaaagctcaaatcaccagccagccaacagctaagaaaagcacaattacag).
  • the circPOLK POLK mRNA is a circular RNA formed by reverse splicing of the first to third exons.
  • the expression of circPOLK has obvious evolutionary conservation of human and mouse, it is resistant to RNase R digestion and actinomycin D treatment, and has high stability.
  • the experiment of the present invention proves that the expression level of circPOLK is significantly different between tumor tissue and paracancerous tissue, therefore, it can be used as a tumor diagnostic biomarker in tumor treatment. Moreover, the prognosis of lung cancer patients with high expression of circPOLK is poor, indicating that circPOLK itself can be used as a marker for evaluating tumor prognosis. Therefore, the present invention provides a kit for tumor treatment targets and diagnostic biomarkers, including molecular probes that are specifically complementary to circPOLK described in the first aspect or used to amplify the circPOLK described in the first aspect Primer pairs for circPOLK.
  • circPOLK is used as a tumor treatment target in anti-tumor.
  • a circPOLK used as a tumor treatment target and a diagnostic biomarker provided by the present invention and its application will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
  • CirPOLK circular RNA
  • circular RNA derived from the POLK gene was determined by using biological information means combined with PCR amplification technology and its biological characteristics were identified.
  • the specific content is as follows:
  • circBase http://www.circbase.org
  • the POLK gene can be spliced to form 8 kinds of circRNAs, namely hsa_circ_0073052, hsa_circ_0129635, hsa_circ_0129636, hsa_circ_0129637, hsa_circ_0129639, hsa_circ_0129640, hsa _circ_0129641 and hsa_circ_0129642.
  • hsa_circ_0073052 is formed by reverse splicing of exon 1 and exon 3 of mRNA encoded by POLK gene (chr5:74842834-74848416), and its mature sequence length is 268bp (See Figure 1).
  • circPOLK is a circular RNA formed at the level of post-transcriptional splicing, its splice site sequence does not exist on the genome, in order to rule out that the observed head-to-tail splicing of circPOLK is produced by trans-splicing, genomic rearrangement, or PCR products and many other possibilities, the designed outward expansion and inward primers were used to amplify circPOLK and POLK mRNA respectively.
  • A549 and NCI-H1299 cell cDNA and genomic DNA (gDNA) were amplified by PCR (amplification procedure as described above) and agarose gel electrophoresis.
  • 3.1 circRNA lacks free 5' and 3' ends, and has its unique closed circular structure, so it can resist the digestion of exonuclease RNase R (RNase R), because RNase R can digest almost all linear RNA molecules, but circRNAs are not easy to digest, so RNase R digestion experiments are widely used in the stability identification experiments of circRNAs to prove that they have a circular structure.
  • RNase R exonuclease RNase R
  • circRNAs are not easy to digest
  • RNase R digestion experiments are widely used in the stability identification experiments of circRNAs to prove that they have a circular structure.
  • total RNA was first extracted from lung cancer cells A549, NCI-H1299 and NCI-H460, digested with RNase R, and then RT-PCR was used to detect the levels of circPOLK and linear POLK mRNA, respectively. The results showed that circPOLK was indeed resistant to digestion by RNase R compared with POLK mRNA (cf. See Figure 3).
  • POLK F ccacgaaggggtccagattt (SEQ ID NO: 3);
  • POLK R tgttggctggtgatttgagc (SEQ ID NO: 4);
  • GAPDH F ggagcgagatccctccaaaat (SEQ ID NO:5);
  • GAPDH R ggctgttgtcatacttctcatgg (SEQ ID NO:6);
  • GAPDH F gaaggtgaaggtcgagtc (SEQ ID NO:7);
  • GAPDH R gaagatggtgatgggatttc (SEQ ID NO: 8).
  • Actinomycin D Actinomycin D was used to inhibit the synthesis of nascent RNA in lung cancer cells A549 and NCI-H1299 to verify the stability of circPOLK.
  • the degradation rate of circPOLK and POLK mRNA was detected by qRT-PCR within 24 h. The results showed that POLK mRNA was almost completely degraded, and the half-life of circPOLK was greater than 24 h, which indicated that circPOLK had high stability (see Figure 4).
  • circPOLK has the biological characteristics of cyclization and can withstand RNase R digestion and actinomycin D treatment, and has high stability.
  • RNA fluorescence in situ hybridization is an important non-radioactive in situ hybridization technique, which can perform relative qualitative, quantitative and localization analysis of circPOLK.
  • a probe (aaacttatctgtaattgtgcttttc, SEQ ID NO: 9) specifically targeting the splicing site of circPOLK was designed and synthesized by Gemma Gene Company, and its 5' and 3' ends were labeled with Cy3 red.
  • the cytoplasm of NCI-H1299 lung cancer cells was detected by cell RNA FISH experiment Expression of circPOLK (see Figure 5). Therefore, cellular RNA FISH experiments showed that circPOLK was mainly distributed in the cytoplasm.
  • the basal expression of circPOLK was detected in the three cell lines A549, NCI-H1299, and NCI-H460 by qRT-PCR, showing that the expression of circPOLK was the highest in A549 cells, and the expression of circPOLK in NCI-H1299 cells was the lowest (see Fig. 7).
  • the circPOLK overexpression plasmid was constructed by introducing the pLC5-ciR vector, and the overexpression efficiency of circPOLK was verified in the lung cancer cell line NCI-H1299, and the results proved that the circPOLK overexpression plasmid was successfully constructed (see Figure 13).
