CN114920806B - Polypeptide and antibody thereof, and application of reagent for detecting polypeptide and antibody in preparation of nasopharyngeal carcinoma diagnosis, prognosis or treatment preparation - Google Patents

Abstract

The invention belongs to the technical field of tumor molecular biology, and particularly relates to a polypeptide and an antibody thereof, and application of a reagent for detecting the polypeptide and the antibody in preparation of a nasopharyngeal carcinoma diagnosis, prognosis or treatment preparation. MTT experiment, matrigel invasion experiment and scratch healing experiment are carried out in nasopharyngeal carcinoma cell lines HNE2, CNE2 and HONE1, the proliferation, invasion and migration ability of the cell of the circSTX6-144aa group is obviously enhanced, namely, the overexpression of the circSTX6-144aa protein promotes proliferation, invasion and migration of the nasopharyngeal carcinoma cells, and the polypeptide can be used as a marker for diagnosing and prognosticating the nasopharyngeal carcinoma and a treatment target. In particular, a polyclonal antibody is designed aiming at 15 specific amino acids at the carboxyl terminal of the circSTX6-144aa, so that the expression of the intracellular circSTX6-144aa protein can be specifically detected. The polyclonal antibody has potential application prospect in clinical detection and clinical treatment of nasopharyngeal carcinoma.

Description

Polypeptide and antibody thereof, and application of reagent for detecting polypeptide and antibody in preparation of nasopharyngeal carcinoma diagnosis, prognosis or treatment preparation

Technical Field

The invention belongs to the technical field of tumor molecular biology, and particularly relates to a cyclic RNA circSTX6-144aa coded protein, an antibody thereof and application thereof in preparation of nasopharyngeal carcinoma diagnosis, prognosis or treatment preparations.

Background

Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is a common malignant tumor of head and neck in south China, and has obvious regional aggregation and good human hair development. The pathogenesis of nasopharyngeal carcinoma mainly comprises genetic factors, EB virus infection, environmental factors and the like. Most nasopharyngeal carcinoma is non-keratinized squamous carcinoma, and the malignant degree is high, and cervical lymph node metastasis can occur in early stage. At present, the treatment method of nasopharyngeal carcinoma mainly comprises radiotherapy, combined chemotherapy, operation treatment and the like, and although the total survival rate of early NPC patients is up to 95% in 5 years, the recurrence rate of nasopharyngeal and cervical lymph nodes is 8.6% -23.7%. This is because the occurrence and development of NPC involves complicated gene regulation and multistage processes, the molecular mechanism of which is not yet clear, and the therapeutic effect of conventional radiotherapy is not very ideal in addition to individual differences caused by tumor heterogeneity and resistance to radiotherapy. Therefore, finding a diagnostic, prognostic molecular marker and therapeutic target for nasopharyngeal carcinoma is of great importance.

Circular RNAs (circrnas) are a class of non-coding RNA molecules formed by reverse splicing of precursors (mRNA precursor messenger RNA, pre-mRNA) that do not have a 5 'end cap and a 3' end poly (a) tail and form a Circular lasso structure by covalent bonds. Previous studies have not found the objective existence of the part of circular RNA due to the limitation of the technical level, and along with the development of deep RNA sequencing and large-scale bioinformatics, researchers really find that a great number of cyclized RNA molecules exist in organisms, and the cyclized RNA is very stable in the organisms due to the formation of a closed loop, so that the circular RNA has the potential of being used as a diagnostic marker and a therapeutic target.

The process of circRNA formation can be divided into two major classes, namely exon cyclisation (exon circularization) and intron cyclisation (intron circularization). Jeck et al propose that exon-derived circRNA (exonic circRNA, ecircRNA) can be divided into two formation modes, namely lasso-driven cyclization (larait-driven circularization) and intron pairing-driven cyclization (intron-pairing-driven circularization), wherein lasso-driven cyclization is a mode that the 3 '-end of an exon is taken as a splice donor (splice donor) to attack a 5' -end splice acceptor (splice acceptor), alu regions are covalently combined to form a lasso structure, and the lasso structure is subjected to internal splicing and then introns are excised to form the circRNA; introns pair driven circularization is the cleavage of an intron to form a circRNA after base-pairing of two introns to form a circular structure. In fact, introns themselves may also be cyclized, and intron-derived circRNA (circular intronic RNA, cisRNA) may be formed. circRNA is a class of non-coding RNA molecules formed by reverse splicing of pre-mRNAs that do not have a 5 'terminal cap and a 3' terminal poly (A) tail and form a closed loop structure in the form of a covalent bond. Has the characteristics of high stability, conservation and specificity and high content. The specific function of the circularized RNA is not clear, and only a few presumptions are made that 1) the circular RNA can be used as a sponge for absorbing miRNA to inhibit the function of the RNA; 2) The circRNA directly regulates the level of other RNA through base complementation pairing; 3) The binding of the circRNA to the protein, inhibition of protein activity, recruitment of components of the protein complex or modulation of protein activity 4) the circRNA may also serve as a template for translation to direct protein synthesis.

In recent years, more and more circrnas have been discovered, and the number of circrnas known so far has reached more than thirty thousand. The circRNA is no longer considered as the wrong RNA transcript, but rather rises as glaring-star in non-coding RNA studies. More novel circRNA and the polypeptide coded by the same are found to be used as biomarkers for tumor diagnosis and prognosis and application of the biomarkers, and the novel circRNA and the polypeptide coded by the same can be well protected in the patent field as early as possible, so that the international competitiveness of China in the technical field can be remarkably improved.

