Gastric cancer pathogenic helicobacter pylori nucleic acid fragment and application thereof

Abstract

The invention provides a nucleic acid fragment of helicobacter pylori (HP) which is pathogenic to gastric cancer and application of the nucleic acid fragment of the helicobacter pylori (HP). In the invention, a novel gastric cancer treatment method is explored based on an HP pathogenic mechanism, a nucleic acid fragment (EC-HP-Fs) causing gastric cancer in HP is determined, and the nucleic acid fragment can participate in an HP-induced gastric cancer process through epigenetic regulation of expression of a host gene to promote the gastric cancer process, and can be used as a new target to treat gastric cancer. The invention also provides nucleic acid inhibitors for targeted modulation. The invention provides a new target for clinical treatment and detection of infectious stomach diseases caused by HP or HP.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbiology and molecular biology, and more particularly, the present application relates to a Helicobacter pylori nucleic acid fragment and application for gastric cancer pathogenesis. BACKGROUND

[0002] Helicobacter pylori (HP) is a non-cardia cancer class 1 carcinogen, and HP infection can cause long-term infiltration of acute and chronic inflammatory cells in the gastric mucosa, leading to the destruction and disappearance of normal glands, followed by atrophy, intestinalization, and dysplasia, and thus is considered a major trigger factor for intestinal-type gastric cancer.

[0003] There are many mechanisms for HP to induce gastric cancer. Existing studies have shown that the carcinogenic effect of Helicobacter pylori on the gastric mucosa is achieved through the complex interaction of bacterial factors, host factors, and environmental factors. Helicobacter pylori can also activate many signaling pathways, such as the NF-κB signaling pathway, the P53 signaling pathway, and others. Some studies have also shown that epigenetic modification, MicroRNAs, oxidative stress, and DNA damage are also involved in the HP-induced gastric cancer process. However, the precise mechanism by which HP regulates cancer development is not yet well understood, and therefore there is an urgent need to explore new pathogenic mechanisms.

[0004] Eradicating HP is a primary prevention measure for gastric cancer, mainly using 10-14 day non-bismuth quadruple therapy and triple therapy based on Vonoprazan, or dual therapy of Vonoprazan and amoxicillin. However, these methods can cause bacterial antibiotic resistance and cross-resistance. And there is currently a lack of effective vaccines that can inhibit HP infection.

[0005] Therefore, there is still a need in the art to find new treatment methods for HP infection. SUMMARY

[0006] The purpose of the present application is to provide a Helicobacter pylori nucleic acid fragment and application for gastric cancer pathogenesis.

[0007] In a first aspect of the present application, the use of a polynucleotide in the preparation of a targeted inhibitor for inhibiting HP infection of gastric disease is provided; wherein the polynucleotide comprises: a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; the polynucleotide is derived from Helicobacter pylori.

[0008] In a preferred embodiment, the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 is applied respectively.

[0009] In another preferred embodiment, two or more of the polynucleotides of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 are combined (e.g., mixed, concatenated, or fused, etc.).

[0010] In another preferred embodiment, the polynucleotides of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 are comprised in an HP fragment, which has the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 6 and / or SEQ ID NO: 10.

[0011] In another preferred embodiment, the HP fragment (nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 6 and / or SEQ ID NO: 10) is used to prepare an expression construct (expression vector).

[0012] In another preferred embodiment, the segment of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 in the HP fragment is the core region.

[0013] In another preferred embodiment, the polynucleotide does not include the full-length viral genome sequence.

[0014] In another preferred embodiment, the H. pylori infectious gastric disease is a disease caused by the interaction (e.g., long-term or persistent interaction) of H. pylori with the infected subject.

[0015] In another preferred embodiment, the H. pylori infectious gastric disease includes: gastric cancer, gastritis.

[0016] In another preferred embodiment, the targeted inhibitor inhibits the proliferation of cancer cells, or inhibits the migration / metastasis of cancer cells.

[0017] In another preferred embodiment, the targeted inhibitor inhibits the expression of a pathogenic gene; more preferably, the pathogenic gene includes UBE2J1, SRSF12, PM20D2, NOL4, or MAPRE2.

[0018] In another preferred embodiment, the gastric cancer includes carcinoma in situ and metastatic carcinoma. In another preferred embodiment, the gastric cancer includes carcinoma in situ and metastatic carcinoma.

[0019] In another preferred embodiment, the targeting inhibitor targets the polynucleotide, and interferes with or inhibits the function of the polynucleotide (e.g., at the DNA level), its transcript (e.g., at the RNA level), or its translation product (e.g., at the protein level); preferably, the targeting inhibitor includes, but is not limited to, an antisense nucleotide (ASO), an interfering molecule (e.g., siRNA, miRNA, shRNA, dsRNA, etc.), a gene editing reagent (e.g., sgRNA, etc.), or a construct capable of expressing or forming the antisense nucleotide, the interfering molecule, or the gene editing reagent; preferably, the targeting inhibitor is an antisense nucleotide; more preferably, the antisense nucleotide has a sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO: 13.

[0020] In another aspect of the present application, a targeting inhibitor for inhibiting H. pylori infectious gastropathy is provided, which targets a polynucleotide; wherein the polynucleotide includes a polynucleotide derived from H. pylori or a homologous polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1, and / or SEQ ID NO: 5; wherein the targeting inhibitor targets the polynucleotide, and interferes with or inhibits the function of the polynucleotide, its transcript, or its translation product; preferably, the targeting inhibitor includes, but is not limited to, an antisense nucleotide (ASO), an interfering molecule (e.g., siRNA, miRNA, shRNA, dsRNA, etc.), a gene editing reagent (e.g., sgRNA, etc.), or a construct capable of expressing or forming the antisense nucleotide, the interfering molecule, or the gene editing reagent; preferably, the targeting inhibitor is an antisense nucleotide; more preferably, the antisense nucleotide has a sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO: 13.

[0021] In another preferred embodiment, the homologous polynucleotide has a sequence identity of 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, compared to the polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1, and / or SEQ ID NO: 5.

