Prediction marker eccDNAcircATP6V0A1 for cis-platinum drug resistance of hypopharyngeal cancer and application of prediction marker eccDNAcircATP6V0A1

Abstract

The invention discloses an eccDNAcircATP6V0A1 serving as a predictive marker of cis-platinum drug resistance of hypopharyngeal cancer and an application of the eccDNAcircATP6V0A1. The nucleotide sequence of the predictive marker eccDNA is as shown in SEQ ID NO. 3. According to the invention, a hypopharyngeal carcinoma cisplatin drug-resistant cell line model FaDu / DDP is cultured for the first time, meanwhile, a differential eccDNA expression profile and a circRNA expression profile in hypopharyngeal carcinoma cells FaDu and hypopharyngeal carcinoma cisplatin drug-resistant cells FaDu / DDP are disclosed and analyzed in a combined manner, and the eccDNA circATP6V0A1 is further screened and verified as a predictive marker of hypopharyngeal carcinoma cisplatin drug resistance through combined analysis. And a new treatment strategy diagnosis and treatment thought is provided for cis-platinum chemotherapy of hypopharyngeal carcinoma patients clinically.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, and specifically to an eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer. circATP6V0A1 and its applications. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignant tumor in the world. According to the latest GLOBOCAN statistics, there were 890,000 new cases and 450,000 deaths in 2018. It is predicted that the number of global cases will reach 1,080,000 by 2030 (Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018. 68(6): p. 394-424). Hypopharyngeal squamous cell carcinoma (HSCC) is a malignant tumor originating from the hypopharynx, with an incidence rate of about 3%-5% of head and neck squamous cell carcinoma (Cooper, JS, et al., National Cancer Database report on cancer of the head and neck: 10-year update. Head Neck, 2009. 31(6): p. 748-58). Due to the hidden site of onset, the symptoms and signs of hypopharyngeal cancer patients are not obvious, making early diagnosis difficult. Most patients are in the locally advanced stage at the first visit and their pathological differentiation is poor, resulting in a high degree of malignancy. The five-year overall survival rate of patients is only 20%-40% (Newman, JR, et al., Survival trends in hypopharyngeal cancer: a population-based review. Laryngoscope, 2015. 125(3): p. 624-9).Head and neck squamous cell carcinoma is a common tumor, ranking ninth in the incidence of malignant tumors in my country (Lewis-Jones, H., S. Colley, and D. Gibson, Imaging in head and neck cancer: United Kingdom National Multidisciplinary Guidelines. J Laryngol Otol, 2016. 130(S2): p. S28-S31), sixth in incidence and seventh in mortality among men (Pan, R., et al., Cancer incidence and mortality: Acohort study in China, 2008-2013. Int J Cancer, 2017. 141(7): p. 1315-1323). China's socio-demographic index (SDI) is at a medium level globally. For a long time to come, the incidence of malignant tumors, including hypopharyngeal cancer, will continue to rise in my country, and the disease burden it causes to my country deserves attention.

[0003] How to improve the treatment level of hypopharyngeal cancer has always been a hot topic and difficulty in this field. At present, surgical treatment is still the main means of treating hypopharyngeal cancer. With the continuous updating and development of technologies such as radiotherapy, chemotherapy, targeted therapy, and immunotherapy, a large number of studies at home and abroad have shown that non-surgical treatment helps improve the quality of life and survival rate of hypopharyngeal cancer. It can be seen that non-surgical treatment plays an increasingly important role in the treatment of hypopharyngeal cancer. According to the latest version of the 2021 Chinese Society of Clinical Oncology (CSCO) and the 2022 National Comprehensive Cancer Network (NCCN) issued guidelines for the diagnosis and treatment of head and neck tumors, in addition to participating in clinical trials, the treatment strategy for locally advanced hypopharyngeal cancer can also be treated by three methods: 1) According to the response, induction chemotherapy is followed by other treatments; 2) According to the pathological risk characteristics, surgery and postoperative chemoradiotherapy are performed; or 3) synchronous chemoradiotherapy. Among them, the preferred systemic drug is high-dose cisplatin. As for targeted therapy led by cetuximab and immunotherapy represented by pembrolizumab, both domestic and foreign guidelines have included them in the treatment of recurrent / metastatic extremely advanced head and neck squamous cell carcinoma and used in combination with cisplatin in the hope of alleviating the disease or prolonging survival. Only when patients are intolerant to chemotherapy or chemotherapy fails will targeted or immunotherapy alone be recommended. It can be seen that for a long time in the future, chemotherapy based on cisplatin will still be one of the core treatments for hypopharyngeal cancer. However, hypopharyngeal cancer is prone to resistance to platinum-based chemotherapy drugs, which limits its further development in the treatment of hypopharyngeal cancer. Although scholars have done a lot of basic research on the molecular mechanism of chemotherapy resistance in head and neck tumors from different angles, such as DNA / RNA damage repair, drug efflux, cell apoptosis inhibition, cell autophagy, cell pyroptosis, etc. mediated by various coding genes and non-coding genes at the transcriptional level or post-transcriptional level, it still cannot completely solve the problem of acquired resistance in clinical chemotherapy. Therefore, exploring new molecular mechanisms of chemotherapy resistance in hypopharyngeal cancer has important clinical significance for improving the treatment level of hypopharyngeal cancer patients.

