Prostatic cancer related chromosome outer circular DNA and application thereof as molecular marker for diagnosing prostatic cancer

Abstract

The invention discloses a prostatic cancer related chromosome outer circular DNA and application of the prostatic cancer related chromosome outer circular DNA as a molecular marker for diagnosing prostatic cancer. The prostatic cancer related chromosome outer circular DNA comprises TXLNA eccDNA1, TXLNA eccDNA2 and TXLNA eccDNA3 which are derived from TXLNA protein, and the nucleotide sequences of the TXLNA eccDNA1, the TXLNA eccDNA2 and the TXLNA eccDNA3 are respectively shown as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. The eccDNA can inhibit proliferation and migration of prostate cancer cells, can be used as a potential molecular marker for clinical diagnosis of prostate cancer, has the potential of being applied to clinical diagnosis of prostate cancer, provides a new idea for diagnosis of prostate cancer, and overcomes the limitation of poor specificity of traditional PSA detection.

Description

(I) Technical field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a molecular marker for specific diagnosis of prostate cancer and its application. (II) Background technology

[0002] Extrachromosomal circular DNA (eccDNA) is free circular DNA outside chromosomes in the cell nucleus. It is a complex structural variation caused by genomic instability and an important molecular marker for many cancers. Small extrachromosomal circular DNA is commonly found in different tissues and cells. Studies have reported that its number and length characteristics in normal tissues are significantly different from those in cancer tissues, suggesting that it has the potential to become a cancer molecular marker.

[0003] There have been many studies on the functions of eccDNA in different cancers. Zou et al. found that eccDNA carrying the miR-17-92 cluster can promote the proliferation and migration of liver cancer cells and thus promote the development of liver cancer (Zou et al., Hepatology, 2023); Jiang et al. proved that eccDNA carrying enhancers or miRNA precursors overexpressed in gastric cancer cells can promote the development of gastric cancer (Jiang et al., Cell Mol. Life Sci., 2023); Luo et al. analyzed the characteristics of eccDNA in cancer tissues and plasma of 25 pairs of cancer patients and showed that eccDNA in plasma is a potential clinical molecular diagnostic marker.

[0004] The functional study of eccDNA has been a research hotspot in recent years, but most studies are limited to describing the characteristics of eccDNA in different tissue cells. The inability to simply and quickly synthesize eccDNA in vitro is a major factor hindering the functional study of eccDNA. The current method for synthesizing eccDNA is mainly LAMA (Ligase Assisted Mini-circle Accumulation) technology, the main principle of which is to synthesize two cross-complementary DNA sequences in vitro according to the target eccDNA sequence, continuously denature and anneal the synthesized DNA sequence, and finally fill the gap with T4 ligase to obtain the target eccDNA. However, the disadvantages of this method are that the synthesis cost of linear DNA is high and the cycle is long; second, this method is mainly synthesized by linear DNA annealing of eccDNA, and the annealing efficiency of long-fragment DNA is difficult to guarantee.

[0005] However, it is unclear whether there is specific eccDNA in prostate cancer (PCa) that plays a role in the development of prostate cancer and serves as a potential molecular marker for clinical diagnosis.

[0006] Therefore, there is an urgent need to develop a prostate cancer-specific eccDNA and a simple, economical and efficient eccDNA synthesis method to promote in-depth research on the function of eccDNA. (III) Summary of the invention

[0007] The purpose of the present invention is to provide an extrachromosomal circular DNA related to prostate cancer and its application as a molecular marker for the specific diagnosis of prostate cancer. The present invention obtains TXLNA-derived extrachromosomal circular DNA specifically expressed in prostate cancer through screening, which can inhibit the proliferation and migration of prostate cancer cells and can be used as a new molecular marker for the clinical diagnosis of prostate cancer. The present invention also provides a simple, economical and efficient eccDNA synthesis method.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides an extrachromosomal circular DNA associated with prostate cancer, comprising three extrachromosomal circular DNAs derived from TXLNA protein (gliding protein α, whose binding dynamics involve interaction with the C-terminal coiled coil region of synaptophysin family members), namely TXLNA eccDNA1, TXLNA eccDNA2, and TXLNA eccDNA3, and the nucleotide sequences are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, respectively.