A391 bp circular RNA circSTX6 was detected. Experiments show that the protein circSTX6-144aa coded by the ORF of the cross-shearing site of the circular RNA is highly expressed in nasopharyngeal carcinoma and can promote invasion and metastasis of the nasopharyngeal carcinoma, and the circular RNA is likely to be used as a diagnosis, prognosis marker and treatment target of the nasopharyngeal carcinoma.

Disclosure of Invention

The invention discovers a section of open reading frame (Open Reading Frame, ORF) with 435 bases open in a cyclic RNA circSTX6 (circID: hsa_circ_23135, sequence shown in SEQ ID NO. 1), and can code short peptide with 144 amino acids, and the invention is named as: the molecular weight of the small peptide was about 16kDa, circSTX6-144aa (FIG. 1). The circSTX6 ORF has a nucleotide sequence shown in SEQ ID NO. 2; the circSTX6-144aa has an amino acid sequence shown in SEQ ID NO. 3.

The invention can achieve the following purposes:

the first object of the present invention is to provide a polypeptide, the name of which is circSTX6-144aa, and the amino acid sequence is shown as SEQ ID NO. 3.

A second object of the present invention is to provide the use of the above-mentioned polypeptide detection reagent for the preparation of a reagent for diagnosis and/or prognosis of nasopharyngeal carcinoma.

Further, the nasopharyngeal carcinoma diagnosis and/or prognosis reagent includes: an antibody that specifically binds to the polypeptide.

Further, antibodies that specifically bind to the polypeptide include: the resulting antibody was designed for 15 specific amino acids at the carboxy terminus of circSTX6-144aa.

A third object of the present invention is to provide a diagnostic and/or prognostic formulation for nasopharyngeal carcinoma, said formulation comprising a reagent for detecting the polypeptide circSTX6-144 aa; the amino acid sequence of the circSTX6-144aa is shown as SEQ ID NO. 3.

Further, the reagent for detecting the polypeptide circSTX6-144aa contains the above-mentioned antibody specifically binding to the polypeptide.

The fourth object of the invention is to provide the application of the antibody designed for 15 specific amino acids at the carboxyl terminal of the circSTX6-144aa in preparing a preparation for treating nasopharyngeal carcinoma, wherein the amino acid sequence of the circSTX6-144aa is shown as SEQ ID NO. 3.

It is a fifth object of the present invention to provide a formulation for treating nasopharyngeal carcinoma, said formulation comprising an antibody as described above which specifically binds to said polypeptide.

The sixth object of the invention is to provide an application of ASO designed for circSTX6-144aa in preparing a preparation for treating nasopharyngeal carcinoma, wherein the amino acid sequence of circSTX6-144aa is shown as SEQ ID NO. 3.

A seventh object of the present invention is to provide a formulation for treating nasopharyngeal carcinoma, said formulation comprising ASO designed for circSTX6-144 aa; preferably the sequence of ASO is shown in SEQ ID NO. 4.

Sequence of ASO: 5'-CAUAUGACCAGUGGACAUGATT-3'

The formulation may also contain one or more pharmaceutically acceptable excipients or pharmaceutically acceptable carriers.

Through research, circSTX6 was found to be a novel variant of the STX6 gene in a circular pattern, which is a molecule that forms RNA in a circular pattern from the STX6 gene precursor transcript RNA by reverse splicing. Variants of the cyclization pattern are formed by head-to-tail cyclization of the 4, 5, 6, 7 exons of the STX6 gene, consisting of 391 nucleotides and named circSTX6 (FIG. 1). After STX6 forms a circular RNA molecule, a complete open reading frame is formed across the splice site, encoding 144 amino acids, the protein having a molecular weight of about 16kDa (FIGS. 2-4), hereinafter referred to as the peptide fragment of circSTX6-144aa or circSTX6-144aa. The small molecule protein has 129 amino acids homology with STX6 full-length protein, and 15 specific amino acid sequences at the C terminal. Through the research of cell biology functions, the over-expression of the circSTX6-144aa can significantly promote proliferation and invasion migration of nasopharyngeal carcinoma cells (figures 6-8); in contrast, knockdown of circSTX6/circSTX6-144aa produced the opposite effect, inhibiting proliferation, invasion and migration of NPC cells (FIGS. 6-8). The reversion experiments further demonstrated that circSTX6-144aa promoted proliferation, invasion and migration of nasopharyngeal carcinoma cells (fig. 9).

Animal experiments prove that the over-expression of the circSTX6-144aa can obviously promote the nodulation and metastasis capability of nasopharyngeal carcinoma cells (shown in fig. 10-11), and the knockdown of the circSTX6/circSTX6-144aa obviously inhibits the nodulation and metastasis capability of the nasopharyngeal carcinoma cells (shown in fig. 10-11). Immunohistochemical experiments examined the expression of circSTX6-144aa in clinical samples of nasopharyngeal carcinoma, and found that high expression of circSTX6-144aa was associated with a shorter survival of nasopharyngeal carcinoma patients (FIG. 12).

In addition, the 15 specific amino acid micro peptide at the C terminal of the synthetic circSTX6-144aa can enter the nasopharyngeal carcinoma cells and can obviously promote proliferation and migration of the nasopharyngeal carcinoma cells (FIG. 13).

Targeting intracellular fragments is more difficult than targeting extracellular fragments. Fortunately, the inventor finds that the transcripts of the intracellular fragments can form a cyclic molecule, and most importantly, the expression of the circSTX6-144aa polypeptide can be very effectively inhibited by blocking the connection of the interface positions of the transcripts to form the cyclic molecule. Such means have been demonstrated by the practice of the present invention for its feasibility and reliability. By, for example, siRNA, ASO, etc., it is possible to smoothly enter the cell to inhibit the formation of circSTX6. This is a particularly preferred solution.