[0004] Extrachromosomal circular DNA (eccDNA) is a closed circular DNA that exists outside the chromosome genome of the cell nucleus and is single-stranded or double-stranded. eccDNA is free from the chromosome genome, and the chromatin is highly open, resulting in unrestricted amplification and expression; the circular structure is more stable and not easily degraded; and a certain number of genes can be carried in its own fragments. Based on its unique function and structure, eccDNA has become a hot topic of research in recent years. More and more studies have reported that eccDNAs are widely involved in biological processes. For example, eccDNAs can mediate intercellular signal transduction and heterogeneity (Libuda, DE and F. Winston, Amplification of histone genes by circular chromosome formation in Saccharomyces cerevisiae. Nature, 2006. 443 (7114): p. 1003-7); promote "genetic compensation effect" and are related to aging (Hull, RM and J. Houseley, The adaptive potential of circular DNA accumulation in ageing cells. Curr Genet, 2020. 66 (5): p. 889-894); transcribe to produce non-coding RNA, which acts as a molecular sponge for transcription molecules to indirectly regulate gene expression (Greenwood, SJ, et al., Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis. Nucleic Acids Res, 2001.29(10):p.2191-8).At the same time, eccDNAs also play an important role in the pathogenesis of malignant tumors, including acting as a carrier of oncogene amplification and playing a role in tumor heterogeneity (Kim, H., et al., Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers. Nat Genet, 2020. 52(9): p. 891-897); making cancer cells resistant by amplifying drug-resistant genes (Curt, GA, et al., Unstable methotrexate resistance in human small-cell carcinoma associated with double minute chromosomes. N Engl J Med, 1983. 308(4): p. 199-202); the abnormal expression of eccDNA in the peripheral blood of cancer patients makes it a promising biomarker (Cheng, C., et al., Quantification of circulating cell-free DNA in the plasma of cancer patients during radiation therapy. Cancer Sci, 2009. 100(2): p. 303-9). Therefore, in-depth research on eccDNA can improve the understanding of the pathogenesis and treatment strategies of malignant tumors and provide new ideas for clinical treatment of cisplatin resistance in hypopharyngeal cancer. Summary of the invention

[0005] At present, there are few studies on molecular biomarkers for chemotherapy resistance in head and neck tumors, and most of them are limited to the research of microRNA, lncRNA, tRNA molecules, etc. The stability of the above molecular types is relatively poor, the expression abundance is not high and they are easily degraded. There is an urgent need for a biomarker with constant expression and stable molecular structure to be applied in clinical practice to predict and evaluate the chemotherapy efficacy of patients with head and neck tumors including hypopharyngeal cancer. The present invention provides an eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer circATP6V0A1 And its application, eccDNA as a molecular marker can predict the efficacy of cisplatin chemotherapy in patients with hypopharyngeal cancer.

[0006] In view of the fact that there is no mature and stable cisplatin-resistant cell line model for hypopharyngeal cancer in China and abroad, the present invention first obtains the hypopharyngeal cancer cell line FaDu for culture, performs STR cell identification on it and the American ATCC cell bank, and then performs gradient induction culture of the chemotherapy drug cisplatin. After repeated culture and screening for up to 6 months, the cisplatin-resistant cell line FaDu / DDP is finally cultivated, and STR cell identification is performed again. At the same time, the resistance of FaDu / DDP cells to the chemotherapy drug cisplatin is verified by CCK8 experiment, and the drug resistance index is confirmed to be greater than 2.

[0007] Two cell lines, FaDu and FaDu / DDP, were used as research objects. We first digested traditional linear DNA and mitochondrial DNA, performed eccDNA rolling circle amplification, and further enriched eccDNA. Then, we sequenced and analyzed the eccDNA. At the same time, we sequenced and analyzed circRNA. The two sequencing results were jointly analyzed according to the sequence information, and 10 eccDNA carrying circRNA (hsa_circ_0043837, hsa_circ_0002632, hsa_circ_0042493, hsa_circ_0022007, hsa_circ_0109315, hsa_circ_0116611, hsa_circ_0042881, hsa_circ_0136407, hsa_circ_0007892, hsa_circ_0008419) were screened out as candidate molecular markers for predicting cisplatin chemotherapy resistance.

[0008] Select eccDNA circATP6V0A1 We used hsa_circ_0043837(circATP6V0A1) to obtain its chromosomal location and gene sequence information, designed and synthesized relevant primers, and used qRT-PCR technology to verify its differential expression. At the same time, we constructed siRNA targeting hsa_circ_0043837(circATP6V0A1), and used cell transfection to down-regulate the expression of hsa_circ_0043837(circATP6V0A1) in cisplatin-resistant cells FaDu / DDP. Then, the CCK8 experiment found that it can partially reverse cisplatin resistance, thereby further clarifying its potential as a molecular marker of chemotherapy resistance.

[0009] The present invention provides an eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer, and the nucleotide sequence of the predictive marker eccDNA is shown in SEQ ID NO.3.