[0010] The present invention provides a primer for specifically detecting the extrachromosomal circular DNA, and the primer is as follows:

[0011] TXLNA eccDNA1

[0012] F: 5'-CCCAAGCTTCCCACCTCAGCCTCCCAAAGT-3' (SEQ ID No. 4),

[0013] R: 5'-CCCAAGCTTCATCTGCAGCTGAATGTCATTC-3' (SEQ ID No. 5);

[0014] TXLNA eccDNA2

[0015] F: 5'-CCCAAGCTTCTGCGAAGAATTTCGAAGTCTG-3' (SEQ ID No. 6),

[0016] R: 5'-CCCAAGCTTGCTGCCGGGACCTTCTGCTTCTA-3' (SEQ ID No. 7);

[0017] TXLNA eccDNA3

[0018] F: 5'-CCTTAATTAACTGCTTGCCCTAGTGAGTTACC-3' (SEQ ID No. 8),

[0019] R: 5'-CCTTAATTAATCCACCTCAGCCTCCCAAAGT-3' (SEQ ID No. 9).

[0020] The present invention provides a method for synthesizing the extrachromosomal circular DNA, the method comprising the following steps:

[0021] S1, using HEK293T genomic DNA (gDNA) as a template, using the primers to perform PCR amplification to obtain sufficient linear DNA;

[0022] The amplification reaction system was: 2*Taq Plus Master MixII 200μL, F-Primer 16μL, R-Primer 16μL, genomic DNA about 500ng, ddH2O supplemented to 400μL;

[0023] PCR reaction conditions were as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 20 s, 72°C extension for 60 s / kb, 35 cycles; 72°C extension for 5 min; 4°C storage;

[0024] S2, digest the S1 amplified fragment with the corresponding restriction endonuclease;

[0025] Enzyme digestion reaction system: 5 μL 10*Reaction buffer, 2 μg amplified fragment, 2 μL restriction endonuclease, add ddH2O to 50 μL;

[0026] The restriction endonuclease for TXLNA eccDNA3 is the restriction endonuclease PacI, and the others are the restriction endonucleases HindIII;

[0027] S3, use T4 DNA ligase to connect the S2 digested fragments into a circle;

[0028] Ligation reaction system: 5 μL 10*T4 DNA ligation Buffer, 40 μL restriction fragment, 5 μL T4 DNA ligase;

[0029] S4. Use DNA exonuclease Plasmid-safe DNase to digest the unconnected linear DNA in the S3 connection product at 37°C for 16 hours to obtain the target eccDNAs, namely TXLNA eccDNA1 (nucleotide sequence as shown in SEQ ID NO.1), TXLNA eccDNA2 (nucleotide sequence as shown in SEQ ID NO.2), and TXLNA eccDNA 3 (nucleotide sequence as shown in SEQ ID NO.3);

[0030] Digestion reaction system: 31 μL ligation product, 4 μL 10*Reaction buffer, 1.6 μL 25 mM ATP, 2 μL Plasmid-safe DNase, and make up to 40 μL with ddH2O.

[0031] The present invention also provides an application of the extrachromosomal circular DNA as a molecular marker for specific diagnosis of prostate cancer.

[0032] The application is to detect the expression level of extrachromosomal circular DNA. The higher the expression level, the higher the possibility of prostate cancer.

[0033] The present invention also provides a detection product using the extrachromosomal circular DNA as a molecular marker for prostate cancer, wherein the detection product detects the expression level of the extrachromosomal circular DNA, and the detection product comprises a kit, a chip or a high-throughput sequencing platform.

[0034] The kit includes a gene detection kit and a protein immunoassay kit; the gene detection kit includes a PCR kit, a RT-PCR kit, and an in situ hybridization kit; the protein immunoassay kit includes an immunoassay kit; the immunoassay kit includes an antibody that specifically binds to extrachromosomal circular DNA, and the in situ hybridization kit includes a probe that hybridizes to the nucleotide sequence of the extrachromosomal circular DNA.