The circSTXT6 nucleic acid fragment inhibitor is a substance for inhibiting transcription or expression of whole or partial circSTX6 nucleic acid fragment; preferably, the substance is one or more of siRNA, ASO, compound and gene editing tool.

The circSTX6 peptide fragment inhibitor is one or more of substances, siRNA, ASO and gene editing tools for inhibiting the generation or activity of whole or partial circSTX6-144aa protein peptide; preferably, the substance inhibiting the generation or activity of the peptide fragment of circSTX6-144aa, either wholly or locally, is one or more of an antibody fusion protein, a polypeptide and a compound.

Such as ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat technique).

ZFN, TALEN and CRISPR/Cas9 are three major gene editing techniques that combine specific DNA targeted recognition with endonuclease-completed DNA sequence changes using non-homologous end-linked pathway (NHEJ) repair and Homologous Recombination (HR) repair. Thus, common to these three editing tools is: contains a recognition region of a target DNA sequence and a DNA cleavage functional region, wherein the ZFN technology has a zinc finger domain capable of recognizing the target DNA, the DNA recognition region of the TALEN is a repeat of repeated variable double residues, and the DNA cleavage region is an endonuclease domain named Fokl. The DNA recognition region of CRISPR is the crrna or guide RNA, and Cas9 protein is responsible for DNA cleavage. When the DNA binding domain recognizes the target DNA sequence, endonuclease or Cas9 protein cleaves DNA, double strand breaks the target DNA, and then the DNA damage repair mechanism is started, so that gene knockout, insertion and the like are realized.

The aim of treating tumors by gene editing is fulfilled by the circSTX 6; especially nasopharyngeal carcinoma;

in the present invention, the terms "polypeptide", "micro peptide" and "short peptide" are understood to have the same meaning and are used to describe amino acid fragments. In addition to having the above-described SEQ ID NO:3, and the remaining polypeptides of the prior art that routinely modify the sequences of the present invention should also be understood to have the tumor suppressors of the present invention. For example, polypeptide therapeutics may have a short circulation half-life and proteolytic degradation and low solubility. To improve the pharmacokinetic and pharmacodynamic properties of the biopharmaceuticals of the present invention, methods such as manipulation of amino acid sequences to reduce or increase immunogenicity and reduce proteolytic cleavage may be performed; peptides may be fused or coupled to immunoglobulins and serum proteins such as albumin; may also be incorporated into drug delivery vehicles for biopharmaceuticals (such as the peptides of the invention) and antibodies to protect and slow release; and coupling to natural or synthetic polymers is also contemplated. Specifically, for synthetic polymer coupling, pegylation or acylation, such as N-acylation, S-acylation, amidation, and the like, is also contemplated.

As used herein, "pharmaceutically acceptable excipient or pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. The use thereof in therapeutic compositions is contemplated unless any conventional medium or agent is incompatible with the active ingredient. Supplementary active ingredients may also be incorporated into the composition. The use of such media and agents for pharmaceutically active substances is well known in the art. The use thereof in therapeutic compositions is contemplated unless any conventional medium or agent is incompatible with the active ingredient. Supplementary active ingredients may also be incorporated into the pharmaceutical compositions of the present invention.

The pharmaceutical compounds may be administered in a convenient manner, for example by oral, intravenous (in the case of water solubility), intramuscular, subcutaneous, intraperitoneal, nasal, intradermal or suppository routes or implantation (e.g., using slow release molecules through the intraperitoneal route or through the use of cells or lentiviruses and subsequent transfer to the recipient. Depending on the route of administration, it may be desirable to encapsulate the active ingredient in a material to protect it from enzymes, acids and other natural conditions that may inactivate the ingredient.

The invention has the beneficial effects that:

1. the invention discovers STX6 variant in nasopharyngeal carcinoma for the first time. The variant is a molecule that forms a circular pattern of RNA from STX6 gene precursor transcript RNA by reverse splicing: circSTX6. The circSTX6 translates into a small molecule protein with a molecular weight of 16 kDa: circSTX6-144aa. The circSTX6-144aa can be used as a new treatment target point of nasopharyngeal carcinoma and can also be used as a marker for diagnosing/screening/prognosis of nasopharyngeal carcinoma.

2. Experiments prove that the proliferation and invasion and metastasis of tumors can be obviously promoted through over-expression of circSTX6-144aa and cell functional experiments and animal experiments.

3. The prepared circSTX6-144aa antibody based on the newly discovered circular RNA circSTX6 and the polypeptide circSTX6-144aa coded by the circular RNA circSTX6 can provide a potential polypeptide medicament, a new thought and a new strategy for clinical treatment or prevention/prevention of nasopharyngeal carcinoma.

Drawings

FIG. 1. Schematic representation of the expression of circSTX6-144aa. The circSTX 6is formed by splicing exons 4 to 7 of STX6 end to end, and E represents exon; the CircSTX6 codes for the CircSTX6-144aa pattern; amino acid sequence of circSTX6-144aa.

FIG. 2 immunoblotting detects endogenous expression of circSTX6-144aa. Schematic of the position of the STX6 antibody epitope; the STX6 antibody detects the expression of the circSTX6-144aa in nasopharyngeal carcinoma cells; STX6-255aa syntaxin-6 isosporm 1 encoded protein; STX6-154aa: syntaxin-6 isoport 2 encoded protein. C. Designing a polyclonal antibody aiming at 15 specific amino acids at the carboxyl terminal of the circSTX6-144 aa; antibodies obtained from immunization of rabbits with 15 amino acids detected the expression of circSTX6-144aa.