[0010] In some embodiments of the present invention, the predictive marker eccDNA is an extrachromosomal circular DNA produced by the genomic fragment chr17:40650941-40653322; wherein: the DNA sequence of the genomic fragment chr17:40650941-40653322 is shown in SEQ ID NO.1.

[0011] In some embodiments of the present invention, the RNA sequence of the genomic fragment chr17:40650941-40653322 is shown as SEQ ID NO.2.

[0012] The present invention also provides an application of the predictive marker eeccDNA in the preparation of a product for diagnosing and / or treating cisplatin resistance in hypopharyngeal cancer.

[0013] In some embodiments of the present invention, the product includes a reagent, a kit and / or a chip.

[0014] The present invention also provides a hypopharyngeal cancer cisplatin resistance diagnosis or treatment chip, the chip includes a solid phase carrier, and an oligonucleotide probe fixed on the solid phase carrier; the oligonucleotide probe includes eccDNA specifically corresponding to the prediction marker.

[0015] The present invention also provides a diagnostic kit for cisplatin resistance in hypopharyngeal cancer, comprising a reagent for detecting the expression level of the predictive marker eccDNA in a subject's sample; if the eccDNA expression level in the subject's sample is significantly increased compared with the predictive marker eccDNA expression level in a normal sample, it is judged that the subject has developed cisplatin resistance.

[0016] The invention also provides a cisplatin-resistant cell line FaDu / DDP, whose preservation number is GDMCC NO:65401.

[0017] The present invention induces the establishment of a cisplatin-resistant cell line FaDu / DDP of hypopharyngeal cancer FaDu cells. The hypopharyngeal cancer cisplatin-resistant cell line is the only known stable resistant cell line in China and internationally.

[0018] This paper first used FaDu and FaDu / DDP cell lines as research objects, digested traditional linear DNA and mitochondrial DNA, performed eccDNA rolling circle amplification, and further enriched eccDNA ， Then eccDNA sequencing and analysis were performed.

[0019] The present invention is the first to use FaDu and FaDu / DDP cell lines as research objects to perform circRNA sequencing analysis.

[0020] In the present invention, circRNA is a relatively stable RNA, and eccDNA is a circular stable DNA. Through the combined analysis of eccDNA sequencing and circRNA sequencing, mature and stable eccDNA carrying circRNA can be screened out. circATP6V0A1 , as a tissue and / or blood molecular marker for cisplatin resistance in hypopharyngeal cancer. circATP6V0A1 As a molecular marker intended to be applied in the field of head and neck malignant tumors or other malignant tumors, the present invention has substantial innovation and clinical transformation value.

[0021] The reagents and raw materials used in the present invention are commercially available.

[0022] The positive and progressive effects of the present invention are:

[0023] The present invention cultivates the hypopharyngeal cancer cisplatin-resistant cell line model FaDu / DDP for the first time, and reveals the differential eccDNA expression profiles and circRNA expression profiles in hypopharyngeal cancer cells FaDu and hypopharyngeal cancer cisplatin-resistant cells FaDu / DDP by joint analysis. Through joint analysis, eccDNAcircATP6V0A1 is further screened and verified as a predictive marker for cisplatin resistance in hypopharyngeal cancer, providing a new treatment strategy diagnosis and treatment idea for cisplatin chemotherapy in clinical patients with hypopharyngeal cancer.

[0024] Biomaterial Deposit Information

[0025] The FaDu / DDP cell line of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on November 1, 2024, with a storage address of 510070, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with a storage number of GDMCC NO: 65401, and a culture name of FaDu / DDP cisplatin-resistant human laryngeal squamous cell carcinoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphology of the parental cells of hypopharyngeal cancer FaDu and the cisplatin-resistant cells of hypopharyngeal cancer FaDu / DDP; Figure 1 A: Morphology of hypopharyngeal cancer parental cells FaDu (100 times); B: Morphology of induced hypopharyngeal cancer cisplatin-resistant cells FaDu / DDP.

[0027] Figure 2 The drug resistance CCK8 test of FaDu / DDP cells constructed had a drug resistance index of 3.1 (greater than 2).

[0028] Figure 3 This is the heat map of eccDNA sequencing of constructed FaDu / DDP cells.

[0029] Figure 4A This is the STR cell identification report for FaDu parental cells.

[0030] Figure 4B This is the constructed STR cell identification report for FaDu / DDP cisplatin-resistant cells (consistent with the FaDu cell gene locus information in the American ATCC cell bank).

[0031] Figure 5 It is a component of Plasmid-Safe NDase reaction solution.

[0032] Figure 6 It is a component of the enzyme digestion reaction solution.

[0033] Figure 7 It is a qPCR reaction system.

[0034] Figure 8 Flow chart of eccDNA data analysis.

[0035] Fig. 9 This is a statistical bar graph of the number of eccDNAs after eccDNA sequencing of FaDu cells and constructed FaDu / DDP cells, as well as the number of coding genes carried by eccDNA.

[0036] Fig.10 Schematic diagram of the combination of genes and eccDNAs types; A: Schematic diagram of the same gene coming from eccDNAs with different sequences and different exon combinations; B: In the eccDNA sequencing of FaDu / DDP cells, 3779 genes came from 1 eccDNA, 833 genes came from 2 eccDNAs, and so on, there was 1 gene from 8 different eccDNAs; C: In the eccDNA sequencing of FaDu cells, 4678 genes came from 1 eccDNA, 1050 genes came from 2 eccDNAs, and so on.