[0022] In another aspect of the present invention, a method for preparing a targeted inhibitor to inhibit Helicobacter pylori-induced gastric disease is provided, comprising: (1) providing isolated polynucleotides, including: polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; (2) designing a targeted inhibitor specifically targeting the polynucleotide of (1) as a target, wherein the targeted inhibitor targets the polynucleotide and interferes with or inhibits the function of the polynucleotide, its precursor, transcript, or its translation product; preferably, the targeted inhibitor comprises (but is not limited to): antisense nucleotides (ASO), interfering molecules (such as siRNA, miRNA, shRNA, dsRNA, etc.), gene editing reagents (such as sgRNA, etc.), or constructs capable of expressing or forming the antisense nucleotide, interfering molecule, or gene editing reagent; preferably, the targeted inhibitor is an antisense nucleotide; more preferably, the sequence of the antisense nucleotide is as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 ... As shown in SEQ ID NO:12 and / or SEQ ID NO:13.

[0023] In another preferred embodiment, one or more sets of targeting inhibitors (for one polynucleotide) are prepared for the target.

[0024] In another preferred embodiment, the targeting inhibitor comprises a modified or unmodified nucleic acid reagent. The modification is performed, for example, using techniques such as nucleic acid backbone modification, and the modification does not substantially alter the binding properties of the oligonucleotide molecule; preferably, modifications that improve the stability of the nucleic acid molecule. For example, the modification is dU modification, thiomodification, or alkyl modification at the 2' position of the ribose.

[0025] In another aspect of the invention, the use of the targeted inhibitor in the preparation of a composition for inhibiting Helicobacter pylori-infected gastric diseases is provided; preferably, the targeted inhibitor inhibits the proliferation of cancer cells or inhibits the migration / metastasis of cancer cells; preferably, the targeted inhibitor inhibits the expression of pathogenic genes; more preferably, the pathogenic genes include UBE2J1, SRSF12, PM20D2, NOL4, or MAPRE2; ​​preferably, the gastric cancer includes carcinoma in situ and metastatic carcinoma.

[0026] In another aspect of the invention, a composition, kit, or reagent kit for inhibiting Helicobacter pylori-infected gastric diseases is provided, wherein the composition comprises the targeted inhibitor; or, the kit or reagent kit comprises the composition.

[0027] In another aspect of the invention, a method for inhibiting Helicobacter pylori (including in vivo or in vitro (e.g., isolated bacteria (cultures)) is provided, comprising: administering an effective amount of the targeted inhibitor or the composition to the object to be inhibited (including bacteria in an individual of an animal or human, or isolated bacteria (cultures)).

[0028] In another preferred embodiment, the method for inhibiting Helicobacter pylori includes non-therapeutic methods.

[0029] In another preferred embodiment, the targeted inhibitor works by inhibiting the proliferation of Helicobacter pylori.

[0030] In another preferred embodiment, the targeted inhibitor works by inhibiting the migration / metastasis of cancer cells.

[0031] In another aspect of the invention, a method is provided for screening potential substances that inhibit Helicobacter pylori or infectious gastric diseases caused by it, the method comprising: (1) treating a system expressing or containing polynucleotides with a candidate substance, said polynucleotides comprising: polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; and (2) detecting the transcription, expression or activity of said polynucleotides in said system; wherein, if said candidate substance can reduce the transcription, expression or activity of said polynucleotides (preferably significantly reduced, such as by more than 20%, more preferably by more than 50%, and more preferably by more than 80%), then the candidate substance is a potential substance for inhibiting Helicobacter pylori or infectious gastric diseases caused by it.

[0032] In another preferred embodiment, step (1) includes adding the candidate substance to a system expressing or containing the polynucleotide in the test group.

[0033] In another preferred embodiment, step (2) includes: detecting the transcription, expression, or activity of the polynucleotide in the test group system and comparing it with a control group system, wherein the control group system is a system without the addition of the candidate substance or containing the polynucleotide.

[0034] In another preferred embodiment, if the transcription, expression, or activity of the polynucleotide in the test group is statistically lower than that in the control group, it indicates that the candidate is a potential substance for inhibiting Helicobacter pylori or infectious gastric diseases caused by it.

[0035] In another preferred embodiment, the system is selected from: cell systems (such as cells expressing the polynucleotide, more particularly such as cells of diseases (such as gastric cancer or gastritis) infected with Helicobacter pylori) (or cell culture systems), subcellular systems, solution systems, tissue systems, organ systems, or animal (model) systems.

[0036] In another preferred embodiment, the candidate substances include (but are not limited to): interfering molecules designed against the polynucleotide, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), small molecule compounds, etc.

[0037] In another preferred embodiment, the system is a cell system, such as a cell system infected with Helicobacter pylori, and the method further includes: further analyzing the proliferative capacity of Helicobacter pylori-infected cells in the system; if the proliferative capacity decreases, then the candidate substance is a potential substance for inhibiting Helicobacter pylori or infectious gastric diseases caused by Helicobacter pylori.

[0038] In another preferred embodiment, the system is a cell system, such as a Helicobacter pylori-infected cell system, and the method further includes: further analyzing the transfer / migration ability of Helicobacter pylori-infected disease cells in the system; if the transfer / migration ability decreases, then the candidate substance is a potential substance for inhibiting Helicobacter pylori or infectious gastric diseases caused by it.

[0039] In another preferred embodiment, the method further includes: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances from the candidate substances that are useful for inhibiting Helicobacter pylori or infectious gastric diseases caused by it.

[0040] In another aspect of the invention, the use of polynucleotides in the preparation of diagnostic reagents or kits for detecting Helicobacter pylori-infected gastric diseases is provided; said polynucleotides include: polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5.

[0041] In another aspect of the invention, the use of a detection reagent for specifically recognizing or amplifying polynucleotides is provided for preparing a kit for detecting Helicobacter pylori-infected gastric diseases; the polynucleotides include: polynucleotides having or homologous to the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; preferably, the detection reagent includes (but is not limited to): primers for specifically amplifying the polynucleotides, probes for specifically recognizing the polynucleotides, or chips for specifically recognizing the polynucleotides.

[0042] In another aspect of the present invention, a method for preparing a detection reagent for detecting Helicobacter pylori-infected gastric diseases is provided, comprising: preparing a reagent that specifically recognizes or amplifies the polynucleotide, using a polynucleotide as the target; the polynucleotide comprising: a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 or a polynucleotide homologous thereto; preferably, the detection reagent comprises (but is not limited to): primers that specifically amplify the polynucleotide, probes that specifically recognize the polynucleotide, or a chip that specifically recognizes the polynucleotide.

[0043] In another preferred embodiment, the detection includes distinguishing Helicobacter pylori-infected gastric diseases (such as gastric cancer or gastritis) from other diseases.