FIG. 3 IP-MS identifies intracellular circSTX6-144aa. A. IP experiments were performed with antibodies prepared with 15 specific amino acids, and then B.Mass Spectrometry was performed to detect the 15 specific amino acids at the C-terminus of circSTX6-144aa and circSTX6-144aa.

FIG. 4.CircSTX6-144aa related vector pattern. The circSTX 6is a loop forming vector and can express the circSTX6-144aa polypeptide; the circSTX6-mut is a vector obtained by mutating the first ATG in the circSTX6 vector, and the circSTX6-144aa polypeptide cannot be expressed; the circSTX6-flag is inserted into the first ATG of the circSTX6 vector, and the flag label can express the circSTX6-144aa-flag polypeptide; the circSTX6-mut-flag is inserted into the first ATG of the circSTX6-mut vector, and a flag tag can not be expressed in the circSTX6-144aa-flag polypeptide; the STX6-144aa vector is a linearization vector simulating the expression of circSTX6-144aa, and can express the same polypeptide as circSTX6-144aa. The STX6-144aa-flag vector is a linearization vector simulating the expression of the circSTX6-144aa-flag, and can express the same protein as the circSTX6-144 aa-flag.

FIG. 5.15aa antibody detects the expression level of circSTX6-144aa. Western blotting detects the expression level of circSTX6-144aa in nasopharyngeal carcinoma cells, including exogenous expression detection and endogenous knock-down detection; B-D immunofluorescence assay to detect expression level of circSTX6-144aa, including exogenous expression detection and endogenous knockdown detection. * p <0.05, < p <0.01, < p <0.001.

FIG. 6.CircSTX6-144aa promotes proliferation of nasopharyngeal carcinoma cells. The MTT assay detects the effect of circSTX6-144aa on proliferation of nasopharyngeal carcinoma cells. * p <0.05, < p <0.01, < p <0.001.

FIG. 7.CircSTX6-144aa promotes invasion of nasopharyngeal carcinoma cells. The effect of circSTX6-144aa on nasopharyngeal carcinoma cell invasion was examined by glue-spread Transwell experiments. * p <0.05, < p <0.01, < p <0.001.

FIG. 8.CircSTX6-144aa promotes migration of nasopharyngeal carcinoma cells. The scratch assay detects the effect of circSTX6-144aa on nasopharyngeal carcinoma cell migration. * p <0.05, < p <0.01, < p <0.001.

FIG. 9A reversion experiment shows that the circSTX6-144aa promotes proliferation, invasion and migration of nasopharyngeal carcinoma cells. MTT recovery experiments demonstrated that circSTX6-144aa promoted proliferation of nasopharyngeal carcinoma cells. B. The Transwell experiment of spreading the gel proves that the circSTX6-144aa promotes the invasion of nasopharyngeal carcinoma cells. C. The scratch experiment proves that the circSTX6-144aa promotes the migration of nasopharyngeal carcinoma cells. * p <0.05, < p <0.01, < p <0.001.

FIG. 10 in vivo experiments demonstrate that circSTX6-144aa promotes proliferation of nasopharyngeal carcinoma cells. STX6-144aa (circSTX 6-144 aa) significantly promoted subcutaneous neoplasia of nasopharyngeal carcinoma cells. D-E. In situ hybridization detects expression of circSTX6 in tumors, immunohistochemical detects expression of circSTX6-144aa and proliferation marker Ki 67. * p <0.05, < p <0.01, < p <0.001.

FIG. 11 in vivo experiments demonstrate that circSTX6-144aa promotes nasopharyngeal carcinoma cell metastasis. A. The average number of nodules on the lung surface of 10 nude mice per group was counted. B. Representative pictures of lung metastasis nodules from six groups of nude mice, circSTX6 and circSTX6-144a.a. obvious tumor cell clusters can be seen. C. Representative pictures of hematoxylin-eosin stained lung tissue sections of six groups of nude mice, rectangular boxes show metastases in lung tissue. * p <0.05, < p <0.01, < p <0.001.

FIG. 12. High expression of circSTX6-144aa is associated with poor prognosis for patients with nasopharyngeal carcinoma. A-B immunohistochemical assay detects the expression of circSTX6-144aa in clinical samples of nasopharyngeal carcinoma. The overall survival time of the nasopharyngeal carcinoma patients with the high expression of the C.circSTX6-144aa is shorter. * p <0.05, < p <0.01, < p <0.001.

FIG. 13A chemical synthetic, circSTX6-144aa C-terminal 15 specific amino acid composition of a micro peptide to promote proliferation and invasive migration of nasopharyngeal carcinoma cells. A.15aa of the micro peptide promotes proliferation of nasopharyngeal carcinoma cells. B.15aa of the micro peptide promotes invasion of nasopharyngeal carcinoma cells. C.15aa of the micro peptide promotes migration of nasopharyngeal carcinoma cells. * p <0.05, < p <0.01, < p <0.001.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

The nasopharyngeal carcinoma cell lines of HNE2, CNE2, HONE1 and the like used in the invention are all preserved by a molecular genetic laboratory of tumor institute of university of middle and south China. The cell culture conditions were: RPMI1640 liquid medium of 10% Fetal Bovine Serum (FBS) and 1% diabody (penicillin, streptomycin), 37 ℃, 5% CO ₂ And (5) growing in an adherent way in a constant-temperature incubator with the concentration.

The circSTX6-144aa antibody of the present invention was prepared by the company gill biochemistry (Shanghai) limited (polyclonal antibody against 15 amino acids). STX6 Antibody (Center) (cat.# AP16043 c) was purchased from Abcepta corporation.

