CN114517204A

CN114517204A - circPOLK for tumor treatment target and diagnosis biomarker and application thereof

Info

Publication number: CN114517204A
Application number: CN202210141590.4A
Authority: CN
Inventors: 张翀; 李杨玲; 曾玲晖; 刘也涵; 凌世生; 董文坤
Original assignee: Hangzhou Anxiu Biotechnology Co ltd; Hangzhou City University
Current assignee: Hangzhou Anxiu Biotechnology Co ltd; Hangzhou City University
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-05-20
Anticipated expiration: 2042-02-16
Also published as: WO2023155857A1; CN114517204B

Abstract

The invention discloses circPOLK for tumor treatment target and diagnosis biomarker and application thereof, belonging to the technical field of tumor diagnosis, wherein the nucleotide sequence of the circPOLK is shown in figure 1 and is a new circular RNA formed by reverse splicing of 1 st-3 th exons of POLK mRNA. Compared with normal patients, the circPOLK in the blood exosomes of the tumor patients is high in expression, the expression of the circPOLK is positively correlated with the migration, invasion and metastasis capacities of the lung cancer cells, and the lung cancer cells can secrete the circPOLK through the exosomes to promote the migration and invasion capacities of vascular endothelial cells. As the most common parts of tumor patients with distant metastasis are lung and liver, circPOLK can be applied to tumor treatment and tumor diagnosis as a treatment target and a diagnosis biomarker of lung cancer and liver cancer, and provides a new idea and a new direction for treatment-related medicines and diagnosis products.

Description

circPOLK for tumor treatment target and diagnosis biomarker and application thereof

Technical Field

The application relates to the technical field of tumor diagnosis, in particular to circPOLK for tumor treatment targets and diagnosis biomarkers and application thereof.

Background

The DNA polymerase kappa (POLK) is a specific DNA polymerase involved in the trans-lesion DNA synthesis (TLS), and belongs to one of the Y family DNA polymerases. The POLK gene is located in

zone

3, 3 sub-zone (5q13.3) of the long arm l region of chromosome 5, has a full length of 87.5kb and encodes a protein Polk with 870 amino acids. POLK replicates DNA under damage through a series of regulatory mechanisms to maintain genome integrity, while repairing through single and double bond breaks affecting oxidative stress effects in vivo. Human cell lines lacking functional copies of the POLK gene have impaired genomic integrity and affected oxidative damage processes, and thus play an important role in DNA repair and oxidative damage processes. The POLK gene is a key gene crossing the pathways involved in damaged DNA synthesis, and when the gene is dysregulated, a series of genomic instability events such as DNA fragmentation, chromosomal abnormality, etc. occur and are associated with tumor progression. Researches show that the genetic variation of the POLK gene is related to the chemotherapy curative effect, adverse reaction and prognosis of the platinum drugs of non-small cell lung cancer patients. However, the research on POLK does not completely reveal the diversity of the gene involved in the process of tumorigenesis, and in general, the research on POLK gene has been focused mainly on the study of DNA damage repair and oxidative stress function, while the research on non-coding RNA generated from POLK gene has been reported rarely.

Only less than 2% of the human genome sequences are protein-coding genes, whereas most of the human transcriptome sequences are non-coding RNAs, i.e., have no protein-coding capacity. circRNA is a novel class of single-stranded closed non-coding RNA molecules formed by reverse splicing of mRNA precursors, without a 5 'cap and a 3' poly (A) tail. With the continuous updating and improvement of RNAseq technology and bioinformatics analysis technology, a large amount of circRNA was found to be widely expressed in eukaryotes, for example, approximately 10% of gene transcripts in eukaryotic cells can be spliced to produce circRNA and exert biological functions. circRNA plays a wide range of biological functions in eukaryotic cells, such as acting on miRNA "sponges", protein "sponges", regulating mRNA stability, regulating parental gene transcription, and in addition, studies have shown that some of the circRNA has the ability to translate functional proteins. Several studies have shown that circRNA plays a crucial role in the development and invasion or metastasis of tumors.