[0037] Fig.11 Schematic diagram of the combination of eccDNAs and gene types; A: Schematic diagram of different types of eccDNA carrying multiple genes; B: In the eccDNA sequencing of FaDu / DDP cells, 2366 types of eccDNAs were "empty circles", that is, they did not carry any meaningful gene sequences, 1958 types of eccDNAs carried 1 gene, and so on, 4 types of eccDNAs carried 8 different genes; C: In the eccDNA sequencing of FaDu cells, 2886 types of eccDNAs were "empty circles", 2418 types of eccDNAs carried 1 gene, 103 types of eccDNAs carried 2 genes, and so on.

[0038] Fig.12Distribution diagram of eccDNA sequence length in eccDNA sequencing; wherein: A: distribution diagram of eccDNA sequence length in FaDu / DDP cell eccDNA sequencing; B: distribution diagram of eccDNA sequence length in FaDu cell eccDNA sequencing.

[0039] Fig.13 This is a heat map of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0040] Fig.14 This is a scatter plot of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0041] Fig.15 This is a volcano map of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0042] Fig.16 The figure is a bar graph showing the statistical numbers of upregulated and downregulated circRNAs in FaDu / DDP cells compared with FaDu cells.

[0043] Fig.17 This is the Venn diagram of eccDNA sequencing data and circRNA sequencing data.

[0044] Fig.18 The differential expression of hsa_circ_0043837 in circRNA sequencing data (left figure) and qRT-PCR validation of its differential expression in FaDu parental cells and FaDu / DDP-resistant cells (right figure) (parental cell data were normalized to 1); **P<0.01.

[0045] Fig.19 Schematic diagram of hsa_circ_0043837 looping, chromosome location, and cross-junction sequence.

[0046] Fig. 20 This is a schematic diagram of the eccDNA [chr17circle 40610146-40675630] circularization, chromosome location, and exon information.

[0047] Fig.21 PCR validation of siRNA down-regulating the expression of circATP6V0A1 in FaDu / DDP-resistant cells; ***P<0.001.

[0048] Fig. 22 The CCK8 experiment found that downregulating the expression of circATP6V0A1 can partially reverse the cisplatin resistance of FaDu / DDP cells.

[0049] Fig.23 Overexpression level of circATP6V0A1 was verified by PCR in FaDu parental cells; **P<0.01.

[0050] Fig.24 The CCK8 experiment found that upregulating the expression of circATP6V0A1 can induce the cisplatin-resistant phenotype of FaDu cells. DETAILED DESCRIPTION

[0051] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0052] Example 1 STR identification of FaDu cells and establishment of cisplatin-resistant FaDu / DDP cell line

[0053] First, the STR identification of FaDu cells was performed and compared with the cell library data of the ATCC cell bank in the United States, proving that the FaDu cell species used in the present invention is completely normal (see Figure 4A Then, the FaDu cells were repeatedly stimulated with increasing concentrations of cisplatin. After half a year of induction and culture, a FaDu / DDP cell model with a stable cisplatin-resistant phenotype was finally cultivated. The FaDu / DDP cells were then subjected to STR identification again. The results showed that the key sequence site information of the FaDu / DDP cells was completely consistent with the FaDu in the ATCC cell bank in the United States (see for details). Figure 4B Sequencing identification report in ).

[0054] The induction process of FaDu / DDP cells with cisplatin-resistant phenotype is as follows:

[0055] (1) FaDu cell culture method was the same as above. When inducing the first generation of FaDu / DDP-resistant cells, cisplatin with a final concentration of 1.5 μM was added to the culture medium to stimulate the cells for 24 hours. After 24 hours, a large number of cells died, and the medium was changed to discard the dead cells. The remaining cells were cultured to more than 50% confluence for the second induction. The final concentration of cisplatin was still 1.5 μM, and the remaining cells continued to be cultured;

[0056] (2) When the cells were induced for the 3rd to 7th time, the final concentration of cisplatin was increased to 3.0 μM, and the rest of the cell treatments were the same as described above;

[0057] (3) The cells after each induction were subjected to a drug-resistant colony formation assay to detect drug resistance. The method is described in the following section. Since the fourth induction, the FaDu cells had a drug resistance index (RI, calculation method see below) of more than 2, which was considered to have a certain degree of drug resistance and was named FaDu / DDP.