[0035] The chip includes a gene chip and a protein chip; the protein chip includes an antibody that specifically binds to extrachromosomal circular DNA; and the gene chip includes a probe that hybridizes with a nucleotide sequence of the extrachromosomal circular DNA.

[0036] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0037] The present invention screens out extrachromosomal circular DNA that is specific to prostate cancer by analyzing circle-seq data of eccDNA in urine of prostate cancer patients and non-cancerous control samples. The eccDNA can inhibit the proliferation and migration of prostate cancer cells and can be used as a potential molecular marker for the clinical diagnosis of prostate cancer. It has the potential to be applied in the clinical diagnosis of prostate cancer, provides a new idea for the diagnosis of prostate cancer, and overcomes the limitation of poor specificity of traditional PSA detection.

[0038] The present invention also provides a method for synthesizing eccDNA in vitro, which uses a cheap PCR reaction to amplify the target eccDNA sequence, then digests the amplified fragment, connects it with T4 ligase, and finally digests the unconnected linear DNA to obtain eccDNA with relatively high purity. The operation is simple and convenient, the cost is low, and it is suitable for more researchers to explore the function of eccDNA. (IV) Description of the drawings

[0039] Figure 1 , flow chart of purification and sequencing of urine eccDNA.

[0040] Figure 2 , identification of prostate cancer-specific eccDNA, a is the frequency statistics of genes that produce eccDNA in different numbers of samples; b is the intersection Venn diagram of genes that produce eccDNA in prostate cancer samples and control samples; c is the gene enrichment pathway diagram that specifically produces eccDNA in prostate cancer.

[0041] Figure 3 , Schematic diagram of in vitro synthesis of eccDNA.

[0042] Figure 4 , gel electrophoresis of eccDNA amplification products.

[0043] Figure 5 , qPCR enrichment verification Ct value graph of eccDNA.

[0044] Figure 6 , schematic diagram of eccDNA transfection.

[0045] Figure 7 , TXLNA eccDNA enrichment and identification after transfection. The left picture shows the enrichment verification of eccDNA in transfected cell DNA, and the right picture shows the enrichment verification of eccDNA transcription products in transfected cell RNA.

[0046] Figure 8 , functional investigation of TXLNA eccDNA in prostate cancer cells, a is a micrograph of the cell scratch experiment, b is a statistical chart of cell healing in the scratch experiment, and c is a CCK-8 experiment. (V) Specific implementation methods

[0047] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0048] Example 1: Screening of genes related to prostate cancer that produce eccDNA

[0049] 1. Sample collection and processing

[0050] Urine samples of prostate cancer patients and non-cancer controls were collected from the Urology Department of an affiliated hospital of Zhejiang University School of Medicine, including 21 urine samples of prostate cancer patients (PCa) and 16 urine samples of non-cancer controls (NC). After collection, urine samples were stored at 4°C and transported to the laboratory within 2 hours. Centrifuged at 3000g for 15 minutes at room temperature, 10mL of urine supernatant was taken and 700μL of urine preservative (from Quick-DNA Urine Kit) was added, mixed by inversion, and stored at room temperature.

[0051] All subjects gave their informed consent to participate in this experiment, and the study was reviewed and approved by the Ethics Committee of an affiliated hospital of Zhejiang University School of Medicine.

[0052] 2. WGS library preparation, high-throughput sequencing and data analysis of eccDNA in prostate cancer urine

[0053] Reference Figure 1 The process is as follows:

[0054] 1) Extract cell free DNA from urine using the Quick-DNA Urine Kit (ZYMO, #D3061).

[0055] 2) The linear DNA was digested with Plasmid-Safe ATP-dependent DNase at 37°C for 16 h, and then the digested DNA was purified with VAHTS DNA Clean Beads.

[0056] 3) Utilize DNA polymerase and random primers were used to amplify the purified DNA obtained in step 2) at 30° C. for 8 h.

[0057] 4) Using a Covaris S220 DNA ultrasonic disruptor, the DNA amplified in step 3) was broken into fragmented DNA with a size of 250 bp-750 bp.