[0044] In another aspect of the present invention, a method for diagnosing Helicobacter pylori-infected gastric disease is provided, comprising: using a detection reagent or kit that specifically recognizes or amplifies polynucleotides to detect the amount of polynucleotides present in an ex vivo sample (such as disease in situ tissue or body fluid tissue (blood or serum, etc.) of a subject.

[0045] In another preferred embodiment, the method includes: extracting total RNA from the sample, reversing it into cDNA, performing fluorescent PCR amplification, and qualitatively or quantitatively analyzing the amplification products.

[0046] In another preferred embodiment, the method includes: extracting total RNA from the sample, reversing it into cDNA, synthesizing and labeling cRNA, hybridization, washing, scanning, and signal analysis.

[0047] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0048] Figure 1 , 5EC-HP-Fs were constructed onto a vector and lentiviruses were prepared. These lentiviruses were then used to infect MKN45 and AGS cells, and the GFP positivity rate was measured in both cell types.

[0049] Figure 2 A. Collect some cells and extract RNA. Detect the expression of EC-HP-Fs in MKN45 cells by real-time quantitative PCR.

[0050] Figure 2 B. Collect some cells and extract RNA, and detect the expression of EC-HP-Fs in AGS cells by real-time quantitative PCR.

[0051] Figure 3 RNA-seq was performed on MKN45 cells overexpressing EC-HP-F002 to analyze genes / pathways that showed significant changes.

[0052] Figure 4 RNA-seq was performed on MKN45 cells overexpressing EC-HP-F003 to analyze genes / pathways that showed significant changes.

[0053] Figure 5 RNA-seq was performed on MKN45 cells overexpressing EC-HP-F004 to analyze genes / pathways that showed significant changes.

[0054] Figure 6 A. The effect of EC-HP-Fs on miR-4268 expression.

[0055] Figure 6 B. Effects of EC-HP-Fs on the expression of ZNF689, RAB6B, HOXB13 and FOXL2 genes.

[0056] Figure 6 C. Effects of EC-HP-Fs on the expression of SRSF12 and UBE2J1 genes.

[0057] Figure 6 D. Effects of EC-HP-Fs on the expression of NOL4 and MAPRE2 genes.

[0058] Figure 6 The effects of E and EC-HP-Fs on the expression of STAG2 and TENM1 genes.

[0059] Figure 7 A. Effects of EC-HP-Fs on the expression of PM20D2 and UBE2J1 genes in AGS cells.

[0060] Figure 7B. Effects of EC-HP-Fs on the expression of NOL4 and MAPRE2 genes in AGS cells.

[0061] Figure 7 C. Effects of EC-HP-Fs on STAG2 gene expression in AGS cells.

[0062] Figure 8 A. Effect of EC-HP-Fs overexpression on the proliferation of MKN45 cells.

[0063] Figure 8 B. Effects of EC-HP-Fs overexpression on the proliferation of AGS cells.

[0064] Figure 8 C. Effects of high expression of EC-HP-Fs on cell clone formation ability.

[0065] Figure 9 Transwell experiments were performed on MKN45 cells overexpressing EC-HP-F001 / 002 / 004 / 005.

[0066] Figure 10 Transwell experiments were performed on AGS cells overexpressing EC-HP-F001 / 002 / 004.

[0067] Figure 11 In MKN45 stable cells, AC and EC-HP-F002 / 003 / 004 inhibitors can significantly reduce the expression level of their pathogenic fragments, while also decreasing the expression of oncogenes such as UBE2J1, NOL4, and STAG2.

[0068] Figure 11 D. The EC-HP-F002 inhibitor significantly reduced the expression of EC-HP-F002 and oncogenes such as UBE2J1 in AGS stable transgenic strains.

[0069] Figure 12 AC and CCK8 results showed that treatment with the EC-HP-F002 / 003 / 004 inhibitor significantly inhibited the proliferation of EC-HP-Fs stable transgenic MKN45 cell lines.

[0070] Figure 13 Transwell assays were performed on MKN45 cells overexpressing EC-HP-F002 / 003 / 004. Detailed Implementation

[0071] This invention explores novel treatment methods for gastric cancer based on the pathogenic mechanism of Helicobacter pylori (HP). Through extensive analysis and research, the inventors identified nucleic acid fragments (EC-HP-Fs) in Helicobacter pylori that induce gastric cancer. These nucleic acid fragments can participate in the HP-induced gastric cancer process and promote its progression by epigenetically regulating the expression of host genes. They can be used as novel targets for the treatment of gastric cancer. This invention also provides nucleic acid inhibitors for targeted regulation. This invention provides new targets for the clinical treatment and detection of HP or infectious gastric diseases caused by HP.

[0072] EC-HP-Fs

[0073] This invention reveals that five nucleic acid fragments (EC-HP-Fs) from the Helicobacter pylori genome are key factors in H. pylori or infectious gastric diseases caused by H. pylori (such as H. pylori-induced gastric diseases), and are named EC-HP16-F001, EC-HP16-F002, EC-HP16-F003, EC-HP16-F004, and EC-HP16-F005, respectively.

[0074] The inventors identified the nucleic acid fragments associated with *Helicobacter pylori* (HP) or infectious gastric diseases induced by it through bioinformatics and experimental analysis. In a specific embodiment, the inventors conducted in-depth experimental verification, constructing stable MKN45 and AGS gastric cancer cell lines containing these fragments. The results showed that EC-HP-Fs significantly enhanced the cell proliferation, colony formation, and migration abilities of the gastric cancer cell lines, suggesting their carcinogenic potential. Subsequently, transcriptome sequencing of MKN45 cells overexpressing EC-HP-F002 / 003 / 004 revealed that EC-HP-Fs significantly enriched transcriptome-expressed genes in non-coding RNA metabolic pathways and cell proliferation-related tumor pathways, suggesting that EC-HP-Fs may promote gastric cancer development by altering gene expression. RT-qPCR results showed that different HP fragments upregulated oncogenes such as ZNF698 and RAB6B. Furthermore, the inventors designed specific inhibitors targeting these EC-HP-Fs fragments. The cell proliferation, colony formation, and migration abilities of Helicobacter pylori cell lines with high EC-HP-Fs expression treated with the inhibitors were significantly reduced, and the upregulation of genes was also significantly reversed.

[0075] Therefore, the EC-HP-Fs are not only important pathogenic substances causing HP-infected gastric diseases (including gastric cancer and gastritis), and their function can be targeted and inhibited for the treatment of HP-infected gastric diseases, but also potential biomarkers for the progression of gastric diseases caused by HP infection, which can be used for clinical assessment of gastric disease progression.