[0058] (4) In the interval between each generation of induction, when culturing the induced drug-resistant cells, a low concentration of 0.3uM cisplatin is added to the culture medium to stimulate the cells to maintain drug resistance; each time the drug-resistant cells are used for qRT-PCR, Western Blot, colony formation experiments, etc., the culture medium without cisplatin needs to be replaced for 2 weeks of culture;

[0059] (5) Drug-resistant colony formation assay:

[0060] ① Digest the cells in the drug-resistant group and the control group with trypsin for 3-5 minutes until the cells are suspended, add 2-3 ml of culture medium to terminate the digestion, count the cells under a microscope using a counting plate, and dilute the cells with complete culture medium to a final concentration of 1×10 3 Cell suspension of 100 cells / ml;

[0061] ②Inoculate the two diluted groups of cells into a 24-well plate, add 1 ml of cell suspension to each well, and add 12 wells to each group of cells;

[0062] ③ 24 hours after inoculation, cells were observed to adhere to the wall, and the culture medium in the wells was replaced with a culture medium containing cisplatin. The final concentration of cisplatin in each of the three wells of the two groups was the same, and a total of three concentration gradients (including 0 nM) were set up;

[0063] ④ After 24 hours of cisplatin stimulation, the cells were washed twice with PBS and replaced with complete medium without cisplatin and cultured for more than one week until the colonies reached the appropriate size;

[0064] ⑤ Wash the cells once with PBS, add an appropriate amount of methanol to each well to fix the cells for about 5 minutes, then discard the methanol, add enough crystal violet stain to cover the bottom surface of the well, let it sit for about 15 minutes, discard the stain, and use PBS to wash the residual stain;

[0065] ⑥ Use ImageJ to count the colonies in each well, calculate the cell inhibition ratio at different concentrations according to the number of colonies, calculate the half maximal inhibitory concentration (IC50) of cells to cisplatin by GraphPad Prism 8, and calculate the resistance index (RI) according to the following formula:

[0066] RI = IC50 drug resistance / IC50 control. If the colonies are too fused, the total area of ​​the colonies is used instead of the number of colonies for calculation.

[0067] Figure 1 The morphology of the parental cell FaDu of hypopharyngeal cancer and the cisplatin-resistant cell FaDu / DDP of hypopharyngeal cancer are shown in Figure 1. Figure 1A: Morphology of hypopharyngeal carcinoma parental cell FaDu (100 times); Figure 1 B: Morphology of induced hypopharyngeal cancer cisplatin-resistant cells FaDu / DDP. Figure 1 It can be seen that FaDu / DDP-resistant cells have become slender in morphology and have more pseudopodia compared with FaDu parental cells, which is a typical cell morphology after epithelial-mesenchymal transition, indicating that while the FaDu / DDP cell line has become resistant to cisplatin, its invasion and metastasis capabilities are also enhanced compared with before.

[0068] Figure 2 The drug resistance of the constructed FaDu / DDP cells was verified by CCK8 assay, and the drug resistance index was 3.1 (greater than 2).

[0069] Figure 4 is the STR cell identification report of FaDu parental cells and FaDu / DDP cisplatin-resistant cells (consistent with the gene locus information of FaDu cells in the American ATCC cell bank).

[0070] Example 2 Analysis of differential expression profiles of circRNAs in FaDu and FaDu / DDP cell lines

[0071] The present invention uses the FaDu cell line and the FaDu / DDP cell line in Example 1 as research objects to perform circRNA sequencing.

[0072] The circRNA sequencing process is as follows:

[0073] 1. Sequencing library construction

[0074] (1) Removal of ribosomal RNA from total RNA;

[0075] (2) using ribonuclease R to break the RNA into fragments of 250 bp to 300 bp;

[0076] (3) Using RNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized under the action of reverse transcriptase;

[0077] (4) After degrading the RNA strand, the second strand of cDNA is synthesized using the first strand of cDNA as a template and purified;

[0078] (5) The double-stranded cDNA is end-repaired, A-tailed, and connected to a sequencing adapter, and then the cDNA of 350 bp to 400 bp is screened using magnetic beads;

[0079] (6) Degrade the second strand of cDNA and obtain the library after PCR amplification.

[0080] 2. Transcriptome Sequencing Library Quality Assessment

[0081] (1) filtering the raw reads obtained by sequencing to obtain valid reads;

[0082] (2) Use Hisat2 software to align valid data to the reference genome. Count the valid reads that have been aligned successfully, and further analyze statistical values ​​such as alignment efficiency. The aligned reads are called aligned reads. The quality of the transcriptome library is evaluated by the base sequencing error rate and the percentage of bases with Phred values ​​greater than 20 and 30 in the total bases.

[0083] 3. Transcript splicing

[0084] Stringtie software was used to assemble the aligned reads into transcripts and perform quantitative analysis. Stringtie-merge was then used to merge the assembled results and add annotation information of the reference genome.

[0085] 4. Construction of CircRNA Expression Profile

[0086] Find-circ software and CIRI software were used to identify Junction reads in Mapping and SAM files that were not aligned to the reference genome, respectively, to preliminarily identify candidate circRNAs. The results of the two software were then analyzed jointly to improve the accuracy of circRNA identification. For the identified circRNAs, TPM was used to normalize the expression levels to eliminate the effects of sequence length and sequencing depth on the gene expression levels of individual samples.

[0087] 5. Screening of differentially expressed circRNAs

[0088] (1) Filter low-abundance circRNAs to avoid bias. In this paper, we define circRNAs with less than 10 valid reads in more than half of the samples as low-abundance expressed circRNAs;

[0089] (2) Differential expression analysis was performed using the DESeq2 R package, and circRNAs with P < 0.05 and absolute fold change > 2 were defined as differentially expressed circRNAs.