[0058] 5) Use VAHTS Universal DNA Library Prep Kit for Illumina V3 (vazyme,

[0059] The fragmented DNA was constructed using a DNA library constructed with a kit (#ND607) and sequenced on an Illumina NovaSeq6000 sequencing platform using a double-end 150bp sequencing strategy. The sequencing work was completed by Tianjin Novogene.

[0060] 6) The sequencing data were processed and analyzed using Circle-Map software, and genes with high credibility that could produce eccDNA were filtered using split reads greater than 5 as the standard. The frequencies of genes that could produce eccDNA in different samples were divided into intervals ( Figure 2 In a), 6,333 genes were found in PCa samples that could produce eccDNA in more than 50% of the samples, while 7,787 were found in NC samples. A Venn diagram of the above gene set was drawn, of which 530 were PCa-specific ( Figure 2 b), the Hallmark gene set was enriched for these 530 genes to obtain the gene set of the androgen response signaling pathway involved in the regulation of prostate cancer development ( Figure 2 (c).

[0061] TXLNA protein belongs to the syntaxin family of synaptic fusion protein binding protein. Studies have reported that it can promote the metastasis and invasion of renal cell carcinoma, and is associated with the malignant potential of hepatocellular carcinoma and the prognosis of pancreatic cancer. TXLNA is located in the 530 genes that specifically produce eccDNA in PCa, and is most correlated with the overall survival analysis of prostate cancer (P=0.0095). Therefore, we selected TXLNA as a target gene to study the function of the eccDNA it produces.

[0062] Example 2: In vitro synthesis of prostate cancer-related eccDNA

[0063] Reference Figure 3 , three TXLNA eccDNAs were synthesized in vitro and transfected into prostate cancer cells PC-3, and the eccDNA enrichment in the transfected cells was verified by qPCR. The specific steps are as follows:

[0064] 1. Design of synthetic primers

[0065] First, based on the sequencing results of eccDNA generated by the TXLNA gene, three eccDNA sequences generated in the TXLNA exon region were selected and primers were designed. At the same time, based on the human embryonic kidney cell HEK 293T genome sequence published in Genbank, the sequence of the intergenic region was selected as the control sequence to design primers, and restriction endonuclease sequences and protective bases CCC or CC were added to the 5' ends of the upstream and downstream primers, respectively. The added restriction endonuclease should not exist in the target eccDNA sequence, and 4 sets of primer pairs were obtained, which were used to synthesize TXLNA eccDNA and intergenic region eccDNA, respectively. The restriction endonuclease used to synthesize TXLNA eccDNA3 was the restriction endonuclease PacI sequence with a protective base of CC, and the rest were restriction endonuclease HindIII sequences and protective bases CCC.

[0066] The primers for in vitro synthesis of TXLNA eccDNA are:

[0067] TXLNA eccDNA1

[0068] F: 5'-CCCAAGCTTCCCACCTCAGCCTCCCAAAGT-3' (SEQ ID No. 4),

[0069] R: 5'-CCCAAGCTTCATCTGCAGCTGAATGTCATTC-3' (SEQ ID No. 5);

[0070] TXLNA eccDNA2

[0071] F: 5'-CCCAAGCTTCTGCGAAGAATTTCGAAGTCTG-3' (SEQ ID No. 6),

[0072] R: 5'-CCCAAGCTTGCTGCCGGGACCTTCTGCTTCTA-3' (SEQ ID No. 7);

[0073] TXLNA eccDNA3

[0074] F:5'-CCTTAATTAACTGCTTGCCCTAGTGAGTTACC-3'(SEQ ID No.8),

[0075] R: 5'-CCTTAATTAATCCACCTCAGCCTCCCAAAGT-3' (SEQ ID No. 9).

[0076] Intergenic eccDNA synthesis primers are as follows:

[0077] F:5'-CCCAAGCTTCCACAGTTCCATGCAAGCATCT-3'(SEQ ID No.10),

[0078] R: 5'-CCCAAGCTTTGCAGAGTTCCATGGATTCCCT-3' (SEQ ID No. 11).

[0079] 2. eccDNA enrichment and verification primer design

[0080] Based on the target eccDNA sequence, qPCR primers spanning the eccDNA breakpoints were designed to detect eccDNA enrichment.