[0076] This invention identifies for the first time novel key targets associated with Helicobacter pylori (HP) or infectious gastric diseases induced by HP. By targeting inhibitory agents (such as molecules that antisense complementarily bind to EC-HP-Fs and thereby block their function), the aim is to treat HP or infectious gastric diseases induced by HP. The EC-HP-Fs comprise polynucleotides having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1, and / or SEQ ID NO: 5.

[0077] Furthermore, the present invention may also include polynucleotides, fragments of polynucleotides, or variants homologous to the sequences described. For example, polynucleotides with variations in the sequences that do not alter the key pathogenicity of the polynucleotides (such as nonsense variations) resulting in fewer differences in the target sequence may also be included in the present invention. For example, the variants or homologous polynucleotides are polynucleotides having sequence identity of 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more compared to the polynucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5. However, the isolated polynucleotides do not include the full-length viral genome sequence.

[0078] Fragments derived from the aforementioned polynucleotides can also be used in this invention.

[0079] Targeted inhibitors and their applications

[0080] Based on the inventors’ new discovery, the use of EC-HP-Fs targeted inhibitors is provided for the preparation of compositions (including pharmaceutical compositions) that inhibit HP or infectious gastric diseases caused by HP.

[0081] As used herein, the targeted inhibitors of EC-HP-Fs include blockers, antagonists, downregulators, inhibitors, etc., and these terms are used interchangeably.

[0082] The targeted inhibitors of EC-HP-Fs include regulatory molecules / reagents that can reduce the stability of EC-HP-Fs, decrease the expression of EC-HP-Fs, and inhibit the transcription / translation of EC-HP-Fs. These regulatory molecules / reagents can all be used in this invention as substances useful for targeted inhibition of EC-HP-Fs, thereby inhibiting HP or infectious gastric diseases induced by it. As a preferred embodiment of this invention, the inhibitors are nucleic acid inhibitors. For example, the targeted inhibitors include interfering RNA molecules or antisense nucleotides (including locked antisense nucleic acids, etc.) that specifically interfere with the expression of EC-HP-Fs.

[0083] In a preferred embodiment of the present invention, antisense compounds that specifically hybridize with one or more nucleic acids encoding EC-HP-Fs are used to regulate EC-HP-Fs expression. The specific hybridization of oligomers with their target nucleic acids interferes with the normal function of the nucleic acids. This regulation of target nucleic acid function by compounds that specifically hybridize with the target nucleic acid is commonly referred to as "antisense".

[0084] Preferably, the antisense compound is an antisense nucleotide. As used herein, "antisense nucleotide" is also known as "antisense nucleic acid" or "antisense oligonucleotide (AS-ONs)" or "antisense drug," referring to DNA molecules, RNA molecules, their modified forms, or their analogues that are approximately 15-30 bases in length and can complement non-coding RNAs such as mRNA or miRNA.

[0085] This invention provides target sequences suitable for designing antisense nucleotides. Furthermore, the target or antisense nucleotides provided according to this invention can be appropriately modified while retaining their activity; these modifications are all applicable to this invention. Various antisense nucleotides capable of inhibiting or silencing the EC-HP-Fs are applicable to this invention, and their type is not limited to DNA or RNA. For example, the antisense nucleotides of the EC-HP-Fs are sequences substantially (preferably completely) complementary to the sequence of the EC-HP-Fs. For example, the sequences of the antisense nucleotides shown in SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO: 13 (or their complementary sequences) have more than 80% identity, preferably more than 85% identity, more preferably more than 90% identity, more preferably more than 95% identity, such as 96%, 97%, 98% or 99% identity; they have the same function as the antisense nucleotides listed in the embodiments of this invention.

[0086] Existing technologies have indicated that certain variations of antisense nucleotides are desirable and can exert inhibitory effects on corresponding target sequences. For example, the literature review "Design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells" (NATUREPROTOCOLS; VOL.3 NO.10; 2008; 1537-1549) summarizes several studies, suggesting that generally, extending some bases on both sides of the antisense nucleotide is acceptable. Furthermore, when the affinity between the antisense nucleotide and miRNA is high (e.g., locked nucleic acid modification), the antisense nucleotide can be truncated to approximately 2 / 3 of its length.

[0087] As an optional approach of this invention, homologous recombination is employed to specifically target EC-HP-Fs, causing expression defects or deletions. For example, Cre and loxp methods can be used to selectively knock out, reduce, or inactivate relevant genes in the genome of animals or cells.

[0088] As another alternative approach of this invention, a CRISPR / Cas (e.g., Cas9) system is used for targeted gene editing to downregulate (including knock out) EC-HP-Fs. Common methods for downregulating EC-HP-Fs include co-transferring sgRNA or nucleic acids capable of forming said sgRNA, Cas mRNA or nucleic acids capable of forming said Cas mRNA to a target region or target cell. After identifying the target site, known methods can be used to introduce sgRNA and Cas into the cell. The nucleic acid capable of forming said sgRNA is a nucleic acid construct or expression vector, or the nucleic acid capable of forming said Cas9 mRNA is a nucleic acid construct or expression vector. These expression vectors are introduced into the cell, thereby forming active sgRNA and Cas9 enzyme within the cell.

[0089] In a preferred embodiment of the present invention, the targeting inhibitor can be an EC-HP-Fs-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.). Those skilled in the art will understand that such interfering RNA molecules can be prepared using the EC-HP-Fs sequence information provided in this invention. There are no particular limitations on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis, in vitro transcription, etc. The interfering RNA can be delivered into cells using appropriate transfection reagents, or it can be delivered into cells using various techniques known in the art.

[0090] In one embodiment of the invention, RNAi is used to inhibit EC-HP-Fs. RNAi is an evolutionarily conserved cellular defense mechanism used to control the expression of exogenous genes in most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA and induces sequence-specific mRNA degradation of single-stranded target RNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), typically produced by the enzymatic cleavage of long dsRNA within the cell. siRNAs are typically about 21 nucleotides long (e.g., 21-23 nucleotides). After the small RNA or RNAi is introduced into the cell, the sequence is believed to be delivered to an enzyme complex called the RISC (RNA-induced silencing complex). The RISC recognizes the target and cleaves it with a nuclease. Notably, if a larger RNA sequence is delivered to the cell, the RNase III enzyme (Dicer) converts the longer dsRNA into 21-23 nt ds-siRNA fragments.