Lung cancer (lung cancer) and liver cancer (liver cancer) are two kinds of malignant tumors with the highest mortality rate in China at present, and are two most common metastatic cancer species with tumor metastasis. Smoking is still a leading cause of lung cancer, and second-hand smoke and third-hand smoke have also become a leading cause of lung cancer in recent years. Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are major risk factors for inducing liver cancer, and excessive drinking of alcohol and aflatoxin are important causes of increased risk of liver cancer. However, the specific pathological mechanism of lung cancer and liver cancer is not clear. The diagnosis of lung and liver cancer relies mainly on imaging diagnosis, serum tumor marker examination and tissue biopsy. Due to the lack of specific symptoms of early lung and liver cancer, most patients do not diagnose until late without regular screening, and the prognosis is generally poor and irreversible. Surgery is a recommended treatment for patients with early stage lung and liver cancer. Radiotherapy and chemotherapy are the main treatment for patients with advanced stages however, radiotherapy and chemotherapy often produce serious side effects that patients cannot tolerate. In recent years, molecular targeted therapy and immunotherapy have become increasingly popular, but only for certain specific types of cancer patients, and there is an urgent need to explore more specific and effective methods for cancer diagnosis and treatment. Because the application of targeted therapy in lung cancer is more prominent and mature, the lung cancer is finally selected for deep research.

In recent years, circRNA has been widely reported to participate in the development of non-small cell lung cancer. The circRNA mainly promotes the non-small cell lung cancer process in a way of adsorbing miRNA or coding protein. Botai Li et al found that circNDUFB2 inhibited the progression of non-small cell lung cancer by destabilizing IGF2BPs and activating anti-tumor immunity. Daishi Chen et al found that hsa _ circ _100395 modulates the miR-1228/TCF21 pathway to inhibit lung cancer progression. In addition, Yongsheng Zhao et al also believe that circCDR1as regulates both sternness and cisplatin resistance in non-small cell lung cancer cells by targeting the miR-641/HOXA9 axis. However, in general, the specific role of most circrnas in the process and pathogenesis of non-small cell lung cancer remains unclear.

Disclosure of Invention

In view of the above, the present invention aims to provide circPOLK for tumor therapy target and diagnostic biomarker and its application.

According to a first aspect of the embodiments of the present invention, there is provided circPOLK for use in tumor therapy target and diagnostic biomarker, the nucleotide sequence of the circPOLK is shown as SEQ ID No. 1.

According to a second aspect of embodiments of the present invention there is provided a kit for diagnosing tumorigenesis comprising a molecular probe specifically complementary paired to the circPOLK of the first aspect or a primer pair for amplifying the circPOLK of the first aspect.

According to a third aspect of the embodiments of the present invention, there is provided a use of circPOLK according to the first aspect in the preparation of a medicament for preventing and/or treating tumors.

Further, the tumor includes liver cancer and lung cancer. Preferably, the tumor comprises non-small cell lung cancer.

Further, the tumor includes primary tumor and metastatic tumor.

According to a third aspect of embodiments of the present invention, there is provided a medicament for preventing and/or treating a tumor, which comprises the circPOLK of the first aspect as an active ingredient.

Further, the tumors include colon cancer and lung cancer.

Further, the tumor includes primary tumor and metastatic tumor.

The technical scheme provided by the embodiment of the application can have the following beneficial effects:

from the above examples, the present application discloses for the first time a circular RNA (circPOLK) formed by reverse splicing exons 1 to 3 of POLK mRNA, wherein the expression of circPOLK has obvious human and mouse evolution conservation, and also has the characteristics of resistance to rnase R digestion and actinomycin D treatment. The expression of the circPOLK is up-regulated in tumor tissues and cells, the prognosis of a circPOLK high-expression tumor patient is poor, and the expression level of the circPOLK is positively correlated with the lymph node metastasis and the distant metastasis of a non-small cell lung cancer patient; the related experiments of the invention all prove that: both a tumor cell line stably expressing circPOLK and an endogenous circPOLK knockout cell line can prove that the high expression of the circPOLK obviously promotes the migration and invasion of non-small cell lung cancer cells, and has no obvious influence on the apoptosis, proliferation and clonogenic capacity. Compared with normal patients, the circPOLK in the blood exosomes of the lung cancer patients is highly expressed, and the lung cancer cells can secrete the circPOLK through the exosomes to promote the migration and invasion capacity of vascular cells. Therefore, the invention provides a new drug target for tumor treatment, especially clinical treatment or prevention of metastatic tumor based on the novel circular RNA (circPOLK) nucleic acid sequence, and provides a new idea and strategy for early diagnosis of tumor.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.