[0081] qPCR-TXLNA-eccDNA1:

[0082] F:AGAAGCGCAAGGAGGTGACCT(SEQ ID No.12)

[0083] R:GGGCCCCTCTGTATATCAGGT(SEQ ID No.13)

[0084] qPCR-TXLNA-eccDNA2:

[0085] F:CCAAAAAGCAGCCCAGGACA(SEQ ID No.14)

[0086] R:CGATGCTGGGTCAGGAGAAGA(SEQ ID No.15)

[0087] qPCR-TXLNA-eccDNA3:

[0088] F:GTAAAAAAGCAAGGCTGGGTG(SEQ ID No.16)

[0089] R:CTCCTCCCATGAACTCACAAT(SEQ ID No.17)

[0090] qPCR-Intergenic-eccDNA:

[0091] F:AGCAGCCCACAACTTAGAGG(SEQ ID No.18)

[0092] R:GGGAGGTATCATCTGGAGGT(SEQ ID No.19)

[0093] 3. In vitro synthesis of eccDNA

[0094] S1. Using the genomic DNA of normal embryonic kidney cell line HEK293T as a template, PCR amplification was performed using the primers synthesized in step 1 to obtain sufficient linear DNA; the amplified product was analyzed by agarose gel electrophoresis. Figure 4 As shown, it shows that the size of the amplified fragment is consistent with the expected value.

[0095] The fragment amplification reaction system was: 2*Taq Plus Master MixII 200μL, F-Primer 16μL, R-Primer 16μL, genomic DNA about 500ng, and ddH2O supplemented to 400μL.

[0096] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 60 s / kb, 35 cycles; extension at 72°C for 5 min; and storage at 4°C.

[0097] S2, digest the S1 amplified fragment with the corresponding restriction endonuclease;

[0098] Enzyme digestion reaction system: 5 μL 10*Reaction buffer, 2 μg amplified fragment, 2 μL restriction endonuclease, add ddH2O to 50 μL.

[0099] S3, use T4 DNA ligase to connect the S2 digested fragments into a circle;

[0100] Ligation reaction system: 5μL 10*T4 DNA ligation Buffer, 40μL restriction fragment, 5μL T4 DNA ligase.

[0101] S4. Use DNA exonuclease Plasmid-safe DNase to digest the linear DNA that was not successfully connected in the S3 connection product, and digest it at 37°C for 16 hours to obtain the target eccDNA, namely TXLNA eccDNA1 (nucleotide sequence as shown in SEQ ID NO.1), TXLNA eccDNA2 (nucleotide sequence as shown in SEQ ID NO.2), TXLNA eccDNA 3 (nucleotide sequence as shown in SEQ ID NO.3) and the intergenic region eccDNA (SEQ ID No.20) as a control.

[0102] Digestion reaction system: 31 μL ligation product, 4 μL 10*Reaction buffer, 1.6 μL 25 mM ATP, 2 μL Plasmid-safe DNase, and make up to 40 μL with ddH2O.

[0103] TXLNA eccDNA1(SEQ ID No.1):

[0104] CCCACCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCTGCCATGTGCCTGCATTTTTCTAGGGGGAGAATCTCACTTGATGTCACCTGATATACAGAGGGGCCCATTGGAACCCGCATTGCACAACATCCTGGAGTCTGGCTACTCCACGCTTTGGGAGCAGGGAGGGCTGTTGGCAGAGACCATCTGTGGACTAGCTGGGGGACCCTTGTGAGGTAGCAGTGGATGATGGCTCTCGGGCTGACTTCTTTGCCCAGGAAGAAGGTGTGCAGCGGGCCCGGGAGGAGGAGGAGAAGCGCAAGGAGGTGACCTCGCACTTCCAGGTGACACTGAATGACATTCAGCTGCAGATG。

[0105] TXLNA eccDNA2(SEQ ID No.2):