  • Figures 14 and 15 demonstrate that the overexpression of circPOLK significantly promotes the migration, invasion and wound healing abilities of lung cancer cells. It was found by western blot that knocking down circPOLK can cause changes in EMT-related proteins, increase the level of E-cadherin, and decrease the level of N-cadherin (see Figure 12). The above results confirmed that circPOLK promoted the migration and invasion of non-small cell lung cancer cells in vitro.
  • si-NC sense uucuccgaacgugucacgutt
  • si-NC antisense acgugacacguucggagaatt
  • si-circPOLK#1 sense: aagcacaauuacagauaagtt;
  • si-circPOLK#1 antisense cuuaucuguaaauugugcuutt
  • si-circPOLK#2 sense:agcacaauuacagauaagutt
  • si-circPOLK#2 antisense acuuaucuguaaauugugcutt.
  • CircPOLK knockdown cells were injected into the tail vein of mice to detect its effect on lung metastasis and liver metastasis (for specific methods, please refer to the literature: doi:10.1038/s41467-019-12651-2.PMID:31619685). The results showed that knocking down circPOLK could promote the ability of lung cancer cells to metastasize to the lung and liver (see Figure 16).
  • circPOLK can promote neovascularization through exosomes:
  • HUVEC cells were co-cultured with lung cancer cells without serum. Lung cancer cells were inoculated in transwell chambers (0.8 ⁇ m), and HUVEC cells were inoculated in 24-well plates. After 24 hours, the wound healing ability of HUVEC cells was observed. It was found that knocking down circPOLK inhibited HUVEC The wound healing of the cells (see Figure 18), this phenomenon was reversed after overexpression of circPOLK (see Figure 20).
  • HUVEC cells were co-cultured with lung cancer cells without serum, HUVEC cells were seeded in transwell chambers, lung cancer cells were seeded in 24-well plates, and the migration ability of HUVEC cells was observed after 24 hours. It was found that knocking down circPOLK inhibited the migration of HUVEC (see Figure 19), and this phenomenon was reversed after overexpressing circPOLK (see Figure 21). The above results indicate that non-small cell lung cancer cells can secrete circPOLK through exosomes to promote the migration and motility of vascular endothelial cells.
  • Human non-small cell lung cancer cells A549, NCI-H1299, NCI-H460 and human umbilical vein endothelial cells HUVEC were cultured in medium with 10% FBS and 90% RPMI 1640. All cells in 5% CO 2 , Cultured in a cell culture incubator at 37°C.
  • Lung cancer cells with high or low expression of circPOLK were serum-starved overnight, digested and counted, and inoculated into transwell chambers at a density of 2 ⁇ 10 4 /well, with 200 ⁇ l of serum-free culture medium per well. Add 600 ⁇ l of culture solution containing 20% serum to the corresponding 24-well plate. After the cells were incubated for 24 hours, the chamber was stained with 1% crystal violet for 30 minutes in the dark, and then the cells inside the chamber were washed with 1 ⁇ PBS, and then the cells migrating to the lower surface of the chamber were photographed and counted with a microscope.
  • Matrigel was thawed at 4°C in advance, and the chamber, 24-well plate and pipette tips were pre-cooled at -20°C. Dilute Matrigel with cell culture medium at 1:20, add 100 ⁇ l of Matrigel to each chamber, place the chamber in a 24-well plate and place it in a 37°C incubator for 30 minutes, take out the 24-well plate, and discard the unsolidified Matrigel in the chamber , and rinse with culture medium.
  • non-small cell lung cancer cells with high or low expression of circPOLK were serum-starved overnight, digested and counted, and inoculated into transwell chambers with Matrigel gel at a density of 3 ⁇ 10 4 /well, with 200 ⁇ l of serum-free culture medium per well. Add 600 ⁇ l of culture solution containing 20% serum to the corresponding 24-well plate. After the cells were incubated for 24 hours, the chamber was stained with 1% crystal violet for 30 minutes in the dark, and then the cells inside the chamber were washed with 1 ⁇ PBS, and the cells that invaded the lower surface of the chamber were photographed and counted with a microscope.
  • the cells were seeded in a six-well plate at a density of 2 ⁇ 10 5 /well, and after the cells adhered to the wall, they were transfected with the required plasmid or siRNA.
  • the confluence of the cells reaches 80-100%, use a pipette to scratch the cell surface, wash off the cells falling on the surface with PBS, replace with a new culture medium, and take a picture of the wound distance at 0h with a microscope. After 24h, wash with PBS
  • the six-well plate was washed to remove non-adherent cells, the wound distance was photographed under a microscope for 24 hours, and the wound healing was calculated.
  • Cells were seeded in 6-well plates (5 ⁇ 10 5 /well). Transfect when the cell density reaches 30-40%, Take 200 ⁇ l of transfection buffer from each well into an RNase-free EP tube, add 2 ⁇ g of target siRNA, mix well, and let stand for 5 minutes. Add 4 ⁇ l For the transfection reagent, mix well and centrifuge, let it stand for 20min, then add the transfection reagent, shake slightly to mix evenly.
  • BSA (2mg/ml) was diluted with deionized water to make a standard curve, and the sample protein was diluted 10 times with deionized water.
  • 1Gel electrophoresis Prepare 500ml of 1 ⁇ Running Buffer, and load protein samples in a certain order and in a certain amount. First adjust the voltage to 70V. When the sample reaches the separation gel, increase the voltage to 110V. When the sample is 1cm away from the bottom of the gel, stop the electrophoresis.