FIG. 1 is a Sanger sequencing of the reverse splice site sequence of circPOLK provided by an embodiment of the present invention;

FIG. 2 is a polyacrylamide gel diagram of the reverse spliced amplification product of circPOLK provided by an embodiment of the present invention;

FIG. 3 is a graph showing the results of digestion experiments with RNase R to verify the stability of circPOLK according to the present invention;

FIG. 4 is a graph showing the results of actinomycin D treatment to determine the half-life of circPOLK according to the embodiment of the present invention;

FIG. 5 is a FISH result chart for detecting the distribution of circPOLK cytoplasm and nucleus provided by the embodiment of the invention;

FIG. 6 is a graph showing the results of analyzing the correlation between circPOLK expression levels and cancer patients according to the present invention;

FIG. 7 is a diagram showing the results of RT-PCR for detecting the basal expression level of circPOLK in lung cancer cells according to the embodiment of the present invention;

FIG. 8 is a diagram showing the results of RT-PCR for detecting the knockout efficiency of circPOLK in lung cancer cells according to the embodiment of the present invention;

FIG. 9 is a diagram showing the results of Transwell testing the effect of knocking down circPOLK on lung cancer migration ability, according to the present invention;

FIG. 10 is a diagram showing the results of Transwell method for detecting the effect of circPOLK knock-down on lung cancer invasion capacity;

FIG. 11 is a graph showing the results of wound healing in testing the effect on lung cancer metastasis after knocking down circPOLK according to the present invention;

FIG. 12 is a western blot result chart for analyzing the effect of knocking down circPOLK on lung cancer metastasis capacity according to the embodiment of the present invention;

FIG. 13 is a diagram showing the results of RT-PCR for detecting the overexpression efficiency of circPOLK in lung cancer cells according to the embodiment of the present invention;

FIG. 14 is a diagram showing the results of Transwell testing the effect of over-expression of circPOLK on lung cancer migration and invasion;

FIG. 15 is a graph of the results of wound healing in testing the effect on lung cancer metastasis after over-expression of circPOLK, provided by an embodiment of the present invention;

FIG. 16 is a graph showing the results of in vivo experiments in mice for testing the effect of circPOLK on lung cancer metastasis and liver metastasis;

FIG. 17 is a schematic diagram showing the effect of circPOLK secreted by exosomes of lung cancer cells on the angiogenic ability according to an embodiment of the present invention;

Fig. 18 is a diagram illustrating the results of wound healing to detect the effect of circPOLK secreted by lung cancer cell exosomes on the angiogenic ability after the circPOLK is knocked down according to the embodiment of the present invention;

FIG. 19 is a diagram showing the results of Transwell testing the effect of circPOLK secreted by lung cancer cell exosomes on the angiogenic ability after knocking down circPOLK according to the present invention;

FIG. 20 is a graph showing the results of wound healing in testing the effect of circPOLK secreted by lung cancer cell exosomes on the angiogenic ability after over-expression of circPOLK provided in the present invention;

FIG. 21 is a diagram showing the results of Transwell testing the effect of circPOLK secreted by lung cancer cell exosomes on the angiogenic ability after over-expression of circPOLK according to the present invention.

Detailed Description

Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

The invention provides circPOLK for tumor therapy target and diagnosis biomarker, and the nucleotide sequence of the circPOLK is shown as SEQ ID NO. 10 (ataagtttataccatggatagcacaaaggagaagtgtgacagttacaaagatgatcttctgcttaggatgggacttaatgataataaagcaggaatggaaggattagataaagagaaaattaacaaaattataatggaagccacgaaggggtccagattttatggaaatgagctcaagaaagaaaagcaagtcaaccaacgaattgaaaatatgatgcaacaaaaagctcaaatcaccagccaacagctaagaaaagcacaattacag).

And the 1 st to 3 rd exons of the circPOLK POLK mRNA are reversely spliced to form circular RNA. The expression of circPOLK has obvious human and mouse evolution conservation, is resistant to digestion by RNase R and treatment by actinomycin D, and has high stability.