[0091] In one embodiment of the present invention, shRNA technology is used for interference. shRNA is an RNA sequence that can rotate a tight hairpin, which can be used to silence gene expression through RNA interference. shRNA is introduced into cells using a vector and utilizes a promoter (such as U6) to ensure that shRNA is always expressed. This vector is typically delivered to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved into siRNA by cellular mechanisms and then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the bound siRNA. shRNA is transcribed by RNA polymerase III.

[0092] In this invention, various nucleic acid inhibitors (such as antisense nucleotides) also include modified forms, wherein the modifications substantially do not alter the activity of the nucleic acid inhibitor, and more preferably, the modifications can improve the activity, stability, or therapeutic effect of the nucleic acid inhibitor. Modifications to nucleic acid inhibitors include, but are not limited to: methoxylation modifications, thiolation modifications, cholesterol modifications, alkyl modifications, locked nucleic acid modifications, peptide nucleic acid modifications, and / or nucleic acid inhibitors in which the phosphate backbone is replaced by phospholipid linkages.

[0093] In one embodiment of the present invention, the targeted inhibitor is a small molecule compound targeting EC-HP-Fs. Those skilled in the art can use methods suitable for screening small molecule compounds to screen such compounds. The screening can rely on various existing or future compound libraries in the art, or can involve establishing new compound libraries independently.

[0094] The above are some representative methods for downregulating EC-HP-Fs. After those skilled in the art understand the overall scheme of the present invention, other methods known in the art or methods under development can also be used to regulate EC-HP-Fs, and these methods are also included in the present invention.

[0095] Particularly preferred, according to the analysis results of the embodiments of the present invention, the antisense nucleotides designed for the EC-HP-Fs of the present invention exert a significant inhibitory effect on HP or infectious gastric diseases induced by HP not only at the cellular level but also at the animal level, and have great clinical value.

[0096] Target-based drug screening

[0097] Knowing that EC-HP-Fs are targets of Helicobacter pylori (HP) or infectious gastric diseases induced by HP, and that their expression (effective, normal, or high expression) is closely related to HP or infectious gastric diseases induced by HP, substances that inhibit EC-HP-Fs can be screened based on this characteristic. Drugs that are truly useful for inhibiting HP or infectious gastric diseases induced by HP can be found among these substances.

[0098] Accordingly, the present invention provides a method for screening potential substances (candidate substances or candidate drugs) that inhibit *Helicobacter pylori* or infectious gastric diseases caused by it. The method includes: treating a system expressing EC-HP-Fs with a candidate substance; and detecting the transcription, expression, or activity of EC-HP-Fs in the system; if the candidate substance can inhibit the transcription, expression, or activity of EC-HP-Fs, it indicates that the candidate substance is a potential substance for inhibiting *Helicobacter pylori* or infectious gastric diseases caused by it.

[0099] The preferred system for expressing EC-HP-Fs is a cell (or cell culture) system, wherein the cells can be endogenously expressing EC-HP-Fs or recombinantly expressing EC-HP-Fs. Furthermore, the usefulness of the potential substance can be assessed by observing the interactions between EC-HP-Fs and its upstream and downstream genes / proteins.

[0100] Based on the inventors' research results, as a preferred method of screening in this invention, the proliferative capacity of disease cells in the system can be further analyzed; if their proliferative capacity decreases, the candidate substance is a potential substance for reducing HP or infectious gastric diseases caused by HP.

[0101] Based on the inventors' research results, as a preferred method of screening in this invention, the ability of disease cells to transfer / migrate in the system can be further analyzed; if their transfer / migration ability decreases, then the candidate substance is a potential substance for reducing the disease.

[0102] In a preferred embodiment of the present invention, during screening, a control group can be set up to more easily observe changes in the transcription, expression, or activity of EC-HP-Fs. The control group can be a system expressing EC-HP-Fs without the addition of the candidate substance. The control group includes, but is not limited to, a blank control without the candidate substance, an empty plasmid control, etc.

[0103] Furthermore, the method may also include: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and identify substances that are truly useful for inhibiting H. pylori or infectious gastric diseases induced by it.

[0104] On the other hand, the present invention also provides potential substances for inhibiting *Helicobacter pylori* or infectious gastric diseases induced by *HP*, obtained using the screening method described above. These initially screened substances can constitute a screening library, from which substances useful for inhibiting the transcription, expression, and activity of EC-HP-Fs, thereby inhibiting *HP* or infectious gastric diseases induced by *HP*, can be ultimately screened.

[0105] Composition

[0106] The present invention also provides a composition (including a pharmaceutical combination) containing an effective amount (e.g., 0.000001-50 wt%; preferably 0.00001-20 wt%; more preferably 0.0001-10 wt%) of the EC-HP-Fs targeting inhibitor, and a pharmaceutically or physiologically acceptable carrier. Preferably, the composition is used to inhibit HP or infectious gastric diseases induced by HP.

[0107] In a preferred embodiment of the present invention, the targeted inhibitor includes, but is not limited to: nucleic acid inhibitors (preferably antisense nucleotides, but may also include siRNA, etc.), reagents for knocking out or silencing EC-HP-Fs, small molecule chemical antagonists or inhibitors targeting EC-HP-Fs, etc. In a more specific embodiment, the targeted inhibitor includes, but is not limited to: CRISPR gene editing reagents targeting EC-HP-Fs, interfering molecules that specifically interfere with the expression of the coding gene of EC-HP-Fs, homologous recombination reagents or site-directed mutagenesis reagents targeting EC-HP-Fs, wherein the homologous recombination reagents or site-directed mutagenesis reagents induce loss-of-function mutations in EC-HP-Fs.

[0108] As used herein, "effective amount" means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0109] Once the use of the target inhibitor of the EC-HP-Fs is known, various methods well known in the art can be used to administer the target inhibitor or its encoding gene, or its pharmaceutical composition, to mammals or humans.

[0110] Preferably, gene therapy can be used. For example, the EC-HP-Fs targeting inhibitor can be directly administered to the subject by means such as injection; or, the expression unit (such as an expression vector or virus, or siRNA) carrying the EC-HP-Fs targeting inhibitor can be delivered to the target site through a certain route, and the EC-HP-Fs targeting inhibitor can be expressed. The specific circumstances depend on the type of targeting inhibitor, all of which are well known to those skilled in the art.