  • 2Membrane transfer Prepare 900ml of 1 ⁇ Transfer Buffer, activate a PVDF membrane of appropriate size with 100ml of absolute ethanol for 1min, and add the prepared Transfer Buffer. According to the protein size required for the experiment, cut the glue into the corresponding size, transfer the membrane according to the method of black glue and white film, set the current at 330mA, and transfer in ice water bath Film 90min.
  • 3Milk blocking and incubation with primary antibody After the transfer time is up, put the PVDF membrane in 5% skimmed milk (prepared in T-PBS) to block at room temperature for 1 hour. Discard the milk, wash the PVDF membrane three times with T-PBS, add the primary antibody required for the experiment, and incubate overnight at 4°C on a shaker.
  • 5Exposure Prepare the exposed things in advance, incubate the strips in the dark with ECL chromogenic solution (A liquid and B liquid 1:1 in the ECL kit), transfer to the exposure box, and press the X-ray film in the dark room , 5-20s for a short time (depending on the fluorescence intensity of the band), and 30min for a long time, take out the film, place it in the developer solution for 1.5min, rinse it with tap water, place it in the fixer solution for 1.5min, and image.
  • ECL chromogenic solution A liquid and B liquid 1:1 in the ECL kit
  • the supernatant was discarded, and the pellet was gently washed with 500 ⁇ l of pre-cooled 75% ethanol, and centrifuged at 8000 rpm at 4°C for 5 min. Wash again, place in a ventilated place, wait for ethanol to evaporate, add 20 ⁇ l of DEPC water to dissolve mRNA, and measure mRNA concentration.
  • the mRNA was reverse transcribed into complementary DNA (cDNA) using the TransStart Top Green qPCR SuperMix (Lot #40426) kit from Quangold.
  • HUVEC cells (serum starved overnight) were seeded into each transwell chamber (0.8 ⁇ m) at a density of 2 ⁇ 10 4
  • pretreated non-small cell lung cancer cells (serum starved overnight) were seeded at a density of 5 ⁇ 10 4 Inoculate in a 24-well plate below the chamber.
  • the chamber was stained with 1% crystal violet for 30 minutes in a dark chamber, the cells in the chamber were washed away with PBS, and the cells migrating to the bottom of the chamber were photographed and counted with a microscope.
  • HUVEC cells (serum starved overnight) were seeded into 24-well plates at a density of 8 ⁇ 10 4 . After the cells adhered to the wall, a 20 ⁇ l pipette tip was used to scratch the cell surface, and the wound distance of the cell 0h was photographed. Then pretreated non-small cell lung cancer cells (serum starved overnight) were seeded into each transwell chamber (0.4 ⁇ m) at a density of 2 ⁇ 10 4 , and the chamber was placed in a 24-well plate containing HUVEC cells. After 24 hours of cell incubation, the wound healing of HUVEC cells was observed and photographed.