The test results of the invention all adopt statistical analysis: the t-test was used to evaluate the difference between the two groups. Chi-square test is used to evaluate differences in clinical parameters of sex, age, tumor stage, clinical stage and metastasis status, whether gene expression or not. p <0.05 was used to represent statistical significance, all p values used a double sided test. Statistical analysis was performed using SPSS 13.0 and Graphpad 7.0 software.

Example 1: sanger sequencing demonstrated that circular RNA was formed

To demonstrate that the circSTX6 formation is circular RNA rather than linear, qRT-PCR products were recovered and sent to sanger sequencing (prime). The sequences returned by the companies are compared by DNASTAR software, and chromas software looks at a peak diagram to judge the quality of the sequencing. The results showed that circSTX6 was indeed formed by the head-to-tail splice cyclization of exons 4-7 of gene STX6 (see fig. 1).

Example 2: prediction and identification of cyclic RNA circSTX6 translation small molecule protein

A short peptide of 144 amino acids, designated as circSTX6-144aa, was encoded by the circRNADb (http:// reprod. Njmu. Edu. Cn/cgi-bin/circRNADb/circRNADb. Php) found in the circular RNA circSTX6 (circID: hsa_circ_23135) as an open reading frame (Open Reading Frame, ORF) of 435 nt. The molecular weight of the predicted protein was about 16kDa by the protein molecular weight prediction software https:// web. Expasy. Org/protparam. Designing a polyclonal antibody which specifically recognizes the micro peptide according to 15 specific amino acid sequences (GHERSDVNFICAGIS) of the carboxyl terminal of the circSTX6-144aa and can be detected by using a western blot and immunofluorescence method (figures 2 and 5); and the presence of the circSTX6-144aa protein was detected by IP-MS using the 15aa antibody (FIG. 3). The specific method for preparing the 15aa antibody is as follows: by a method of chemically synthesizing the polypeptide, 15 specific amino acid sequences GHERSDVNFICAGIS at the carboxyl end of the circSTX6-144aa are synthesized as immunogens, new Zealand white rabbits are injected and immunized, and then the target polyclonal antibody is purified for detecting the intracellular circSTX6-144aa protein.

Example 3: immunofluorescence assay

The cultured cells were fixed with 4% paraformaldehyde for 10 minutes and then blocked with 5% BSA and 0.1% Triton X-100 in PBS for 30 minutes at room temperature. Immunostaining was performed using appropriate primary and secondary antibodies (Invitrogen, USA). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). An image was taken using a laser confocal microscope (fig. 5).

Vector: refers to an empty vector pcDNA3.1 (+) CircRNA Mini Vectors

circSTX6-mut: is a vector which mutates the first ATG in the circSTX6 vector and can not express the circSTX6-144aa polypeptide;

circSTX6: is a circSTX6 over-expression vector, and can express the circSTX6-144aa polypeptide;

STX6-144aa: is a linearization vector simulating the expression of the circSTX6-144aa, and can express the polypeptide which is the same as the circSTX6-144 aa;

ASO-ctrl: is a control sequence (5 '-3'): GAGAACGGGAUAGCAUCGACTT, as shown in SEQ ID NO. 5;

ASO-circSTX6: refers to the ASO sequence (5 '-3') of circSTX6: CAUAUGACCAGUGGACAUGATT

The circSTX6 overexpressing plasmid was constructed by amplifying and cloning exons 4-7 of STX6 into pcDNA3.1 (+) CircRNA Mini Vectors, both ends of the insert sequence comprising tandem repeats to aid in the circRNA loop. The circSTX6-mut plasmid was constructed using the point mutation kit using the circSTX6 plasmid as a template. STX6-144aa plasmid was constructed using pcDNA3.1 (+) as the backbone. Plasmid was transfected with necect. Hiperfect transfects ASO.

Example 4: MTT assay to detect cell proliferation

Firstly, using Neofect to transiently transfect nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 with endotoxin-free plasmids pcDNA3.1 (+) CircRNA Mini Vector and circSTX6 over-expression vector, circSTX6-mut vector and STX6-144aa vector, and after culturing for 24 hours, performing MTT experiment to verify the effect on cell proliferation. As a result, it was found that STX6-144aa and circSTX6 significantly promoted proliferation of three cell lines, whereas circSTX6-mut did not significantly promote proliferation of three cell lines, indicating that the circSTX6-144aa polypeptide acted and that circSTX6 did not exert a carcinomatous effect (FIGS. 6 and 9). ASO-ctrl: is a control sequence (5 '-3'): GAGAACGGGAUAGCAUCGACTT; ASO-circSTX6: refers to the ASO sequence (5 '-3') of circSTX6: CAUAUGACCAGUGGACAUGATT; ASO-circSTX6+STX6-144aa: refers to the transfection of STX6-144aa plasmid after knockdown of circSTX6 in nasopharyngeal carcinoma cells, to see if the cell proliferation capacity is restored (FIG. 9).

1) Preparation: tip head, D-hanks sterilized at high temperature and high pressure; sterilizing with alcohol such as a pipetting gun, a marker pen, a 15mL centrifuge tube, etc., placing in a biosafety cabinet, irradiating with ultraviolet for 30min, and ventilating for 10min.

2) Plating and transfection: the previous day will be 25cm ² Cells in good condition in the cell flask are digested and planted into a 6-well plate, and ASO circSTX 6is transfected when the cell density is about 70% or an overexpression vector is transfected when the cell density is about 80-90%.

3) After 12 hours of transfection, the cell supernatant was aspirated, D-hanks was washed 3 times, 100. Mu.L of pancreatin was added to each well of the 6-well plate, after the cells were digested in round form, the cells were blown down and transferred to a 15mL centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, 1-2 mL of medium was added, and mixed well, 10. Mu.L was added to a cell counting plate for cell counting. Based on the cell count results, the cells were diluted to 5000 cells/mL.