[0090] Fig.13 This is a heat map of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0091] Fig.14 This is a scatter plot of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0092] Fig.15 This is a volcano map of circRNA sequencing using FaDu cells and FaDu / DDP cells as research objects.

[0093] Fig.16The figure is a bar graph showing the statistical numbers of upregulated and downregulated circRNAs in FaDu / DDP cells compared with FaDu cells.

[0094] The results showed that 96 circRNAs were expressed at higher levels in FaDu / DDP cisplatin-resistant cell lines than in FaDu parental cells, and 121 circRNAs were expressed at lower levels in FaDu / DDP cisplatin-resistant cell lines than in FaDu parental cells. The above results showed that there were significant differences in the expression profiles of non-coding circRNAs between the two cell lines. Therefore, further comprehensive analysis of the above sequencing results is needed to narrow the scope of the research in order to find predictive molecules or therapeutic targets for cisplatin resistance in hypopharyngeal cancer.

[0095] Example 3 FaDu and FaDu / DDP cell lines digest traditional linear DNA and mitochondrial DNA, perform eccDNA rolling circle amplification, further enrich eccDNA, and then perform eccDNA sequencing and analysis

[0096] 1. The present invention uses the FaDu cell line and the FaDu / DDP cell line in Example 1 as research objects, and first removes mitochondrial DNA and linear DNA:

[0097] (1) Take 5 μg of purified DNA from each sample (cultured cell sample) and put it into a 1.5 mL centrifuge tube. In order to promote the specific digestion of linear DNA by nuclease exonuclease, the purified DNA was treated with restriction endonuclease PacI (R0547S, New England Biolabs) and supplemented with sterile water to a total volume of 25 μL. Incubate at 37°C for 16 h to digest mitochondrial DNA.

[0098] (2) Heat inactivate the endonuclease PacI at 70°C for 30 min.

[0099] (3) Prepare the Plasmid-Safe DNase reaction system (see Figure 5 ) to remove the remaining linear DNA.

[0100] (4) The digestion reaction solution was incubated at 37°C for 7 days, during which fresh digestion reaction solution was added once a day until the linear DNA was completely digested. The components of the digestion reaction solution are shown in Figure 6 .

[0101] (5) Quantitative real-time PCR (qPCR) was performed on the linear gene COX5B to detect the removal effect of linear DNA.

[0102] a. Amplification primers: COX5B, forward primer 5'-GGGCACCATTTTCCTTGATCAT-3'( SEQ ID NO.4 ), the reverse primer was 5'-AGTCGCCTGCTCTTCATCAG-3' (SEQ ID NO.5) , and the final target gene fragment size was 119 bp.

[0103] b. qPCR reaction system preparation see Figure 7 .

[0104] c. The reaction conditions are as follows: 95°C for 5 min; 95°C for 10 s, 58°C for 20 s, 72°C for 20 s, and cycle 40 times.

[0105] d. After amplification, the product was subjected to 1% agarose gel electrophoresis to detect the size of the target band.

[0106] (6) The exonuclease was inactivated by heating at 70°C for 30 min.

[0107] 2.eccDNA rolling circle amplification:

[0108] (1) Perform eccDNA rolling circle amplification according to the instructions of REPLI-g Mini Kit (150025, Qiagen). Prepare sufficient amount of denaturation buffer (Buffer D1) and neutralization buffer.

[0109] (2) Use 5 μL of enriched and purified eccDNA sample as template, add 5 μL of denaturation buffer, mix well and let stand at room temperature for 3 min. Add 10 μL of neutralization buffer and mix well on an oscillator.

[0110] (3) Add 29 μl REPLI-g Mini reaction buffer and 1 μl REPLI-g Mini DNA polymerase and incubate at 30°C for 16 h.

[0111] (4) Heat REPLI-g Mini DNA polymerase at 65°C for 3 min to inactivate the enzyme.

[0112] (RCA was performed using phi29 DNA polymerase in 20 uL reactions at 30°C for 14 hours, followed by inactivation at 65°C for 10 minutes.)

[0113] (5) The amplified eccDNA was added to AMPure XP beads (Beckman, A63880) at a ratio of 1:1.8 and incubated at room temperature for 5 min. The PCR tube was placed on the Agencourt SPRIPlate 96R circular magnetic plate. Under the action of the magnetic field, the magnetic beads were separated from the solution. When the solvent was clear, the liquid was aspirated with a sterile pipette.

[0114] (6) Wash the magnetic beads twice with 70% ethanol, aspirate the ethanol, remove the PCR tube from the magnetic plate, add 40 μL of elution buffer, mix carefully, and incubate at room temperature for 4 minutes.

[0115] (7) Place the PCR tube back on the magnetic plate and carefully collect the eluted eccDNA.

[0116] (8) Take 1 μL of the reaction solution and measure the DNA concentration using the Qubit dsDNA HS Assay kit.

[0117] 3. Finally, eccDNA sequencing was performed on the machine. The raw sequence data was generated by IlluminaNovaSeq 6000. The raw reads were fine-tuned using fastp software (V0.23.2). The trimmed reads were aligned with the human genome (UCSC HG38) using BWA software (V0.7.17). Circle Map V1.1.4 software was used to detect and screen eccDNA from the sequencing data. The eccDNA data analysis flow chart is shown in Figure 8 .