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Abstract

本发明公开了用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用，属于肿瘤诊断技术领域，其核苷酸序列如SEQ ID NO: 10所示，为POLK mRNA第1-3外显子反向剪接形成的环状RNA。相比于正常患者，肿瘤患者血液外泌体中circPOLK呈现高表达，circPOLK的表达与肺癌细胞的迁移、侵袭和转移的能力呈现正相关，肺癌细胞能经外泌体分泌circPOLK促进血管内皮细胞的迁移和侵袭能力。因此，circPOLK可以作为肿瘤的治疗靶点和诊断生物标志物，为治疗药物和诊断产品的开发提供了新思路和新方向。

Description

一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用 技术领域
本申请涉及肿瘤诊断技术领域，尤其涉及一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用。
背景技术
DNA聚合酶kappa(POLK)是参与跨损伤DNA合成(TLS)的特殊的DNA聚合酶，属于Y家族DNA聚合酶的一种。POLK基因位于5号染色体长臂l区3带3亚带(5q13.3)，全长87.5kb，编码具有870个氨基酸的蛋白Polk。POLK通过系列的调控机制使DNA在损伤的情况下复制，以维持基因组的完整性，同时通过单键及双键断裂修复影响体内氧化应激作用。缺乏POLK基因功能拷贝的人细胞系基因组完整性受损，氧化损伤过程受到影响，故其在DNA的修复过程和氧化损伤过程中发挥重要作用。POLK基因是跨损伤DNA合成相关通路的一个关键基因，当该基因失调后，会发生DNA断裂、染色体异常等一系列使基因组不稳定的事件，并且与肿瘤进展有关。有研究发现POLK基因的遗传变异与非小细胞肺癌患者铂类药物的化疗疗效、不良反应以及预后相关。尽管如此，目前对POLK的探索并不能完全揭示该基因参与肿瘤发生发展过程的多样性，总的来说，以往对POLK基因的研究主要集中在探讨DNA损伤修复和氧化应激功能上，而对POLK基因产生的非编码RNA的研究鲜有报道。
只有不到2％的人类基因组序列是蛋白质编码基因，而人类转录组大多数序列是非编码RNA，即没有编码蛋白质的能力。circRNA是一类由mRNA前体反向剪接形成的不具有5’帽子和3’poly(A)尾的单链闭合的新型非编码RNA分子。随着RNAseq技术和生物信息学分析技术的不断更新和完善，大量的circRNA被发现广泛表达于真核生物中，例如在真核细胞中就有近10％的基因转录可以剪接产生circRNA并发挥生物学功能。circRNA在真核细胞中发挥着 广泛的生物学功能，例如作用于miRNA“海绵”、蛋白质“海绵”、调节mRNA的稳定性、调节亲本基因转录，此外，有研究表明部分circRNA具有翻译功能蛋白的能力。多项研究表明，circRNA在肿瘤的发生发展和侵袭或转移中起着至关重要的作用。
肺癌(lung cancer)和肝癌(liver cancer)是目前我国死亡率最高的两种恶性肿瘤，同时也是肿瘤转移最常见的两大转移性癌种。吸烟仍然是诱发肺癌的主要原因，近年来二手烟和三手烟也逐渐成为肺癌的诱发因素。乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)是诱发肝癌的主要危险因素，同时过量饮酒和黄曲霉毒素也是导致肝癌风险增加的重要原因。但肺癌和肝癌的具体病理机制尚不清楚。肺癌和肝癌的诊断主要依靠影像学诊断、血清肿瘤标志物检查和组织活检。由于早期肺癌和肝癌缺乏特异性症状，大多数患者在没有定期筛查的情况下直到晚期才确诊，预后一般较差且不可逆。手术是早期肺癌和肝癌患者的推荐治疗方法。对于晚期患者，放疗和化疗是主要的治疗手段然而，放疗和化疗往往会产生严重的副作用，使患者无法耐受。近年来，分子靶向治疗和免疫治疗日益流行，但只适用于某些特定类型的癌症患者，迫切需要探索更具体、更有效的癌症诊断和治疗方法。由于靶向治疗在肺癌中的应用更为突出和成熟，因此我们最终选择肺癌进行深入探究。
近年来，circRNA被广泛报道参与非小细胞肺癌的发生发展。circRNA主要以吸附miRNA或编码蛋白的方式促进非小细胞肺癌进程。Botai Li等人发现circNDUFB2通过去稳定IGF2BPs和激活抗肿瘤免疫从而抑制非小细胞肺癌的进展。Daishi Chen等人发现hsa_circ_100395调控miR-1228/TCF21通路来抑制肺癌进展。另外，Yongsheng Zhao等人也认为circCDR1as通过靶向miR-641/HOXA9轴调控非小细胞肺癌细胞的干性和顺铂耐药。但总的来说，大多数circRNA在非小细胞肺癌的过程和发病机制中的具体作用仍不清楚。
发明内容
有鉴于此，本发明的目的在于提供一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用。
根据本发明实施例的第一方面，提供了一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK，所述circPOLK的核苷酸序列如SEQ ID NO:1所示。
根据本发明实施例的第二方面，提供了一种用于诊断肿瘤发生的试剂盒，包括与第一方面所述circPOLK特异性互补配对的分子探针或用于扩增权第一方面所述的circPOLK的引物对。
根据本发明实施例的第三方面，提供第一方面所述circPOLK在制备预防和/或治疗肿瘤的药物中的应用。
进一步地，所述肿瘤包括肝癌和肺癌。优选的，所述肿瘤包括非小细胞肺癌。
进一步地，所述肿瘤包括原发瘤和转移瘤。
根据本发明实施例的第三方面，提供一种用于预防和/或治疗肿瘤的药物，所述药物以第一方面所述circPOLK为活性成分。
进一步地，所述肿瘤包括结肝癌和肺癌。