4) Adding the diluted cell suspension into a 96-well plate, adding 200 mu L of the cell suspension into each well, adding 200 mu L of Dank's buffer solution into the outermost round of the 96-well plate, and placing into an incubator for continuous culture.

5) After about 6-8 h of cell adherence, adding 20 mu L of MTT into each hole, continuously culturing for 4 hours, carefully sucking and discarding culture supernatant in each hole, adding 200 mu L of DMSO into each hole, placing the mixture on a shaking table for shaking for 10min, selecting 490nm wavelength on an ELISA detector to detect the absorbance value of the DMSO loading hole, and recording the result. The absorbance was then measured daily at the same time point with MTT and DMSO, and after 6 days of continuous measurement, statistical analysis was performed.

Example 5: cell transwell invasion assay

The method comprises the steps of firstly, transiently transfecting nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2 with endotoxinfree plasmids pcDNA3.1 (+) CircRNA Mini Vector and circSTX6 over-expression vector, circSTX6-mut vector and STX6-144aa vector by Neofect, and carrying out a transwell experiment after culturing for 24 hours to verify the influence of the cells on cell invasion. As a result, it was found that STX6-144aa and circSTX6 significantly promoted invasion of three cell lines, whereas circSTX6-mut did not significantly promote invasion of three cell lines, indicating that the circSTX6-144aa polypeptide acted and that circSTX6 did not act to promote invasion (FIGS. 7 and 9). ASO-ctrl: is a control sequence (5 '-3'): GAGAACGGGAUAGCAUCGACTT; ASO-circSTX6: refers to the ASO sequence (5 '-3') of circSTX6: CAUAUGACCAGUGGACAUGATT; ASO-circSTX6+STX6-144aa: refers to the transfection of STX6-144aa plasmid after knockdown of circSTX6 in nasopharyngeal carcinoma cells, to see if the cell invasive capacity is restored (FIG. 9).

1) Preparing matrigel: the BD Matrigel glue frozen at-20 ℃ is placed in a refrigerator at 4 ℃ to be melted into liquid state in advance one day, and the tip head and the EP tube for diluting the glue are placed at-20 ℃ overnight, so that the Matrigel glue is not solidified too fast when being spread during the next day of operation;

2) And (3) matrigel dilution: BD Matrigel gum: serum-free medium=1:8, i.e. 20 μl matrigel plus 160 μl 1640 medium were gently mixed;

3) Adding diluted Matrigel into 100 μl of transwell chamber, sucking 80 μl out along edge, sequentially spreading, placing into 37 deg.C incubator, incubating for 2-3 hr, and displaying that the liquid Matrigel gel is solid when the spreading layer is seen to be white;

4) Digesting the transfected cells for 24 hours, washing the transfected cells with a serum-free medium for 2 times, and then re-suspending the cells with the serum-free medium for cell counting, and adjusting the cell concentration to 2 ten thousand cells in 200 mu L;

5) 800. Mu.L of 1640 medium containing 20% FBS is added into the lower chamber, and the 24-well plate is inclined at 45 DEG when the medium is placed into the chamber, so as to avoid bubbles generated between the chamber and the liquid surface during the process of placing the medium into the chamber;

6) 200. Mu.L of the counted cell suspension was added to the transwell upper chamber, and the 24-well plate was returned to the 37℃incubator, and incubated for about 24-48 hours depending on the cell state and the cell invasion rate.

7) The 24-well plate was taken out, washed twice with PBS or D-hanks, immersed in 4% paraformaldehyde for 10min, and washed 3 times with clean water.

8) Dyeing: dripping 0.1% crystal violet into the bottom of a transwell chamber, standing at room temperature for 5-10min, washing with PBS for 2-3 times, and carefully wiping off matrigel on the chamber with a cotton swab;

9) Distilled water 800 μl was added to a 24-well plate, distilled water approximately 200 μl was added to a transwell upper chamber, and then photographed under an inverted microscope, optionally with 5 different fields of view, and the significance of the differences was analyzed by image J software for counting and statistics.

Example 6: cell scratch healing migration experiment

Firstly, using Neofect to transiently transfect the endotoxin-free plasmid pcDNA3.1 (+) CircRNA Mini Vector and the circSTX6 over-expression vector, the circSTX6-mut vector and the STX6-144aa vector into nasopharyngeal carcinoma cells HONE1, HNE2 and CNE2, and after culturing for 24 hours, performing a scratch healing experiment to verify the influence of the scratch healing experiment on cell migration. As a result, it was found that STX6-144aa and circSTX6 significantly promoted migration of three cell lines, whereas circSTX6-mut did not significantly promote migration of three cell lines, indicating that the circSTX6-144aa polypeptide acted and that circSTX6 did not act to promote migration (FIGS. 8 and 9). ASO-ctrl: is a control sequence (5 '-3'): GAGAACGGGAUAGCAUCGACTT; ASO-circSTX6: refers to the ASO sequence (5 '-3') of circSTX6: CAUAUGACCAGUGGACAUGATT; ASO-circSTX6+STX6-144aa: refers to the transfection of STX6-144aa plasmid after knockdown of circSTX6 in nasopharyngeal carcinoma cells, to see if the cell migration ability was restored (FIG. 9).