The experiment proves that the expression level of circPOLK is obviously different in tumor tissues and tissues beside cancer, so that the circPOLK can be used as a biomarker for tumor diagnosis in tumor treatment. And the prognosis of the lung cancer patient with high circPOLK expression is poor, which shows that the circPOLK can be used as a marker for evaluating the tumor prognosis effect. Accordingly, the present invention provides a kit for tumor therapy targets and diagnostic biomarkers comprising a molecular probe specifically complementary paired to the circPOLK of the first aspect or a primer pair for amplifying the circPOLK of the first aspect.

The experiment proves that the high expression of the circPOLK obviously promotes the migration and invasion of the lung cancer cells, and has no obvious influence on the apoptosis, proliferation and clone forming capability, so the circPOLK is applied to the anti-tumor as a tumor treatment target.

The experiment proves that the expression level of circPOLK is positively correlated with lymph node metastasis and distant metastasis of a lung cancer patient.

The following examples are provided to illustrate the circPOLK for tumor therapy target and diagnostic biomarker and its application in detail, but they should not be construed as limiting the scope of the present invention.

Example 1

Discovery and identification of circular RNA derived from POLK Gene (circPOLK):

in this embodiment, the presence of circular rna (circpolk) derived from the POLK gene is determined by using a bioinformatic approach in combination with a PCR amplification technique and the biological characteristics thereof are identified as follows:

discovery of circular RNA derived from POLK Gene (circPOLK)

Using circBase (http:// www.circbase.org) database analysis, it was found that the POLK gene could be spliced to form 8 types of circRNA, 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, respectively.

Unlike the conventional linear splicing of POLK mRNA, hsa _ circ _0073052 is formed by reverse splicing of exon 1 and exon 3 of the mRNA encoded by the POLK gene (chr5: 74842834-748416), and its mature sequence length is 268bp (see FIG. 1).

Further, by designing a primer for external amplification (Divergent primer F: cagctaagaaaagcacaattacaga, SEQ ID NO:1, R: agtcccatcctaagcagaaga, SEQ ID NO:2) spanning the circularized linker site, a sequence of about 200bp in length near the linker was successfully amplified by PCR and Sanger sequencing in NCI-H1299 cells, and 3 'and 5' reverse-spliced sequences of the 1 st and 3 rd exons of POLK were detected (see FIG. 1). As can be seen, this example successfully identified the presence of hsa _ circ _0073052, which was named circPOLK (SEQ ID NO:10) because the circRNA was derived from the POLK gene.

And (3) amplification reaction conditions:

(1)95℃15min；

(2)94℃30s；

(3)63℃30s；

(4)72℃30s；

(2) - (4)39 cycles.

Cyclic identification and conservation analysis of circular RNA derived from the POLK Gene (circPOLK)

First, primers for external amplification spanning the splice site (Divergent primer, SEQ ID NO:1 and SEQ ID NO:2) were designed for identification and specific detection of circPOLK, while primers for internal contraction (Convergent primer, POLK F: ccacgaaggggtccagattt, SEQ ID NO: 3; POLK R: tgttggctggtgatttgagc, SEQ ID NO:4) were designed on exon 3 for detection of POLK mRNA. Since circPOLK is a circular RNA formed at the post-transcriptional splicing level, its splice site sequence is not present on the genome, and in order to exclude the possibility that the observed head-to-tail splicing of circPOLK is due to trans-splicing, genomic rearrangement, or PCR products, circPOLK and POLKmRNA were amplified with designed exo-and endo-primers, respectively. Amplification by PCR (amplification procedure as described above) and agarose gel electrophoresis in A549 and NCI-H1299 cell cDNA and genomic DNA (gDNA).

The results showed that the exo-primers amplified a band only on cDNA and not on gDNA, while the endo-primers amplified the POLK gene on both cDNA and gDNA (see FIG. 2). These results indicate that circPOLK is not formed by genomic rearrangement, but by reverse cleavage of circular RNA at post-transcriptional level, and that circPOLK can be specifically detected using an exo-ampling primer.