[0111] The effective amount of the EC-HP-Fs targeted inhibitor described in this invention can vary depending on the administration method and the severity of the disease to be treated. The preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the EC-HP-Fs targeted inhibitor, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0112] In specific embodiments of the present invention, some dosing regimens for animals such as mice are provided. Converting animal dosages (e.g., mouse) to human dosages is readily achievable by those skilled in the art, for example, using the Meeh-Rubner formula. It should be understood that dosage conversions may vary depending on the drug and clinical circumstances, and may be subject to assessment by an experienced pharmacist.

[0113] The present invention also provides a kit containing the pharmaceutical composition described above or a targeted inhibitor directly containing the EC-HP-Fs described above. Furthermore, the kit may also include instructions on how to use the medication contained therein.

[0114] Detection of infectious gastric diseases caused by Helicobacter pylori

[0115] The term "marker" or "label" refers to a gene whose expression level in a tissue or cell is altered compared to the expression level in a normal or healthy cell or tissue.

[0116] Those skilled in the art will understand that the utility of this invention is not limited to quantifying the gene expression of any specific variant of the biomarker gene of this invention. As a non-limiting example, the biomarker gene may be the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5. In some embodiments, it has a sequence that is at least 85% identical or similar to these sequences, such as at least 80%, 85%, 90%, 92%, 95%, 96%, 98%, 99%, 99.5%, or 99.8% identical or similar to the sequences listed above.

[0117] Based on the inventors' new discovery, EC-HP-Fs can be used as biomarkers for measuring HP or infectious gastric diseases (such as gastritis) caused by HP. By analyzing the expression of EC-HP-Fs in the test sample, the subject's disease status can be determined, providing a basis for disease diagnosis or prognosis. In a preferred embodiment, the test sample is a patient's tissue sample or body fluid.

[0118] This invention can determine gene expression using any method known in the art. Those skilled in the art will understand that the means of determining gene expression are not a key aspect of this invention. The expression level of biomarkers can be detected at the transcriptional level.

[0119] In some embodiments, the expression level of the biomarker is detected at the transcriptional level. Various methods for specific DNA and RNA measurements using nucleic acid hybridization techniques are known to those skilled in the art. Some methods involve electrophoretic separation (e.g., Southern blotting for DNA detection and Northern blotting for RNA detection), but DNA and RNA measurements can also be performed without electrophoretic separation (e.g., by dot blotting). Southern blotting of genomic DNA (e.g., from humans) can be used to screen for restriction fragment length polymorphisms (RFLPs) to detect the presence of genetic conditions affecting the polypeptides of the present invention. All forms of RNA can be detected.

[0120] Various techniques in the art can be used to detect the expression of EC-HP-Fs, and these techniques are all included in this invention. Existing techniques that can be used to detect nucleic acids include (but are not limited to): gene chip technology, probe hybridization technology, polymerase chain reaction (PCR), Northern blotting, etc.

[0121] This invention provides reagents for specifically recognizing or amplifying EC-HP-Fs. Any reagent capable of recognizing EC-HP-Fs is included in this invention. The reagents for specifically recognizing EC-HP-Fs include, but are not limited to, primers for specifically amplifying EC-HP-Fs or probes for specific recognition.

[0122] The reagent that specifically recognizes EC-HP-Fs can also be immobilized on test paper, glass slide, or other solid-phase carriers to prepare immunogold test paper or similar detection materials.

[0123] As one option of this invention, the expression status and level of EC-HP-Fs in a sample are analyzed by quantitative or semi-quantitative polymerase chain reaction (PCR) to make a judgment. Preferably, detection is achieved by real-time quantitative PCR. The reagents used are primers that specifically amplify EC-HP-Fs. After knowing the nucleotide sequence of EC-HP-Fs, primers can be designed based on this sequence.

[0124] As an alternative approach to this invention, gene chip technology can be used for the detection of EC-HP-Fs. Once the nucleotide sequence of EC-HP-Fs is known, probes can be easily designed based on this sequence. For example, if the solid support is a modified glass slide or silicon wafer, and the 5' end of the probe contains an amino-modified polydT string, the oligonucleotide probe can be prepared into a solution, then spotted onto the modified glass slide or silicon wafer using a spotting instrument, arranged into a predetermined sequence or array, and then fixed by incubation overnight to obtain the gene chip of this invention. If the oligonucleotide probe does not contain amino modification, its preparation method can also refer to existing known techniques.

[0125] Specifically, suitable probes can be designed using the EC-HP-Fs described in this invention and immobilized on a solid support to form an "oligonucleotide array". The "oligonucleotide array" refers to an array with addressable sites (i.e., sites characterized by distinctive, accessible addresses), each addressable site containing a characteristic oligonucleotide linked to it. The oligonucleotide array can be divided into multiple subarrays as needed. The solid support may include plastic products, microparticles, membrane carriers, etc.

[0126] The present invention also provides a kit for detecting *Helicobacter pylori* or infectious gastric diseases caused by it, the kit comprising: a reagent specifically recognizing EC-HP-Fs, or a carrier loaded with said reagent. The reagent specifically recognizing EC-HP-Fs is, for example, primers specifically amplifying EC-HP-Fs, probes specifically recognizing EC-HP-Fs, or a chip specifically recognizing EC-HP-Fs.

[0127] The kit may also contain markers for labeling RNA samples, and corresponding substrates. Furthermore, the kit may contain auxiliary reagents: nucleic acid extraction reagents (such as nucleic acid extraction buffer, phenol, chloroform, isoamyl alcohol, NaCl, etc.); and / or polymerase chain reaction reagents (such as dNTPs, Taq enzyme, PCR buffer, DNA polymerase, etc.); and / or enzyme chain immunoassay reagents (such as chromogenic solutions or hybridization solutions, etc.).

[0128] In addition, the kit also includes an instruction manual and / or chip image analysis software.

[0129] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0130] Materials and methods

[0131] 1. Cell Culture

[0132] Human gastric adenocarcinoma cells AGS and MKN45: purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0133] AGS cells and MKN45 culture medium: cultured in RPMI 1640 containing 10% fetal bovine serum.

[0134] The culture conditions for all the above cells were: 37℃, 95% air, 5% CO2 concentration, passage every 2-3 days, and density maintained at 85%-90%.

[0135] The cell cryopreservation conditions were as follows: cells were resuspended in serum-free, unprogrammed cell cryopreservation solution, stored in a cryopreservation box at -80°C for 2 days, and then transferred to a liquid nitrogen tank after gradient cooling.

[0136] 2. Construction of expression vector

[0137] Five plasmids were constructed targeting five HP fragments. First, approximately 150 bp HP genome fragments were obtained by PCR amplification.