1) Illumination of the cell table: 1000 mu L/10 mu L Tip, D-Hank's sterilized by high temperature and high pressure, ruler, 1000 mu L/10 mu L pipette, marker pen, etc., sterilizing with alcohol, and then placing in an ultra clean bench for irradiation with ultraviolet for 30 minutes;

2) Respectively transfecting ASO and NC groups or transfecting plasmids when the cell grows to about 50% -70%;

3) The next day after the cells grow to the bottom of the plate, scratching starts: compared with a straight ruler, the 10 mu L gun head is perpendicular to the bottom of the 6-hole plate, so that cross or cross scratches are quickly carried out, the gun head does not need to be inclined, and the strength is consistent, so that the scratch widths are consistent as much as possible;

4) The culture solution was aspirated, gently washed 3 times with D-hanks, and the broken cells due to scratches were washed off as much as possible;

5) 1640 medium with 1% double antibody and 2% fetal bovine serum is added;

6) Photographing and recording the width of the scratch beside the cross at the moment, and recording the width as 0h;

7) Placing the 6-hole plate back into the incubator for culturing, shooting the same position at intervals of 12h, and recording as 12h;

8) The same position was photographed again at 24h intervals until the scratch healed, all the pictures were sorted, and statistical analysis was performed.

Example 7: animal experiment

We established a BALB/c nude mouse model. Male mice of 4 weeks old were kept in SPF barriers at the center of animals in the university of south China (China Changsha). Tumor xenograft studies (including care, injection, sample collection, dissection) were performed according to approved animal committee protocols. Mice were randomly divided into 6 groups (n=4), each nude mouse was transfected by subcutaneous injection with Vector/circSTX 6-144aa/circSTX6-mut/ASO-NC/ASO-circSTX6, 6×10 ⁶ CNE2 cells. Mice were sacrificed 4 weeks later and the orthotopic tumors were removed for fixation, dehydration and photography. The size of the tumor volume in situ was recorded and embedded in 10% paraffin. Overexpression of circSTX6 and STX6-144aa significantly promoted tumor growth (FIG. 10A-C) Overexpression of circSTX6-mut did not significantly promote tumor growth (FIGS. 10A-C). In contrast, inhibition of circSTX6 significantly inhibited growth of subcutaneous tumors (fig. 10A-C). Next, the present invention confirmed that circSTX6 was highly expressed in mouse subcutaneous tumor tissue by in situ hybridization experiments (FIG. 10D), and IHC experiments showed that circSTX6-144aa was also highly expressed in mouse subcutaneous tumor (FIG. 10D). Ki67 staining was stronger in tumor tissues with high expression of circSTX6 and STX6-144aa (FIGS. 10D-E), whereas Ki67 staining was weaker in tumor tissues with knockdown of circSTX6 (FIGS. 10D-E).

Mice were randomly divided into 6 groups (n=10), each nude mouse was transfected by tail vein injection with Vector/circSTX 6-144aa/circSTX6-mut/ASO-NC/ASO-circ STX6 2×10 ⁶ CNE2 cells. After 8 weeks the mice were dissected and lung tissue was removed for fixation, dehydration and photography. The lung surface nodules were recorded, embedded in 10% paraffin, and treated with hematoxylin and eosin (H&E) Dyeing. H&E staining was used to evaluate its malignancy pathologically. Overexpression of circSTX6 and STX6-144aa effectively increased the number of lung metastases, and overexpression of circSTX6-mut did not effectively increase the number of metastases (FIGS. 11A-C). In contrast, knocking down circSTX6 reduced lung metastases (fig. 11A-C).

Example 8: immunohistochemical experiments

In the invention, 95 cases of clinical tissue samples of nasopharyngeal carcinoma were collected, NPE (paracancerous tissue) =24, npc (nasopharyngeal carcinoma tissue) =95. Expression of circSTX6-144aa in paraffin-embedded nasopharyngeal carcinoma tissue and paracancerous tissue was examined using immunohistochemical experiments (antibody detection using 15 specific amino acid designs at the carboxy terminus of circSTX6-144 aa). Briefly, paraffin-embedded tissue sections were dewaxed in xylene and rehydrated in gradient ethanol. Endogenous peroxidase was blocked with 3% peroxide and then autoclaved for antigen retrieval. Slides were washed 3 times with PBS, blocked with goat serum for 1 hour, and then incubated with primary antibodies overnight at 4 ℃. After washing with PBS, the slides were incubated with secondary antibodies for 1 hour at 37 ℃ and then stained with DAB and hematoxylin. Finally, the sections were dehydrated in gradient alcohol, sealed and observed under an optical microscope (Olympus, japan). The intensity of staining was scored according to the degree of staining (0, no staining; 1, weak staining; 2, strong staining). (2) ratio-based fraction positive signal to total cell number: 0, no positive cells; 1,0-25%; 2. the positive rate is 25-50%; 3. a positive rate of 50-70%; 4. the positive rate is 70-100%. The present invention uses immunohistochemical method to detect the expression of circSTX6-144aa in nasopharyngeal carcinoma tissue and paracancerous non-tumor tissue, and finds that circSTX6-144aa is highly expressed in nasopharyngeal carcinoma tissue (FIG. 12A). When the high and low expression of circSTX6-144aa in NPE (paracancerous tissue) and NPC (nasopharyngeal carcinoma tissue) were analyzed simultaneously, the positive rate of circSTX6-144aa was defined as high expression, and the distribution of the high and low expression of circSTX6-144aa in NPE and NPC was obtained (FIG. 12B). In addition, overall Survival (OS) curves were generated using Kaplan-Meier survival analysis, and it was found that overall survival was significantly shortened in patients with higher levels of circSTX6-144aa in nasopharyngeal tissues (FIG. 12C). When the overall survival rate of nasopharyngeal carcinoma patients is analyzed and counted, the definition of the positive rate of the circSTX6-144aa in the nasopharyngeal carcinoma is lower than 50% and the definition of the positive rate of the circSTX6-144aa is higher than 50% is that of the low expression of the circSTX6-144aa. In the circSTX6-144aa low expression group, 33 cases were dead, 21 cases were alive, and the overall survival rate of the nasopharyngeal carcinoma patients was 39%. In the circSTX6-144aa high expression group, 35 cases of death, 6 cases of survival, and the overall survival rate of nasopharyngeal carcinoma patients was 14.6% (fig. 12C).