Stability verification of circPOLK

3.1 the circRNA lacks free 5 'and 3' ends and has a unique closed loop structure and is thus resistant to digestion by the exonuclease RNase R (RNase R), which digests nearly all linear RNA molecules but does not readily digest circRNA, so the RNase R digestion assay is widely used in the stability characterization assay of circRNA to demonstrate its circular structure. In the experiment, total RNA is extracted from lung cancer cells A549, NCI-H1299 and NCI-H460, and after digestion treatment by RNA enzyme R, the mRNA levels of circPOLK and linear POLK are respectively detected by an RT-PCR method. The results show that circPOLK is indeed able to tolerate digestion by rnase R compared to POLK mRNA (see figure 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 Actinomycin D (actinomycin D) is used for inhibiting the synthesis of newborn RNA of lung cancer cells A549 and NCI-H1299, so as to verify the stability of circPOLK. The degradation rate of circPOLK and POLK mRNA was measured by qRT-PCR over 24h and the results showed that POLK mRNA had almost completely degraded, while the half-life of circPOLK was greater than 24h, indicating that circPOLK has high stability (see figure 4).

The experiments further prove that the circPOLK has the cyclization biological characteristics, can resist digestion of RNA enzyme R and treatment of actinomycin D, and has higher stability.

Subcellular localization of circPOLK

Since the function of circRNA is closely related to its localization, exon-derived circRNA is usually localized to the cytoplasm, whereas intron-derived circRNA is mostly localized to the nucleus. Since the above experimental results demonstrate that circPOLK is exon-derived circRNA, which is presumed to be likely to localize in the cytoplasm, in order to verify this inference and explore the specific subcellular localization of circPOLK, the experiment was conducted with a cellular RNA FISH experiment, as follows.

Fluorescence In Situ Hybridization (FISH) of RNA is an important nonradioactive in situ hybridization technique, which can perform relative qualitative, quantitative and localized analysis on circPOLK. In this experiment, probes (aaacttatctgtaattgtgcttttc, SEQ ID NO:9) specific to the circPOLK splice site were designed and synthesized by Gima Gene Corp, and the 5 'and 3' ends thereof carried with Cy3 red markers. The expression of circPOLK, abundant in the cytoplasm of NCI-H1299 lung cancer cells, was detected by cellular RNA FISH assay (see FIG. 5). Therefore, the cellular RNA FISH experiment shows that the circPOLK is mainly distributed in the cell pulp.

Example 2

Expression of circPOLK in serum exosomes of hepatocellular carcinoma patients:

The relation between the expression level of circPOLK in hepatocellular carcinoma patients and normal human serum exosomes is further analyzed in the experiment (see figure 6), and the result indicates that the circPOLK is probably a circRNA with cancer promotion property and is involved in the occurrence and development of cancer, and further the circPOLK is possibly one of potential indexes for judging the prognosis condition of lung cancer patients.

Example 3

1. Cell level detection of migration and invasion capacity of circPOLK in promoting non-small cell lung cancer

The basic expression level of circPOLK was detected by qRT-PCR in three cell lines, A549, NCI-H1299 and NCI-H460, indicating that the expression level of circPOLK in A549 cells was the highest and the expression level of circPOLK in NCI-H1299 cells was the lowest (see FIG. 7). The siRNA sequence design of circPOLK is successful by designing the siRNA sequence (SEQ ID NO:10-15) of the circPOLK and detecting the knockdown efficiency of the circPOLK in lung cancer cell lines A549, NCI-H1299 and NCI-H460 by a qRT-PCR method (see figure 8), the influence of the circPOLK on the migration and invasion capacity of lung cancer cells is detected by Transwell, and the result proves that the migration and invasion capacity of the lung cancer cells are remarkably inhibited after the circPOLK is knocked down (see figures 9 and 10) and the wound healing capacity of the lung cancer cells is also remarkably inhibited after the circPOLK is knocked down (see figure 11). The construction of the circPOLK overexpression plasmid was confirmed by introducing pLC5-ciR vector and verifying the overexpression efficiency of circPOLK in the lung cancer cell line NCI-H1299, as a result, the construction of the circPOLK overexpression plasmid was successful (see FIG. 13). Fig. 14, 15 demonstrate that over-expression of circPOLK significantly promotes the migratory invasion capacity and wound healing capacity of lung cancer cells. It was found by western blot that upon knockdown of circPOLK, it can cause a change in EMT-related proteins, increasing the level of E-cadherin and decreasing the level of N-cadherin (see fig. 12). The above results all confirm that circPOLK promotes migration and invasion of non-small cell lung cancer cells in vitro.