[0106] CTGCGAAGAATTTCGAAGTCTGGAAAATAGCAACTGTGTTTGTTTCTAAAGGATCTTCTCCTGACCCAGCATCGCTCATCACAATGAAGAACCAAGACAAAAAGAACGGGGCTGCCAAACAATCCAATCCAAAAAGCAGCCCAGGACAACCGGAAGCAGGACCCGAGGGAGCCCAGGAGCGGCCCAGCCAGGCGGCTCCTGCAGTAGAAGCAGAAGGTCCCGGCAGC。

[0107] TXLNA eccDNA3(SEQ ID No.3):

[0108] CTGCTTGCCCTAGTGAGTTACCATTTCTCTGTCCCTACGGTGGAGCCTTTGGGGT

[0109] TATTGTGAGTTCATGGGAGGAGCGTGTAAGCACCGGCACAGCATCAGCCCATGA

[0110] GAGTGCTCCTGGCCTGAGAGGGTAAGGGTCAGGGCAGCTCAGGAGACCCTAGA

[0111] CCTGCATAGTGATCCCCCCACCAGGAAGGCCCCACAAGATGCTCACCTGCCCTC

[0112] CCTATCCCTGTCCCCAGCTGGAGGAGCACCGGAATTCACAGAAGCAGATGAAG

[0113] CTCCTACAGAAAAAGCAGAGCCAGCTGGTGCAAGAGAAGGACCACCTGCGCG

[0114] GTGAGCACAGCAAGGCCGTCCTGGCCCGCAGCAAGCTTGAGAGCCTATGCCGT

[0115] GAGCTGCAGCGGCACAACCGCTCCCTCAAGGTAGGCCTGGGCCCCCTGGAACA

[0116] GGTGACTCTGGTTTCCTTGACTTCCACTTAATGTTTCTTTCATGGGCTTTCCTCTT

[0117] AAAAAGTAGTGCAGGCTAGGGCCAGGCGCAGTGGCACACATAAGTGATTAAAA

[0118] ATCTTCTGGCCACTAAAAAACAGAAATTAATTTTAGTAATATACTTAACCCAATAT

[0119] CCAAAACATTACAATTTCAACATGAAATCAGTGTAAAAAAGCAAGGCTGGGTGTGGTGGCTCACACCTGTAATCCCAACACTTTGGGAGGCTGAGGTGGA。

[0120] Intergenic eccDNA(SEQ ID No.20):

[0121] CCACAGTTCCATGCAAGCATCTCGGGGGCCTTCGTAGAGTCCTGCCTACCACAACCCTGATCAACTCTAACTTTCAGTCTCTCCCACCTCCAGATGATACCTCCCCAAAAACCAAGTTCATTTTCCTAAAATGCA CCCTCTGTCCTGTGCCATCCCAACTAAAGGCTATGCCACGGTCAGCTGTGTCCCTAAACAATGAGTCAAACACTCATAAGATAAGGGGAGTTCTGTTCTCAGAGAGCATAACCGTACTTAAAAATTCCTATTTAT AGTTCCTGAAAATCCTGTGTTCCTGGGGTCCAGGGACCCTTTGCCATGACTAAGGACCTAGGCTGGGGTACAGTTGATGGTGTGGGTGCCCAGGGGGATTGAGGAAATTGTCAGGCAGCTGCCCTTTCCAGTGGCCACTCAGCATGACCCCAAGCAGCCCACAACTTAGAGGAAATAGCAGGGAATCCATGGAACTCTGCA.

[0122] 4. eccDNA enrichment verification

[0123] qPCR amplification was performed using primers spanning the eccDNA breakpoints designed in step 2 to verify the enrichment of eccDNA in the digestion product of step 3. The genomic DNA of the normal embryonic kidney cell line HEK293T was used as a control. Compared with the control, the obtained target eccDNA had a lower Ct value, indicating that the synthesized eccDNA was effectively enriched ( Figure 5 ).

[0124] The qPCR reaction system was: 5 μL Taq Pro Universal SYBR qPCR Master Mix, 0.2 μL F-primer, 0.2 μL R-primer, 20.2 μL eccDNA, and made up to 10 μL with ddH2O.