进一步地，所述肿瘤包括原发瘤和转移瘤。
本申请的实施例提供的技术方案可以包括以下有益效果：
由上述实施例可知，本申请首次公开POLK mRNA第1-3外显子反向剪接形成的环状RNA(circPOLK)，circPOLK的表达具有明显的人鼠进化保守性，同时还具有耐受RNA酶R消化及放线菌素D处理的特点。circPOLK在肿瘤组织和细胞中表达上调，且circPOLK高表达的肿瘤患者预后较差，circPOLK的表达水平与非小细胞肺癌患者淋巴结转移和远处转移呈正相关；本发明相关实验均证明：无论是稳定表达circPOLK的肿瘤细胞系，还是内源circPOLK敲除细胞系，均可以证明circPOLK高表达显著促进非小细胞肺癌细胞的迁移和侵袭，而对细胞凋亡、增殖和克隆形成能力无显著影响。相比于正常患者，肺癌患者血液外泌体中circPOLK呈现高表达，肺癌细胞能经外泌体分泌circPOLK促进血管细胞的迁移和侵袭能力。因此，本发明基于这一新型环状RNA(circPOLK)核酸序列，可以为肿瘤治疗，尤其是转移瘤的临床治疗或预防方面 提供一种新的药物靶点，为提前诊断肿瘤的发生提供了新的思路和策略。
应当理解的是，以上的一般描述和后文的细节描述仅是示例性和解释性的，并不能限制本申请。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分，示出了符合本申请的实施例，并与说明书一起用于解释本申请的原理。
图1为本发明实施例提供的circPOLK反向剪接位点序列的Sanger测序图；
图2为本发明实施例提供的circPOLK反向剪接的扩增产物聚丙烯酰胺凝胶图；
图3为本发明实施例提供的验证circPOLK的稳定性的RNA酶R消化实验结果图；
图4为本发明实施例提供的检测circPOLK的半衰期的放线菌素D处理结果图；
图5为本发明实施例提供的检测circPOLK细胞浆和细胞核分布情况的FISH结果图；
图6为本发明实施例提供的分析circPOLK表达水平与癌症患者相关性的结果图；
图7为本发明实施例提供的检测circPOLK在肺癌细胞中基础表达水平的RT-PCR结果图；
图8为本发明实施例提供的检测circPOLK在肺癌细胞中敲除效率的RT-PCR结果图；
图9为本发明实施例提供的检测敲低circPOLK后对肺癌迁移能力影响的Transwell结果图；
图10为本发明实施例提供的检测敲低circPOLK后对肺癌侵袭能力影响的Transwell结果图；
图11为本发明实施例提供的检测敲低circPOLK后对肺癌转移能力影响的伤口愈合结果图；
图12为本发明实施例提供的分析敲低circPOLK后对肺癌转移能力影响的westernblot结果图；
图13为本发明实施例提供的检测circPOLK在肺癌细胞中过表达效率的RT-PCR结果图；
图14为本发明实施例提供的检测过表达circPOLK后对肺癌迁移和侵袭能力影响的Transwell结果图；
图15为本发明实施例提供的检测过表达circPOLK后对肺癌转移能力影响的伤口愈合结果图；
图16为本发明实施例提供的检测circPOLK对肺癌肺转移及肝转移能力影响的小鼠体内实验结果图；
图17为本发明实施例提供的经肺癌细胞外泌体分泌的circPOLK对血管形成能力影响的示意图；
图18为本发明实施例提供的检测敲低circPOLK后经肺癌细胞外泌体分泌的circPOLK对血管形成能力影响的伤口愈合结果图；
图19为本发明实施例提供的检测敲低circPOLK后经肺癌细胞外泌体分泌的circPOLK对血管形成能力影响的Transwell结果图；
图20为本发明实施例提供的检测过表达circPOLK后经肺癌细胞外泌体分泌的circPOLK对血管形成能力影响的伤口愈合结果图；
图21为本发明实施例提供的检测过表达circPOLK后经肺癌细胞外泌体分泌的circPOLK对血管形成能力影响的Transwell结果图。
具体实施方式
这里将详细地对示例性实施例进行说明，其示例表示在附图中。下面的描述涉及附图时，除非另有表示，不同附图中的相同数字表示相同或相似的要素。 以下示例性实施例中所描述的实施方式并不代表与本申请相一致的所有实施方式。相反，它们仅是与如所附权利要求书中所详述的、本申请的一些方面相一致的装置和方法的例子。
在本申请使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。在本申请和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。还应当理解，本文中使用的术语“和/或”是指并包含一个或多个相关联的列出项目的任何或所有可能组合。
本发明提供了一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK，所述circPOLK的核苷酸序列如SEQ ID NO:10(ataagtttataccatggatagcacaaaggagaagtgtgacagttacaaagatgatcttctgcttaggatgggacttaatgataataaagcaggaatggaaggattagataaagagaaaattaacaaaattataatggaagccacgaaggggtccagattttatggaaatgagctcaagaaagaaaagcaagtcaaccaacgaattgaaaatatgatgcaacaaaaagctcaaatcaccagccaacagctaagaaaagcacaattacag)所示。
所述circPOLK POLK mRNA第1～3外显子反向剪接形成的环状RNA。circPOLK的表达具有明显的人鼠进化保守性，耐受RNA酶R消化及放线菌素D处理，具有较高的稳定性。
本发明实验证明，circPOLK的表达水平在肿瘤组织与癌旁组织中呈显著性差异，因此，可以作为肿瘤诊断生物标志物应用于肿瘤治疗中。且circPOLK高表达的肺癌患者预后较差，说明circPOLK本身可以作为评估肿瘤预后作用的标志物。因此，本发明提供了一种用于肿瘤治疗靶点和诊断生物标志物的试剂盒，包括与第一方面所述circPOLK特异性互补配对的分子探针或用于扩增权第一方面所述的circPOLK的引物对。
本发明实验证明，circPOLK高表达显著促进肺癌细胞的迁移和侵袭，而对细胞凋亡、增殖和克隆形成能力无显著影响，因此，circPOLK作为肿瘤治疗靶点应用于抗肿瘤中。