si-NC sense:uucuccgaacgugucacgutt；

si-NC antisense:acgugacacguucggagaatt；

si-circPOLK#1sense:aagcacaauuacagauaagtt；

si-circPOLK#1antisense:cuuaucuguaauugugcuutt；

si-circPOLK#2sense:agcacaauuacagauaagutt；

si-circPOLK#2antisense:acuuaucuguaauugugcutt。

2. Mouse cell level detection of migration and invasion capacity of circPOLK in promoting non-small cell lung cancer

Next, the regulation of lung cancer metastasis by circPOLK in an in vivo environment was examined. The influence of the CircPOLK knockout cell on lung metastasis and liver metastasis is detected by tail vein injection mice (the concrete method is shown in the literature: doi:10.1038/s41467-019-12651-2.PMID: 31619685). The results show that knocking down circPOLK can promote lung metastasis and liver metastasis ability of lung cancer cells (see fig. 16).

Example 3

circPOLK can promote neovascularization by exosomes:

HUVEC cells were co-cultured with lung cancer cells in serum-free, the lung cancer cells were seeded in a transwell chamber (0.8 μm), the HUVEC cells were seeded in a 24-well plate, and the wound healing ability of the HUVEC cells was observed after 24 hours, and as a result, it was found that the knockdown of circPOLK inhibited the wound healing of the HUVEC cells (see fig. 18), and this phenomenon was reversed after overexpression of circPOLK (see fig. 20).

HUVEC cells and lung cancer cells were subjected to serum-free co-culture, the HUVEC cells were inoculated in a transwell chamber, the lung cancer cells were inoculated in a 24-well plate, and the migration ability of the HUVEC cells was observed 24 hours later. As a result, it was found that lowering circPOLK inhibited the migration of HUVEC (see fig. 19), and that this phenomenon was reversed after overexpression of circPOLK (see fig. 21). The results show that the non-small cell lung cancer cell can secrete circPOLK through an exosome so as to promote the migration and movement capacity of the vascular endothelial cell.

The experimental method comprises the following steps:

1. cell culture

Human non-small cell lung carcinoma 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 were in 5% CO₂And culturing at 37 ℃ in a cell culture box.

Transwell experiment

(1) Cell migration assay

Lung cancer cells expressing circPOLK at high and low levels were serum-starved overnight, digested and counted, and inoculated into transwell chamber at a density of 2X 10⁴Per well, 200. mu.l of serum-free medium per well. To the corresponding 24-well plate, 600. mu.l of a culture medium containing 20% serum was added. After 24 hours of cell incubation, the chamber was stained with 1% crystal violet for 30min in the dark, and then the cells inside the chamber were washed with 1 × PBS, and then the cells migrated to the lower surface of the chamber were photographed with a microscope and counted.

(2) Cell invasion assay

The Matrigel was thawed by placing it at 4 ℃ in advance, and the chamber, 24-well plate and tip used were placed at-20 ℃ for pre-cooling. Matrigel was mixed with cell culture medium at a ratio of 1: 20 dilution, 100. mu.l of Matrigel was added to each chamber, the chambers were placed in a 24-well plate and incubated at 37 ℃ for 30min, the 24-well plate was removed, the unsolidified Matrigel in the chamber was discarded, and the cells were rinsed once with the culture solution. Meanwhile, the non-small cell lung cancer cells expressing circPOLK at high and low levels were serum-starved overnight, digested and counted, and inoculated into a transwell chamber with Matrigel gel at a density of 3X 10 ⁴Per well, 200. mu.l of serum-free medium per well. To the corresponding 24-well plate, 600. mu.l of a culture medium containing 20% serum was added. After 24 hours of cell incubation, the chamber was stained with 1% crystal violet for 30min in the dark, and then the cells inside the chamber were washed with 1 × PBS, and then the cells invading the lower surface of the chamber were photographed with a microscope and counted.

3. Wound healing experiments

Cells were plated at 2X 10⁵The density per well was seeded in six well plates and after cells attached to the wall, desired plasmids or sirnas were transfected. And scratching the cell surface by using a pipette when the cell confluence reaches 80-100%, washing off the cells falling from the surface by using PBS, replacing with new culture solution, patting the cells with a microscope for a wound distance of 0h, washing the six-hole plate by using PBS after 24h to remove non-adherent cells, patting the cells with a microscope for a wound distance of 24h, and calculating the wound healing condition.