[0118] 4. The eccDNA sequencing results showed that there were significant differences in the expression profile of eccDNA between the two cell lines, including the size, number, linear chromosome location of eccDNA, and the number and type of genes that may be amplified by eccDNA (see Fig. 9 , Fig.10 , Fig.11 , Fig.12 The above results suggest that eccDNA plays an important role in cisplatin resistance in hypopharyngeal carcinoma and deserves further study.

[0119] Figure 3 This is a heat map of eccDNA sequencing.

[0120] Fig. 9 This is a statistical bar graph of the number of eccDNAs after eccDNA sequencing of FaDu cells and constructed FaDu / DDP cells, as well as the number of coding genes carried by eccDNA.

[0121] Fig.10 Schematic diagram of the combination of genes and eccDNAs types; wherein:

[0122] A: Schematic diagram of the same gene coming from eccDNAs with different sequences and different exon combinations; B: In the eccDNA sequencing of FaDu / DDP cells, 3779 genes came from 1 eccDNA, 833 genes came from 2 eccDNAs, and so on, there was 1 gene coming from 8 different eccDNAs; C: In the eccDNA sequencing of FaDu cells, 4678 genes came from 1 eccDNA, 1050 genes came from 2 eccDNAs, and so on.

[0123] Fig.11 Schematic diagram of the combination of eccDNAs and gene types; wherein:

[0124] A: Schematic diagram of different types of eccDNA carrying multiple genes; B: In the eccDNA sequencing of FaDu / DDP cells, 2366 types of eccDNAs were "empty circles", that is, they did not carry any meaningful gene sequences, 1958 types of eccDNAs carried 1 gene, and so on, 4 types of eccDNAs carried 8 different genes; C: In the eccDNA sequencing of FaDu cells, 2886 types of eccDNAs were "empty circles", 2418 types of eccDNAs carried 1 gene, 103 types of eccDNAs carried 2 genes, and so on.

[0125] Fig.12 Distribution diagram of eccDNA sequence length in eccDNA sequencing; wherein: A: distribution diagram of eccDNA sequence length in FaDu / DDP cell eccDNA sequencing; B: distribution diagram of eccDNA sequence length in FaDu cell eccDNA sequencing.

[0126] Example 4 Combined analysis of eccDNA differential expression profile and circRNA differential expression profile

[0127] The present invention takes the intersection of eccDNA sequencing data and circRNA sequencing data, and screens out 10 candidate circRNAs with the same gene sequences in the two databases and upregulated expression in cisplatin-resistant cell lines.

[0128] Fig.17 The figure shows the Venn diagram of eccDNA sequencing data and circRNA sequencing data. The two data sets were intersected to screen out 10 candidate circRNAs with upregulated expression in drug-resistant cells and the same gene sequences, among which hsa_circ_0043837 (circATP6V0A1) had the most significant difference in the sequencing data.

[0129] Example 5 Chromosomal localization of circATP6V0A1 (hsa_circ_0043837) and acquisition of gene sequence information

[0130] Fig.18 The differential expression of hsa_circ_0043837 in circRNA sequencing data (left figure) and qRT-PCR validation of its differential expression in FaDu parental cells and FaDu / DDP-resistant cells (right figure) (parental cell data were normalized to 1); **P<0.01.

[0131] Fig.19 The following is a schematic diagram of the hsa_circ_0043837 loop, chromosome location, and cross-junction sequence. The following SEQ ID NO.1 corresponds to the chromosome gene sequence of circATP6V0A1 (hsa_circ_0043837).

[0132] Genomic location:chr17:40650941-40653322

[0133] Genomic length: 2381bp

[0134] >hg19_dnarange=chr17:40650941-406533225'pad=03'pad=0strand=+

[0135] repeatMasking=none

[0136] SEQ ID NO.1

[0137]

[0138] Example 6 Acquisition of circATP6V0A1-RNA sequence information

[0139] hsa_circ_0043837(circATP6V0A1)

[0140] RNA sequence:

[0141] SEQ ID NO.2

[0142] ATTTGGAACATTGCTACCAATAAACTGACGTTTCTGAACTCCTTTAAGATGAAGATGTCTGTTATCCTTGGTATCATCCATATGCTGTTTGGAGTCAGCCTGAGTCTGTTCAACCATATCTATTTCAAGAAGCCCCTGAATATCTACTTTGGATTTATTCCTGAAATAATCTTCATGACCTCTTTGTTTTGGCTATTTGGTTATCCTATTTTTTTACAAGTGG ACGGCCTATGATGCTCATACCTCTGAGAATGCACCAAGCTTCTGATCCATTTCATAAACATGTTCCTCTTTTCCTACCCAGAGTCTGGTTATTCAATGTTGTATTCTGGACAGAAAGGAATTCAGTGTTTCCTGGTAGTGGTTGCACTACTGTGTGTACCTTGGATGCTGCTGTTTAAACCATTGGTCCTTCGCCGTCAGTATTTGAGGAGAAAGCATTTG

[0143] Example 7 eccDNA circATP6V0A1 Chromosome location and sequence information (first and last 500bp)

[0144] Fig. 20 This is a schematic diagram of eccDNA [chr17circle 40610146-40675630] circularization, chromosome location, and exon information.