本发明实验证明，circPOLK的表达水平与肺癌患者淋巴结转移和远处转移呈正相关。
下面结合实施例对本发明提供的一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
POLK基因来源的环状RNA(circPOLK)的发现与鉴定：
在本实施案例中，利用生物信息手段结合PCR扩增技术确定POLK基因来源的环状RNA(circPOLK)的存在并对其进行生物学特征的鉴定，具体内容如下：
1.POLK基因来源的环状RNA(circPOLK)的发现
利用circBase(http://www.circbase.org)数据库分析发现，POLK基因可剪接形成8种的circRNA，分别是hsa_circ_0073052、hsa_circ_0129635、hsa_circ_0129636、hsa_circ_0129637、hsa_circ_0129639、hsa_circ_0129640、hsa_circ_0129641和hsa_circ_0129642。
与POLK mRNA的传统线性剪接不同的是，hsa_circ_0073052是由POLK基因(chr5:74842834-74848416)编码mRNA的第1外显子和第3外显子通过反向剪接而成，其成熟序列长度为268bp(参见图1)。
进一步通过设计跨环化接头位点的外扩型引物(Divergent primer F:cagctaagaaaagcacaattacaga，SEQ ID NO:1，R:agtcccatcctaagcagaaga，SEQ ID NO:2)，在NCI-H1299细胞中通过PCR成功扩增出接头附近长约200bp的序列并进行Sanger测序，检测到POLK第1外显子和第3外显子的3’端和5’端反向剪接序列(参见图1)。可见，本实施例成功鉴定了hsa_circ_0073052的存在，由于该circRNA来源于POLK基因，将其命名为circPOLK(SEQ ID NO:10)。
扩增反应条件：
(1)95℃15min；
(2)94℃30s；
(3)63℃30s；
(4)72℃30s；
(2)-(4)39个循环。
2.POLK基因来源的环状RNA(circPOLK)环化鉴定和保守性分析
首先，设计跨剪接位点的外扩型引物(Divergent primer，SEQ ID NO:1和SEQ ID NO:2)用于鉴定及特异性检测circPOLK，同时在第3外显子上设计内收型引物(Convergent primer，POLK F:ccacgaaggggtccagattt，SEQ ID NO:3；POLK R:tgttggctggtgatttgagc，SEQ ID NO:4)用于检测POLK mRNA。因为circPOLK是在转录后剪接水平形成的环状RNA，所以其剪接位点序列不存在于基因组上，为了排除观察到的circPOLK的头尾剪接是由反式剪接、基因组重排或PCR产物产生的等诸多可能性，用设计的外扩型及内收型引物分别扩增circPOLK和POLKmRNA。在A549和NCI-H1299细胞cDNA及基因组DNA(gDNA)中通过PCR扩增(扩增程序如上所述)及琼脂糖凝胶电泳。
结果显示，外扩型引物仅能在cDNA上扩增出条带，在gDNA上不能扩增出条带，而内收型引物在cDNA和gDNA上均能扩增出POLK基因(参见图2)。这些结果说明circPOLK不是通过基因组重排形成的，而是在转录后水平通过反向剪切形成的环状RNA，而且应用外扩型引物可以特异性检测circPOLK。
3.circPOLK的稳定性验证
3.1 circRNA缺乏游离的5’末端和3’末端，有其独特的闭合环状结构，因而能够抵抗核酸外切酶RNA酶R(RNase R)的消化作用，因RNA酶R能够消化几乎所有的线性RNA分子，但不易消化circRNA，故RNA酶R消化实验广泛用于circRNA的稳定性鉴定实验，以证明其具有环形结构。本实验首先在肺癌细胞A549、NCI-H1299和NCI-H460中提取总RNA，应用RNA酶R消化处理后，再应用RT-PCR方法分别检测了circPOLK以及线性POLK mRNA的水平。结果表明，与POLK mRNA相比，circPOLK的确能够耐受RNA酶R的消化(参 见图3)。
circPOLK F:cagctaagaaaagcacaattacaga(SEQ ID NO:1)；
circPOLK R:agtcccatcctaagcagaaga(SEQ ID NO:2)；
POLK F:ccacgaaggggtccagattt(SEQ ID NO:3)；
POLK R:tgttggctggtgatttgagc(SEQ ID NO:4)；
GAPDH F:ggagcgagatccctccaaaat(SEQ ID NO:5)；
GAPDH R:ggctgttgtcatacttctcatgg(SEQ ID NO:6)；
divergent GAPDH F:gaaggtgaaggtcgagtc(SEQ ID NO:7)；
divergent GAPDH R:gaagatggtgatgggatttc(SEQ ID NO:8)。
3.2用放线菌素D(Actinomycin D)抑制肺癌细胞A549和NCI-H1299新生RNA的合成，以此来验证circPOLK的稳定性。通过qRT-PCR检测circPOLK和POLK mRNA的24h内降解速率，结果表明，POLK mRNA已几乎降解完全，而circPOLK的半衰期大于24h，这说明circPOLK具有很高的稳定性(参见图4)。
以上实验进一步证实，circPOLK具有环化生物学特征和能够耐受RNA酶R消化及放线菌素D处理，具有较高的稳定性。
4.circPOLK的亚细胞定位
由于circRNA功能的发挥与其定位密切相关，外显子来源的circRNA通常定位于细胞浆，而内含子来源的circRNA多数定位于细胞核。因为上述实验结果证明，circPOLK是外显子来源的circRNA，推测其可能定位于细胞浆，为了验证此推论并探究circPOLK的具体亚细胞定位情况，本实验进行了细胞RNA FISH实验，具体步骤如下。