4. Transfection

Cells were seeded in 6-well plates (5X 10)⁵Hole/bore). And (3) transfecting when the cell density reaches 30-40%, taking 200 mu l of transfection buffer per hole to an EP tube of RNase free, adding 2 mu g of target siRNA, uniformly mixing, and standing for 5 min. Adding 4 μ l

And (3) uniformly mixing and centrifuging the transfection reagent, standing for 20min, then adding the transfection reagent, and slightly shaking to uniformly mix the transfection reagent.

Western blot method

(1) Protein sample extraction and quantification

Firstly, extraction: the cells that had been previously treated were collected, centrifuged at 2000rpm for 4min, the supernatant was discarded, the pellet was transferred to an EP tube, centrifuged at 2000rpm for 4min, and the supernatant was discarded. Adding whole cell lysate according to the cell number, vortexing once every 10min,

the process is carried out for 3 times, the cells and the lysate are fully and uniformly mixed, the mixture is centrifuged for 30min at 12000rpm and 4 ℃, and the supernatant is collected, namely the protein sample.

Quantifying: BSA (2mg/ml) was diluted in deionized water for a half-fold dilution as a standard curve and the sample protein was diluted 10-fold with deionized water. Separately, 5. mu.l of BSA solution and sample protein with different concentrations were added to 200. mu.l of the BCA reagent mixture (solution A: solution B ═ 50: 1), mixed well, and then placed at 37 ℃ for 30min, and the absorbance at 562nm was read with a microplate reader. And calculating the concentration of the protein of the sample according to the formula of the obtained standard curve, and calibrating the protein according to a certain amount.

③ direct loading method

Discarding the pretreated cell supernatant, washing twice with PBS, adding 2.5 × loading according to cell density, directly stirring to collect cells, centrifuging, boiling in boiling water for 10min to inactivate protein, cooling, centrifuging, and storing at-20 deg.C.

(2) Western blotting

Gel electrophoresis: 500ml of 1 XRunning Buffer was prepared and protein samples were loaded in a certain order and in a certain amount. The voltage is adjusted to 70V, when the sample runs to the separation gel, the voltage can be increased to 110V, and when the sample is 1cm away from the bottom end of the gel, the electrophoresis is stopped.

Secondly, film turning: 900ml of 1 × Transfer Buffer was prepared, and a PVDF membrane of an appropriate size was activated with 100ml of absolute ethanol for 1min, and the prepared Transfer Buffer was added. According to the size of the protein required by the experiment, the gelatin is cut into corresponding size, the membrane is rotated in a black gelatin and white membrane mode, the transverse constant current of 330mA is carried out, and the membrane is rotated in ice water bath for 90 min.

③ sealing and incubating the milk with primary antibody: after the membrane-switching time, the PVDF membrane was placed in 5% skim milk (prepared in T-PBS) and sealed for 1 hour at room temperature. Milk was discarded, PVDF membrane was washed three times with T-PBS, primary antibody required for the experiment was added, and incubated overnight at 4 ℃ on a shaker.

Incubation of secondary antibody: primary antibody was recovered and the bands were washed 3 times with T-PBS for 10min each time. Adding a secondary antibody corresponding to the primary antibody and labeled by horseradish peroxidase, incubating for 90min in a shaking table at room temperature, discarding the secondary antibody, and washing for 3 times by using T-PBS.

Exposure: preparing the exposed matter in advance, incubating the strips in the dark for 1min by using ECL developing solution (solution A and solution B in ECL kit: 1), transferring the strips into an exposure box, tabletting X-ray films in a dark room for 5-20s (according to the fluorescence intensity of the strips), taking out the films for 30min for a short time, placing the films in the developing solution for 1.5min, washing the films with tap water, placing the films in a fixing solution for 1.5min, and imaging.

RNA extraction and reverse transcription

The supernatant of the pre-treated six-well plate cells was discarded, and the cells were rinsed twice with pre-cooled PBS, 1ml of Trizol was added to each well, and the solution was repeatedly blown up until it was clear. The cells were transferred to an EP tube containing 1.5ml of RNase free, 200. mu.l of chloroform was added thereto, and the mixture was vigorously shaken for 30 seconds, allowed to stand at 4 ℃ for 2min, and centrifuged at 12000rpm for 10min at 4 ℃. Aspirate 450. mu.l of the supernatant into a fresh EP tube, add the same amount of isopropanol, gently invert 5 times, stand on ice for 10min, 12000rpm, and centrifuge at 4 ℃ for 15 min. The supernatant was discarded and the pellet was gently washed with 500. mu.l of pre-cooled 75% ethanol and centrifuged at 8000rpm at 4 ℃ for 5 min. The washing was repeated, the place was left to stand in a ventilated place, and after ethanol was evaporated, 20. mu.l of DEPC water was added to dissolve mRNA, and the concentration of mRNA was measured.

mRNA was reverse transcribed to complementary DNA (cDNA) using the TransStart Top Green qPCR SuperMix (Lot #40426) kit with all-gold.