[0138] These fragments were cloned into the pCDH-CMV-MCS-EF1-copGFP lentiviral vector using homologous recombination.

[0139] 3. RNA extraction, reverse transcription, and real-time quantitative PCR

[0140] Cell samples were collected and RNA extracted using RNA-easy™ Isolation Reagent (Vazyme #R701), and the RNA was reverse transcribed into cDNA. Real-time quantitative PCR was performed using the Real Universal Color PCR Premix Kit from Tiangen Pharmaceuticals. The quantitative PCR instrument used was a Lightcycler R 480 / 384. GAPDH was used as an internal control.

[0141] 4. Lentiviral Packaging and Infection

[0142] Packaging: Seed HEK293T cells at 1.2 million per well into 6-well plates. When the cell density reaches 80% the next day, transfect the plasmid (recombinant pCDH plasmid carrying a potential HP pathogenic fragment) using EXfect transfection reagent. After culturing for 6-8 hours, replace with 2-3 mL of complete culture medium and continue culturing for 48-72 hours. Collect the virus solution and filter it through a 0.22 μm filter membrane.

[0143] Infection: 500,000 gastric cancer cells per well were seeded into a 6-well plate, and 500 μL of lentivirus was added. After 12-24 hours of infection, the medium was replaced with fresh medium, and the cells were cultured for another 24 hours. Cell fluorescence was observed, and cells were collected for later use after sufficient fluorescence was observed.

[0144] In addition, gastric cancer cell lines stably expressing the HP fragment were prepared using cells infected with lentiviruses.

[0145] 5. HP fragment function blocking

[0146] EC-HP-Fs expression was silenced by synthesizing inhibitors. Inhibitors were added to stable cell lines, and RNA was extracted from samples 72 hours later. The effect of EC-HP-Fs dysfunction on target genes was detected by quantitative real-time PCR.

[0147] 6. Cell proliferation, colony formation, and cell migration (Transwell assay)

[0148] Cells infected with a virus or treated with an antisense nucleic acid inhibitor were seeded into 96-well plates at 2000 cells per well. Detection was performed at 24h, 48h, and 72h. 10 μl of CCK-8 reagent was added to each well, and the cells were cultured for another 1-4 hours before the absorbance was measured at 450 nm using a microplate reader. Cells infected with a virus or treated with an inhibitor were digested and resuspended to form a single-cell suspension. 500 cells were seeded into 6-well plates and cultured for one week until visible colonies formed. Culture was then terminated, and the cells were stained with 0.25% crystal violet. The cells were photographed, and the number of colonies was counted.

[0149] Similarly, in the cell migration experiment, a chamber was placed in a 24-well plate, and culture medium containing 20% ​​serum was added to the lower layer. The digested cells were resuspended in the chamber with serum-free culture medium. After culturing the cells for 24 hours, they were stained with crystal violet, and five different fields of view were photographed under a microscope and statistically analyzed.

[0150] Example 1: Establishment of an EC-HP-Fs expression system for gastric cancer cells

[0151] After extensive screening and in-depth analysis, the inventors identified five Helicobacter pylori (HP) carcinogenic sequences (EC-HP-Fs), which are closely related to the carcinogenic characteristics of Helicobacter pylori. The sequences of these target fragments are shown in Table 1.

[0152] Table 1

[0153]

[0154] First, the inventors constructed an HP fragment containing any EC-HP-Fs into the pCDH vector, with the specific sequence as follows:

[0155]

[0156]

[0157] Lentiviral viruses were prepared using the recombinant plasmids described above and used to infect MKN45 and AGS cells. Figure 1 The GFP positivity rate in both cell types was above 90%.

[0158] Subsequently, the inventors collected some cells and extracted RNA. The expression of EC-HP-Fs in these cells was detected by real-time quantitative PCR. The expression of EC-HP-Fs in MKN45 cells exceeded 500-fold. Figure 2 A), expression in AGS is nearly 200-fold ( Figure 2 B).

[0159] These results suggest that EC-HP-Fs were successfully expressed in AGS and MKN45 cell lines, and a gastric cancer cell line expressing EC-HP-Fs was successfully established.

[0160] Example 2: EC-HP-Fs alter the transcriptome of gastric cancer cells and upregulate oncogenes

[0161] To investigate the effects of the HP fragment on the host transcriptome, the inventors randomly selected MKN45 cells overexpressing EC-HP-F002 / 003 / 004 for RNA-seq. For example... Figures 3-5 The significantly altered genes in EC-HP-F002 / 003 / 004 were significantly enriched in tumor proliferation-related pathways such as cell division and cell cycle, suggesting that the HP fragment is associated with the occurrence and development of gastric cancer.

[0162] Furthermore, these differentially expressed genes were significantly enriched in non-coding RNA and RNA metabolic processing pathways. According to literature reports, miR-4268 blocks gastric cancer formation by inhibiting KRT80 (PMID: 35748914). Consistent with this, the inventors found that EC-HP-F001 / 002 significantly downregulated miR-4268. Figure 6 A).

[0163] In addition, the inventors verified the upregulated oncogenes in RNA-seq, such as... Figure 6 B, EC-HP-F002 / 003 / 004 can all increase ZNF698, RAB6B, HOXB13 and FOXL2.

[0164] At the same time, these fragments can also activate other oncogenes ( Figure 6 CE), such as EC-HP-F002, can also promote SRSF12 and UBE2J1.

[0165] In addition, EC-HP-F002 / 003 / 004 can also upregulate cancer-related genes such as PM20D2 and STAG2 in AGS cells. Figure 7 Therefore, EC-HP-Fs have the potential to promote gastric cancer formation and progression by activating oncogenes.

[0166] Example 3: EC-HP-Fs can promote the proliferation, growth, and migration of gastric cancer cells.

[0167] To further determine the carcinogenic effect of EC-HP-Fs, the inventors conducted CCK8, clonogenic assays and Transwell assays to evaluate their effects on cell proliferation, growth and migration.

[0168] like Figure 8In both MKN45 and AGS cell lines, compared to the control group, high expression of EC-HP-Fs significantly increased cell proliferation. Consistent with this, high expression of EC-HP-F001 / 003 / 004 significantly promoted the clonogenic ability of MKN45 cells. Figure 8 C) suggests that EC-HP-Fs can promote the proliferation and growth of gastric cancer cells.