RNA荧光原位杂交(fluorescence in situ hybridization，FISH)，是一种重要的非放射性原位杂交技术，可以对circPOLK进行相对定性、定量和定位分析。本实验通过吉玛基因公司设计合成了特异性针对circPOLK剪接位点的探针(aaacttatctgtaattgtgcttttc，SEQ ID NO:9)，其5’和3’端带有Cy3红色标记。通过细胞RNA FISH实验检测到NCI-H1299肺癌细胞的胞浆中有丰富的 circPOLK的表达(参见图5)。因此，细胞RNA FISH实验表明circPOLK主要分布在细胞浆。
实施例2
circPOLK在肝细胞癌患者血清外泌体中的表达：
本实验进一步分析了肝细胞癌患者与正常人血清外泌体中circPOLK表达水平的关系(参见图6)，结果提示circPOLK可能是一种具有促癌性质的circRNA并参与癌症的发生和发展，进而它可能作为判断肺癌患者预后情况的潜在指标之一。
实施例3
1.细胞水平检测circPOLK促进非小细胞肺癌的迁移和侵袭能力
通过qRT-PCR法在A549、NCI-H1299、NCI-H460三个细胞系中检测circPOLK的基础表达量，表明A549细胞中circPOLK的表达量最高，NCI-H1299细胞中circPOLK的表达量最低(参见图7)。通过设计circPOLK的siRNA序列(SEQ ID NO:10-15)，再通过qRT-PCR法在肺癌细胞系A549、NCI-H1299和NCI-H460中检测circPOLK的敲降效率，证明circPOLK的siRNA序列设计成功(参见图8)，通过Transwell检测了circPOLK对肺癌细胞迁移和侵袭能力的影响，结果证明了敲低circPOLK后显著抑制了肺癌细胞的迁移和侵袭能力(参见图9、10)，也证明了敲低circPOLK后显著抑制了肺癌细胞的伤口愈合能力(参见图11)。通过导入pLC5-ciR载体构建circPOLK过表达质粒，在肺癌细胞系NCI-H1299中验证circPOLK的过表达效率，结果证明circPOLK过表达质粒构建成功(参见图13)。图14、15证明了过表达circPOLK后显著促进了肺癌细胞的迁移侵袭能力和伤口愈合能力。通过western blot发现敲低circPOLK后，能够引起EMT相关蛋白的变化，可升高E-cadherin的水平，降低N-cadherin的水平(参见图12)。以上结果都证实circPOLK在体外促进非小细胞肺癌细胞的迁移和侵袭。
si-NC sense:uucuccgaacgugucacgutt；
si-NC antisense:acgugacacguucggagaatt；
si-circPOLK#1 sense:aagcacaauuacagauaagtt；
si-circPOLK#1 antisense:cuuaucuguaauugugcuutt；
si-circPOLK#2 sense:agcacaauuacagauaagutt；
si-circPOLK#2 antisense:acuuaucuguaauugugcutt。
2.小鼠细胞水平检测circPOLK促进非小细胞肺癌的迁移和侵袭能力
接下来检测了体内环境下，circPOLK对肺癌转移的调控。CircPOLK敲低细胞尾静脉注射小鼠检测其对肺转移及肝转移的影响(具体方法参见文献：doi:10.1038/s41467-019-12651-2.PMID:31619685)。结果显示，敲低circPOLK可促进肺癌细胞肺转移以及肝转移的能力(参见图16)。
实施例3
circPOLK可通过外泌体促进新生血管形成：
将HUVEC细胞与肺癌细胞无血清共培养，肺癌细胞接种于transwell小室(0.8μm)，HUVEC细胞接种于24孔板，24小时后观察HUVEC细胞的伤口愈合能力，结果发现敲低circPOLK后抑制了HUVEC细胞的伤口愈合(参见图18)，过表达circPOLK后这一现象被逆转(参见图20)。
将HUVEC细胞与肺癌细胞无血清共培养，HUVEC细胞接种于transwell小室，肺癌细胞接种于24孔板，24小时后观察HUVEC细胞的迁移能力。结果发现敲低circPOLK后抑制了HUVEC的迁移(参见图19)，过表达circPOLK后这一现象被逆转(参见图21)。以上结果表明非小细胞肺癌细胞能经外泌体分泌circPOLK从而促进血管内皮细胞的迁移运动能力。
实验方法：
1.细胞培养
人非小细胞肺癌细胞A549，NCI-H1299，NCI-H460和人脐静脉内皮细胞HUVEC培养于具有10％FBS和90％RPMI 1640培养基中。所有细胞在5％CO2， 37℃的细胞培养箱中培养。
2.Transwell实验
(1)细胞迁移实验
将高低表达circPOLK的肺癌细胞血清饥饿过夜，消化计数，接种到transwell小室中，密度为2×104/孔，每孔无血清培液200μl。相对应的24孔板中加入600μl含有20％血清的培养液。细胞孵育24小时后，用1％结晶紫对小室进行避光染色30min，接着用1×PBS清洗小室内部的细胞，然后用显微镜拍摄迁移到小室下表面的细胞并计数。
(2)细胞侵袭实验
Matrigel提前放置4℃解冻，小室，24孔板和所用枪头放置-20℃预冷。用细胞培养液将Matrigel按1：20稀释，每个小室中加入100μl的Matrigel，将小室放在24孔板中置于37℃孵箱30min，取出24孔板，弃去小室中未凝固的Matrigel，并用培养液润洗一遍。与此同时，高低表达circPOLK的非小细胞肺癌细胞血清饥饿过夜，消化计数，接种到有Matrigel胶的transwell小室中，密度为3×104/孔，每孔无血清培养液200μl。相对应的24孔板中加入600μl含有20％血清的培养液。细胞孵育24小时后，用1％结晶紫对小室进行避光染色30min，接着用1×PBS清洗小室内部的细胞，然后用显微镜拍摄侵袭到小室下表面的细胞并计数。
3.伤口愈合实验
将细胞以2×105/孔的密度接种在六孔板中，待细胞贴壁后，转染所需质粒或siRNA。待细胞汇合度达到80-100％，用移液器在细胞表面划痕，用PBS洗去表面掉落的细胞，换上新的培养液，显微镜拍下0h的伤口距离，24h后，用PBS清洗六孔板以除去非贴壁细胞，显微镜拍下24h的伤口距离，计算伤口愈合情况。
4.转染
将细胞接种在6孔板中(5×105/孔)。待细胞密度达到30-40％进行转染， 每孔取200μl转染buffer至RNA酶free的EP管中，加入2μg的目标siRNA，混匀，静置5min。加入4μl转染试剂，混匀离心，静置20min，然后加入转染试剂，轻微晃动使其混合均匀。