7.qRT-PCR

The qRT-PCR system was 10. mu.l, 5. mu.l of 2 XQuantiTect SYBR Green PCR Master Mix, 1. mu.l of template cDNA, 0.6. mu.l of each target forward and reverse primer, and 2.8. mu.l of DEPC water. The reaction conditions are pre-denaturation at 95 ℃ for 15min, denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 30s and 39 cycles.

8. Cell co-culture

HUVEC cells (serum starved overnight) were cultured at 2X 10⁴Was inoculated into each transwell chamber (0.8 μm) while pre-treated non-small cell lung cancer cells (serum starved overnight) were incubated at 5X 10⁴Is seeded under the chamberIn a 24-well plate. After 24 hours of cell incubation, the chamber was stained with 1% crystal violet for 30min, cells in the chamber were washed out with PBS, and cells migrating to the bottom of the chamber were photographed with a microscope and counted.

HUVEC cells (serum starved overnight) were cultured at 8X 10⁴Was seeded in 24-well plates. After the cells adhered to the wall, the surface of the cells was scratched with a 20. mu.l pipette tip, and the cells were photographed for a wound distance of 0 h. The pretreated non-small cell lung cancer cells (serum starved overnight) were then treated at 2X 10⁴Was seeded into each transwell chamber (0.4 μm) and the chambers were placed in 24-well plates containing HUVEC cells. After 24 hours of cell incubation, HUVEC cells were observed for wound healing and photographed.

9. Statistical analysis

Using t-test to test statistical analysis, P <0.05 was considered as a significant difference in experimental results (. P <0.05,. P <0.01 and. P < 0.001). Each experiment was repeated at least three times.

Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.

It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Sequence listing

<110> Zhejiang City college

Hangzhou anxiu Biotechnology Co.,Ltd.

<120> circPOLK for tumor therapy target and diagnosis biomarker and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagctaagaa aagcacaatt acaga 25

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agtcccatcc taagcagaag a 21

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccacgaaggg gtccagattt 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgttggctgg tgatttgagc 20

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggagcgagat ccctccaaaa t 21

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggctgttgtc atacttctca tgg 23

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaaggtgaag gtcgagtc 18

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaagatggtg atgggatttc 20

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaacttatct gtaattgtgc ttttc 25

<210> 10

<211> 268

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ataagtttat accatggata gcacaaagga gaagtgtgac agttacaaag atgatcttct 60

gcttaggatg ggacttaatg ataataaagc aggaatggaa ggattagata aagagaaaat 120

taacaaaatt ataatggaag ccacgaaggg gtccagattt tatggaaatg agctcaagaa 180

agaaaagcaa gtcaaccaac gaattgaaaa tatgatgcaa caaaaagctc aaatcaccag 240

ccaacagcta agaaaagcac aattacag 268

Claims

1. A circPOLK for use in tumor therapy targets and diagnostic biomarkers, wherein the nucleotide sequence of the circPOLK is shown in figure 1.

2. A kit for diagnosing tumorigenesis, comprising a molecular probe specifically complementary-paired to the circPOLK of claim 1 or a primer pair for amplifying the circPOLK of claim 1.

3. The use of circPOLK according to claim 1 for the preparation of a medicament for the prevention and/or treatment of tumors.

4. The use of claim 3, wherein the tumor comprises liver cancer and lung cancer.

5. The use of claim 4, wherein the tumor comprises primary and metastatic tumors.

6. A drug for preventing and/or treating tumors, which comprises the circPOLK according to claim 1 as an active ingredient.

7. The medicament of claim 6, wherein the tumor comprises a colon cancer and a lung cancer.

8. The medicament of claim 7, wherein the tumor comprises primary tumor and metastatic tumor.