[0125] Example 3: Transfection of prostate cancer-related eccDNA

[0126] Reference Figure 6 The three TXLNA eccDNAs and Intergenic eccDNA synthesized in vitro in Example 2 were transfected into prostate cancer cells PC-3, and the eccDNA enrichment in the transfected cells was verified using the qPCR primers in Example 2. The specific steps are as follows:

[0127] 1. Transfection

[0128] One day before transfection, prostate cancer cells PC-3 were seeded in a six-well plate with 2 mL of complete medium, with approximately 8×10 cells per well. 5 Cells were cultured in a 37°C, 5% CO2 incubator until the cells grew to a confluence of 70%-80%.

[0129] Intergenic eccDNA was transfected with PEI alone, and three types of TXLNA eccDNA were mixed in equal moles as one DNA sample for transfection. Three replicate wells of cells were prepared for each DNA sample for transfection, and water was used instead of DNA sample as blank control ( Figure 6 ). 2 μg of each DNA sample was diluted with 50 μL of fetal bovine serum-free basal medium and mixed thoroughly to form a DNA diluent; 3 μL of PEI 40000 transfection reagent was immediately added to the 50 μL DNA diluent and gently mixed; incubated at room temperature for 15 min to form a DNA-PEI cationic nucleic acid transfection reagent complex; during the formation of the complex, the complete medium in the culture well was removed and 2 mL of fresh complete medium preheated at 37°C was added to each well; 50 μL of DNA-PEI nucleic acid-PEI complex was directly added to the cells, the culture plate was shaken and gently mixed; cultured in a 37°C, 5% CO2 incubator for 24 h.

[0130] Basal culture medium: Ham's F-12K GlutaPlus purchased from Sevier Company, supplemented with 1% penicillin-streptomycin.

[0131] Complete medium: refers to Ham's F-12K, GlutaPlus supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0132] 2. Validation of eccDNA enrichment in transfected cells

[0133] First, DNA and RNA were co-extracted from transfected cells using a DNA / RNA co-extraction kit (Tian Gen, #DP422), and eccDNA enrichment and identification was performed using a Roche 480II real-time fluorescence quantitative PCR instrument, the qPCR primers of Example 2, the reaction system of Table 1, and the reaction conditions of Table 2.

[0134] Table 1. qPCR reaction system

[0135] 2×Taq Pro Universal SYBR qPCR Master Mix 5μl F Primer 0.2μl R Primer 0.2μl DNA template 2μl(1ng) Nuclease free water 2.6μl

[0136] Table 2. qPCR reaction conditions

[0137]

[0138] According to the qPCR fluorescence quantitative data analysis, we can know that:

[0139] Compared with the empty control water, the intergenic region eccDNA and TXLNA eccDNA have high eccDNA or eccDNA transcription products in the DNA and RNA of transfected cells ( Figure 7 ).

[0140] Example 4: Functional study of eccDNA in prostate cancer cells

[0141] The transfected cells of Example 3 were subjected to a cell scratch assay and a CCK-8 cell proliferation assay to investigate the effect of eccDNA on the proliferation and migration of prostate cancer cells.

[0142] 1) Cell scratch assay:

[0143] First, draw five evenly divided parallel lines on the bottom of a 6-well plate in a clean bench, and inoculate approximately 6e-5 PEI-transfected cells from Example 3 into a six-well plate with 2 mL of complete culture medium, and culture in a 37°C, 5% CO2 incubator until the confluence is 100%. Then use a sterile ruler to compare the vertical parallel lines, and use a 10 μL sterile gun tip to vertically scratch the cells. After scratching, wash three times with 1ⅹPBS and culture at 37°C in Ham's F-12K without FBS. Take images of the scratched area at 0h and 24h. Use ImageJ software (National Institutes of Health, Bethesda, MD) to analyze and quantify at least 5 random non-overlapping images for each experiment. The results are shown in Figure 2. Figure 8 a, b in Chinese.

[0144] 2) CCK-8 cell proliferation assay:

[0145] Inoculate the suspension of the PEI-transfected cells in Example 2 with complete culture medium in a 96-well plate, 5000 cells per well, about 100 μL, and repeat 5 times. Add 10 μL of CCK-8 solution to each well, gently shake the culture plate to mix, place the culture plate in an incubator at 37°C for 3 hours, and measure the absorbance (OD) at 450nm with an enzyme reader. Perform cell proliferation activity detection according to the instructions of the CCK-8 kit ( Figure 8 (c).