5.Western blot法
(1)蛋白样本提取和定量
①提取：收集提前处理过的细胞，2000rpm离心4min，弃上清，将沉淀转移至EP管中，2000rpm离心4min，弃上清。根据细胞数量加入全细胞裂解液，每隔10min涡旋一次，
进行3次，充分将细胞和裂解液混合均匀，12000rpm，4℃离心30min，收集上清，上清即蛋白样本。
②定量：BSA(2mg/ml)用去离子水倍半稀释作标准曲线，样本蛋白用去离子水稀释10倍。不同浓度的BSA溶液和样本蛋白分别取5μl，加入200μl现配的BCA试剂混合液(A液：B液＝50：1)，充分混匀后置于37℃，30min，用酶标仪读取波长为562nm处的吸光度。根据所得的标准曲线的公式，计算出样本蛋白的浓度，按一定量标定蛋白。
③直接loading法
将预处理的细胞上清弃去，用PBS清洗两遍，根据细胞的密度加入相应量的2.5×loading直接搅拌收细胞，离心，沸水煮10min使蛋白失活，冷却离心，-20℃保存。
(2)蛋白质免疫印迹
①凝胶电泳：配制500ml的1×Running Buffer，蛋白样本按一定顺序和一定量上样。先调节电压70V，待样本跑至分离胶时，可将电压增加至110V，样本离胶底端1cm时，停止电泳。
②转膜：配制900ml的1×Transfer Buffer，将大小合适的PVDF膜用100ml的无水乙醇激活1min，加入配制好的Transfer Buffer。根据实验所需的蛋白大小，将胶切成相应大小，按黑胶白膜方式转膜，330mA横定电流，冰水浴转 膜90min。
③牛奶封闭及孵育一抗：转膜时间到后，将PVDF膜放入5％脱脂牛奶(T-PBS配制)中室温封闭1h。弃去牛奶，用T-PBS清洗PVDF膜三遍，加入实验所需的一抗，摇床4℃孵育过夜。
④孵育二抗：回收一抗，用T-PBS洗条带3次，每次10min。加入一抗对应的辣根过氧化物酶标记的二抗，室温摇床孵育90min，弃去二抗，T-PBS洗3次。
⑤曝光：提前将曝光的东西准备好，条带用ECL显色液(ECL试剂盒中A液与B液1：1)暗处孵育1min，转移至曝光盒中，暗房中X光胶片压片，短时间5-20s(根据条带荧光强度而定)，长时间30min，将胶片取出，显影液中放置1.5min，自来水冲洗，置于定影液中1.5min，成像。
6.RNA提取和逆转录
将预先处理过的六孔板细胞弃去上清，用预冷的PBS漂洗两次，每孔加入1ml的Trizol，反复吹打至溶液透明。将细胞转移至1.5ml RNA酶free的EP管中，加入200μl的氯仿，剧烈震荡30s，4℃静置2min，12000rpm，4℃离心10min。吸取450μl的上清至新的EP管中，加入等量异丙醇，轻柔地上下颠倒5次，冰上静置10min，12000rpm，4℃离心15min。弃去上清，沉淀用500μl预冷的75％乙醇轻柔清洗，8000rpm，4℃离心5min。重复清洗一遍，放置通风处，等乙醇挥干后，加入20μl的DEPC水溶解mRNA，测定mRNA浓度。
使用全式金的TransStart Top Green qPCR SuperMix(Lot#40426)试剂盒将mRNA逆转录为互补DNA(cDNA)。
7.qRT-PCR
qRT-PCR体系为10μl，分别是5μl 2×QuantiTect SYBR Green PCR Master Mix，1μl模板cDNA，目标正反向引物各0.6μl，DEPC水2.8μl。其反应条件为95℃预变性15min，94℃变性30s，63℃退火30s，72℃延伸30s， 39个循环。
8.细胞共培养
将HUVEC细胞(血清饥饿过夜)以2×104的密度接种到每个transwell小室(0.8μm)中，同时将预处理过的非小细胞肺癌细胞(血清饥饿过夜)以5×104的密度接种在小室下面的24孔板中。细胞孵育24小时后，将小室用1％结晶紫暗室染色30min，PBS洗去小室内的细胞，用显微镜拍摄迁移至小室底部的细胞并计数。
将HUVEC细胞(血清饥饿过夜)以8×104的密度接种到24孔板中。待细胞贴壁后，使用20μl移液器吸头在细胞细胞表面划痕，拍摄细胞0h的伤口距离。然后将预处理过的非小细胞肺癌细胞(血清饥饿过夜)以2×104的密度接种到每个transwell小室(0.4μm)中，将小室放在含有HUVEC细胞的24孔板中。细胞孵育24小时后，观察HUVEC细胞的伤口愈合情况并拍照。
9.统计学分析
使用t检验来检验统计分析，p<0.05被认为实验结果具有显著性差异(*P<0.05，**P<0.01和***P<0.001)。每个实验至少重复三次。
本领域技术人员在考虑说明书及实践这里公开的内容后，将容易想到本申请的其它实施方案。本申请旨在涵盖本申请的任何变型、用途或者适应性变化，这些变型、用途或者适应性变化遵循本申请的一般性原理并包括本申请未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的，本申请的真正范围和精神由权利要求指出。
应当理解的是，本申请并不局限于上面已经描述并在附图中示出的精确结构，并且可以在不脱离其范围进行各种修改和改变。本申请的范围仅由所附的权利要求来限制。

Claims (8)

  1. 一种用于肿瘤治疗靶点和诊断生物标志物的circPOLK，其特征在于，所述circPOLK的核苷酸序列如图1所示。
  2. 一种用于诊断肿瘤发生的试剂盒，其特征在于，包括与权利要求1所述circPOLK特异性互补配对的分子探针或用于扩增权利要求1所述circPOLK的引物对。
  3. 权利要求1所述circPOLK在制备预防和/或治疗肿瘤的药物中的应用。
  4. 根据权利要求3所述应用，其特征在于，所述肿瘤包括肝癌和肺癌。
  5. 根据权利要求4所述应用，其特征在于，所述肿瘤包括原发瘤和转移瘤。
  6. 一种用于预防和/或治疗肿瘤的药物，其特征在于，所述药物以权利要求1所述circPOLK为活性成分。
  7. 根据权利要求6所述药物，其特征在于，所述肿瘤包括结肝癌和肺癌。
  8. 根据权利要求7所述药物，其特征在于，所述肿瘤包括原发瘤和转移瘤。
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