[0145] >GRCh37 / hg19_dna range=chr17:40610146-406756305'pad=03'pad=0

[0146] strand=+repeatMasking=none

[0147] SEQ ID NO.3

[0148] CGTGGTGCGTGCCTGTAATCTCAGCTACTAGGGAGGCTGAGGCAGGAGAATCCCCTGAACCCGGGAGGCGGAGGCTGCAGTGAGCCGAGAACGCGCCACTGCATTCCCGCCTGGGAAACAGACCATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCCGGGCGTGGTGGCTCACGCTTGTAATCCCAACACTTTGGGAGGCCGAGGCGGGTGGATCACGAGGTCAGAATTCGAGACTCAGCCTGGCCAACATAGTGAAACCCCGTCTCTACTTAAAAAAAAAAAAAATACAAAAATTAGCCGGGCATGGTGGTGCGTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGTGGAGGTTGCAGTGGGCCGAGATCACGCCACTGCACTCCTGCTTGAGCAACAGAATGAGACTTCAAAAAAAAAAAAAGAAAGAGAAAAGACAACCTACCCCAGTATAAGGGGTAAGAAAAAGGCTAGTGAGGCACTGCATTCTCGGTACCCTCCCTCTTCACTCCTCCCTCCAGGGGCCTCCAGGGCAGGGGCTGCCCAGGAATGTGACCTGGGCCCCAGACAGGTGAAGGGGCCACAACCCCCTGTTCATTCCATGTTCCAGCCTAACCTAAAGCCCTTCCTCCTCCCTAACAGTGCTACCTGTCCCTGGTGTCGGCCCTGAGAGGCCCACCCCACCCAGCTCCTCAAAGATAAGGTCCCGAGTGGAAACTCTGGTCATTGAGGTCTCAGCTTCTGCTCAGTGGGAAGGGAAGGCCTCATAAGAATAGATATTTGGAATCATTGGATTCGAATTGAGAGTCCAGGAATAAATCCTAACATTTATGGTCACTTGATTTTTGATAAAGGGGTCAAGATAATTCAATGAGGAAAGATCTCTCTTTTTCAACAAATGACACTGGTACAATGGGGTTTCCACATGCAAAGAAACAAATTTGGGCCACTATCC

[0149] Example 8 Verification of ccDNA differential expression profile and circRNA differential expression profile

[0150] Fig.21 PCR validation of siRNA down-regulating the expression of circATP6V0A1 in FaDu / DDP-resistant cells; ***P<0.001.

[0151] Fig. 22 The CCK8 experiment found that downregulating the expression of circATP6V0A1 can partially reverse the cisplatin resistance of FaDu / DDP cells.

[0152] Fig.23 Overexpression level of circATP6V0A1 was verified by PCR in FaDu parental cells; **P<0.01.

[0153] Fig.24 The CCK8 experiment found that upregulating the expression of circATP6V0A1 can induce the cisplatin-resistant phenotype of FaDu cells.

[0154] FaDu and FaDu / DDP in Example 1 were used as research objects, and qRT-PCR was used to verify the differential expression levels of candidate circRNAs in the two cell lines. It was found that the expression level of hsa_circ_0043837 (circATP6V0A1) was the highest in drug-resistant cells. Subsequently, we transfected siRNA into the drug-resistant cells FaDu / DDP to downregulate the expression of circATP6V0A1 and found that its cisplatin resistance could be partially reversed.

[0155] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. An eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer, the nucleotide sequence of the predictive marker eccDNA is shown in SEQ ID NO.

3.

2. The eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer according to claim 1, characterized in that The prediction marker eccDNA is an extrachromosomal circular DNA produced by the genomic fragment chr17:40650941-40653322; wherein: The DNA sequence of the genomic fragment chr17:40650941-40653322 is shown in SEQ ID NO.

1.

3. The eccDNA as a predictive marker for cisplatin resistance in hypopharyngeal cancer according to claim 2, characterized in that The RNA sequence of the genomic fragment chr17:40650941-40653322 is shown in SEQ ID NO.

2.

4. An application of the predictive marker eeccDNA as described in any one of claims 1 to 3 in the preparation of a product for diagnosing and / or treating cisplatin resistance in hypopharyngeal cancer.

5. A chip for diagnosing or treating cisplatin resistance in hypopharyngeal cancer, characterized in that: The chip includes a solid phase carrier, and an oligonucleotide probe fixed on the solid phase carrier; the oligonucleotide probe includes an eccDNA specifically corresponding to the prediction marker as described in any one of claims 1-3.

6. A diagnostic kit for cisplatin resistance in hypopharyngeal cancer, characterized in that: It includes a reagent for detecting the expression level of the predictive marker eccDNA as described in any one of claims 1 to 3 in a subject sample; if the eccDNA expression level in the subject sample is significantly increased compared with the predictive marker eccDNA expression level in a normal sample, it is judged that the subject has developed cisplatin resistance.

7. A cisplatin-resistant cell line FaDu / DDP, whose accession number is GDMCC NO: 65401.

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