[0146] from Figure 8 The results showed that compared with the transfection water control and intergenic region eccDNA control, transfection of TXLNA eccDNA can significantly inhibit the proliferation and migration of prostate cancer cells PC-3. This indicates that TXLNA eccDNA plays a negative regulatory role in the development of prostate cancer and can be used as a specific molecular marker for prostate cancer.

[0147] The above are merely embodiments of the present invention, which are intended to more clearly illustrate the present invention and are not intended to limit the patent scope of the present invention. Any other different forms of changes or modifications made on the basis of the present invention are within the scope of protection of the present invention.

Claims

1. A prostate cancer-related extrachromosomal circular DNA, characterized in that: The extrachromosomal circular DNA includes TXLNA eccDNA1, TXLNA eccDNA2, and TXLNA eccDNA3 derived from TXLNA protein, and the nucleotide sequences are shown as SEQ ID No.1, SEQ ID No.2, or SEQ ID No.3, respectively.

2. A primer for specifically detecting the extrachromosomal circular DNA according to claim 1, characterized in that: The primers are as follows: TXLNA eccDNA1 F: 5'-CCCAAGCTTCCCACCTCAGCCTCCCAAAGT-3', R: 5'-CCCAAGCTTCATCTGCAGCTGAATGTCATTC-3'; TXLNA eccDNA2 F: 5'-CCCAAGCTTCTGCGAAGAATTTCGAAGTCTG-3', R: 5'-CCCAAGCTTGCTGCCGGGACCTTCTGCTTCTA-3'; TXLNA eccDNA3 F: 5'-CCTTAATTAACTGCTTGCCCTAGTGAGTTACC-3', R: 5'-CCTTAATTAATCCACCTCAGCCTCCCAAAGT-3'.

3. A method for synthesizing the extrachromosomal circular DNA according to claim 1, characterized in that: The method comprises the following steps: S1. Using genomic DNA of embryonic kidney cell line HEK293T as a template, PCR amplification is performed using the primers described in claim 2 to obtain sufficient linear DNA; S2, digesting the amplified fragment in step S1 with the corresponding restriction endonuclease; S3, using T4 DNA ligase to connect the digested fragments in step S2 into a circle; S4. Digest the ligation product of step S3 with DNA exonuclease Plasmid-safe DNase at 37°C for 16 h to obtain the target eccDNAs, namely TXLNA eccDNA1, TXLNA eccDNA2, and TXLNA eccDNA3.

4. The method according to claim 3, characterized in that In step S1, the amplification reaction system is: 2*Taq Plus Master MixII 200 μL, F-Primer 16 μL, R-Primer 16 μL, genomic DNA 500 ng, ddH2O supplemented to 400 μL; The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 20 s, extension at 72°C for 60 s / kb, 35 cycles; extension at 72°C for 5 min; and storage at 4°C.

5. The method according to claim 3, characterized in that In step S2, the enzyme digestion reaction system: 5 μL 10*Reaction buffer, 2 μg amplified fragment, 2 μL restriction endonuclease, and ddH2O to make up to 50 μL.

6. The method according to claim 3, characterized in that In step S2, the restriction endonuclease for TXLNA eccDNA3 is the restriction endonuclease PacI, and the others are the restriction endonucleases HindIII.

7. The method according to claim 3, characterized in that In step S3, the ligation reaction system: 5 μL 10*T4 DNA ligation Buffer, 40 μL restriction fragment, 5 μL T4 DNA ligase.

8. The method according to claim 3, characterized in that In step S4, the digestion reaction system: 31 μL ligation product, 4 μL 10*Reaction buffer, 1.6 μL 25 mM ATP, 2 μL Plasmid-safe DNase, and add ddH2O to 40 μL.

9. Use of the extrachromosomal circular DNA according to claim 1 as a molecular marker for specific diagnosis of prostate cancer.

10. A detection product using the extrachromosomal circular DNA as claimed in claim 1 as a molecular marker for prostate cancer.

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