Example 9: chemically synthesized 15-specific amino acid polypeptide for promoting proliferation, invasion and migration of nasopharyngeal carcinoma cells

To further investigate the biological function of the C-terminal 15 specific amino acids of circSTX6-144aa, the addition of a chemically synthesized 15 amino acid peptide to the culture medium stimulated nasopharyngeal carcinoma cells in the present invention, which was found to significantly increase proliferation capacity of HNE2, CNE2 and HONE1 cells (FIG. 13A). Also, the invasion and migration ability of tumor cells showed similar results, as demonstrated by matrigel-plated Transwell and scratch experiments (fig. 13B-C). Taken together, these results indicate that it is likely that the C-terminal 15 specific amino acids of circSTX6-144aa play a key role in nasopharyngeal carcinoma proliferation, invasion and migration.

Sequence listing

<110> university of south-middle school

<120> polypeptide and antibody thereof, and application of reagent for detecting same in preparation of nasopharyngeal carcinoma diagnosis, prognosis or treatment preparation

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 391

<212> RNA

<213> Homo sapiens (Homo sapiens)

<400> 1

gacaugaaag aucagauguc aacuucaucu gugcaggcau uagcugaaag aaaaaauaga 60

caggcacugc ugggagacag uggcagccag aacuggagca cuggaacaac agauaaauau 120

gggcgucugg accgagagcu ccagagagcc aauucucauu ucauugagga gcagcaggca 180

cagcagcagu ugaucgugga acagcaggau gagcaguugg agcuggucuc uggcagcauc 240

ggggugcuga agaacauguc ccagcgcauc ggaggggagc uggaggaaca ggcaguuaug 300

uuggaagauu ucucucacga auuggagagc acucaguccc ggcuggacaa ugugaugaag 360

aaacuugcaa aaguaucuca uaugaccagu g 391

<210> 2

<211> 435

<212> RNA

<213> Homo sapiens (Homo sapiens)

<400> 2

augaaagauc agaugucaac uucaucugug caggcauuag cugaaagaaa aaauagacag 60

gcacugcugg gagacagugg cagccagaac uggagcacug gaacaacaga uaaauauggg 120

cgucuggacc gagagcucca gagagccaau ucucauuuca uugaggagca gcaggcacag 180

cagcaguuga ucguggaaca gcaggaugag caguuggagc uggucucugg cagcaucggg 240

gugcugaaga acauguccca gcgcaucgga ggggagcugg aggaacaggc aguuauguug 300

gaagauuucu cucacgaauu ggagagcacu cagucccggc uggacaaugu gaugaagaaa 360

cuugcaaaag uaucucauau gaccagugga caugaaagau cagaugucaa cuucaucugu 420

gcaggcauua gcuga 435

<210> 3

<211> 144

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 3

Met Lys Asp Gln Met Ser Thr Ser Ser Val Gln Ala Leu Ala Glu Arg

1 5 10 15

Lys Asn Arg Gln Ala Leu Leu Gly Asp Ser Gly Ser Gln Asn Trp Ser

20 25 30

Thr Gly Thr Thr Asp Lys Tyr Gly Arg Leu Asp Arg Glu Leu Gln Arg

35 40 45

Ala Asn Ser His Phe Ile Glu Glu Gln Gln Ala Gln Gln Gln Leu Ile

50 55 60

Val Glu Gln Gln Asp Glu Gln Leu Glu Leu Val Ser Gly Ser Ile Gly

65 70 75 80

Val Leu Lys Asn Met Ser Gln Arg Ile Gly Gly Glu Leu Glu Glu Gln

85 90 95

Ala Val Met Leu Glu Asp Phe Ser His Glu Leu Glu Ser Thr Gln Ser

100 105 110

Arg Leu Asp Asn Val Met Lys Lys Leu Ala Lys Val Ser His Met Thr

115 120 125

Ser Gly His Glu Arg Ser Asp Val Asn Phe Ile Cys Ala Gly Ile Ser

130 135 140

<210> 4

<211> 22

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cauaugacca guggacauga tt 22

<210> 5

<211> 22

<212> DNA/RNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gagaacggga uagcaucgac tt 22

<210> 6

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 6

Gly His Glu Arg Ser Asp Val Asn Phe Ile Cys Ala Gly Ile Ser

1 5 10 15

Claims (4)

1. Application of polypeptide detection reagent in preparing reagent for diagnosing and/or prognosis of nasopharyngeal carcinoma; the name of the polypeptide is circSTX6-144aa, and the amino acid sequence is shown as SEQ ID NO. 3.

2. The use according to claim 1, wherein the nasopharyngeal carcinoma diagnosis and/or prognosis agent comprises: an antibody that specifically binds to the polypeptide.

3. The use according to claim 2, characterized in that: antibodies that specifically bind to the polypeptide include: the resulting antibody was designed for 15 specific amino acids at the carboxy terminus of circSTX6-144aa.

4. Application of ASO designed for circSTX6-144aa in preparation of preparation for treating nasopharyngeal carcinoma, wherein the amino acid sequence of circSTX6-144aa is shown in SEQ ID NO. 3; the sequence of ASO is shown as SEQ ID NO. 4.