[0169] Transwell assays showed that overexpression of EC-HPF001 / 002 / 003 / 004 / 005 significantly increased the number of MKN45 cells that passed through the chambers. Figure 9 This suggests that EC-HP-Fs can promote MKN45 migration. To verify this result, the inventors randomly selected AGS cells overexpressing EC-HP-F001 / 002 / 004 for Transwell experiments. Figure 10 Overexpression of EC-HP-Fs also promotes AGS cell migration.

[0170] These results suggest that EC-HP-Fs can promote the growth and metastasis of gastric cancer cells and are carcinogenic.

[0171] Example 4: Targeting EC-HP-Fs antisense nucleic acid can inhibit oncogenes by reducing HP fragment expression.

[0172] To further evaluate the therapeutic effect of antisense small nucleic acid drugs targeting EC-HP-Fs, the inventors prepared MKN45 cell lines stably expressing EC-HP-F002 / 003 / 004, and also prepared AGS cell lines stably expressing EC-HP-F002. Antisense nucleic acid inhibitors targeting EC-HP-F002 / 003 / 004 were designed, and negative control small nucleic acids (NCs) were designed using meaningless sequences. The sequences of the inhibitors are shown in Table 2.

[0173] Table 2

[0174]

[0175] The inventors transfected NC and inhibitors into gastric cancer cell lines that stably expressed EC-HP-Fs, respectively, and then extracted RNA and used RT-qPCR to detect the effects of inhibitors on HP fragment expression and oncogenes.

[0176] The results are as follows Figure 11In MKN45 stable transgenic cells, compared with the control group, EC-HP-F002 / 003 / 004 inhibitors significantly reduced their expression levels, while also decreasing the expression of oncogenes such as UBE2J1, NOL4, and STAG2.

[0177] Consistent with this, the EC-HP-F002 inhibitor also significantly reduced the expression of EC-HP-F002 and oncogenes such as UBE2J1 and PM20D2 in AGS stable variants. Therefore, this suggests that small nucleic acids targeting EC-HP-Fs have an inhibitory effect on HP infection-related gastric cancer.

[0178] Example 5: Targeting EC-HP-Fs antisense nucleic acids significantly inhibits the growth and migration of gastric cancer cells.

[0179] To further determine the potential therapeutic effects of Inhibitors, the inventors conducted a functional evaluation of MKN45 stable transfected cells transfected with Inhibitors and NC groups.

[0180] like Figure 12 CCK8 results showed that treatment with EC-HP-F002 / 003 / 004 inhibitor significantly inhibited the proliferation of EC-HP-Fs stable MKN45 cell lines (***p<0.001 compared with the NC group).

[0181] Transwell assays were performed, and the results showed that, compared with the control group, inhibitor treatment significantly reduced the migration ability of EC-HP-F002 / 003 / 004 cells stably expressing MKN45. Figure 13 .

[0182] These results indicate that small nucleic acids targeting EC-HP-Fs can reverse the increased proliferation and migration of gastric cancer cells caused by them.

[0183] Therefore, the design of antisense small nucleic acids targeting EC-HP-Fs has the potential to deactivate the carcinogenic activation caused by HP infection and reduce the growth and metastasis of gastric cancer cells, thus providing a new treatment option for HP infection-related gastric cancer.

[0184] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of polynucleotides in the preparation of targeted inhibitors against Helicobacter pylori-induced gastric diseases; among which, The polynucleotides include: polynucleotides having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; said polynucleotides are derived from Helicobacter pylori.

2. The application as described in claim 1, characterized in that, Helicobacter pylori-related gastric diseases include: gastric cancer, gastritis; Preferably, the targeted inhibitor inhibits the proliferation of cancer cells or inhibits the migration / metastasis of cancer cells; Preferably, the targeting inhibitor inhibits the expression of the pathogenic gene; more preferably, the pathogenic gene includes UBE2J1, SRSF12, PM20D2, NOL4, or MAPRE2. Preferably, the gastric cancer includes carcinoma in situ and metastatic carcinoma.

3. The application as described in claim 1, characterized in that, The targeted inhibitor targets the polynucleotide and interferes with or inhibits the function of the polynucleotide, its transcript, or its translation product. Preferably, the targeted inhibitor comprises an antisense nucleotide, an interfering molecule, a gene editing reagent, or a construct capable of expressing or forming the antisense nucleotide, the interfering molecule, or the gene editing reagent. Preferably, the targeted inhibitor is an antisense nucleotide. More preferably, the sequence of the antisense nucleotide is as shown in SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO:

13.

4. A targeted inhibitor that inhibits Helicobacter pylori-induced gastric disease, targeting polynucleotides; among which, The polynucleotides include: polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; The targeted inhibitor targets the polynucleotide and interferes with or inhibits the function of the polynucleotide, its transcript, or its translation product. Preferably, the targeting inhibitor comprises: an antisense nucleotide, an interfering molecule, a gene editing reagent, or a construct capable of expressing or forming the antisense nucleotide, the interfering molecule, or the gene editing reagent; Preferably, the targeting inhibitor is an antisense nucleotide; more preferably, the sequence of the antisense nucleotide is as shown in SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO:

13.

5. A method for preparing a targeted inhibitor to inhibit Helicobacter pylori-induced gastric disease, comprising: (1) Provides isolated polynucleotides, including: polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; (2) Using the polynucleotide of (1) as a target, design a targeting inhibitor that specifically targets the target, wherein the targeting inhibitor targets the polynucleotide and interferes with or inhibits the function of the polynucleotide, its precursor, transcript or its translation product. Preferably, the targeting inhibitor comprises: an antisense nucleotide, an interfering molecule, a gene editing reagent, or a construct capable of expressing or forming the antisense nucleotide, the interfering molecule, or the gene editing reagent; preferably, the targeting inhibitor is an antisense nucleotide; more preferably, the sequence of the antisense nucleotide is as shown in SEQ ID NO: 11, SEQ ID NO: 12 and / or SEQ ID NO:

13.

6. The use of the targeted inhibitor according to claim 4 in the preparation of a composition for inhibiting Helicobacter pylori-infected gastric diseases; Preferably, the targeted inhibitor inhibits the proliferation of cancer cells or inhibits the migration / metastasis of cancer cells; Preferably, the targeting inhibitor inhibits the expression of the pathogenic gene; more preferably, the pathogenic gene includes UBE2J1, SRSF12, PM20D2, NOL4, STAG2, or MAPRE2. Preferably, the gastric cancer includes carcinoma in situ and metastatic carcinoma.

7. A composition, kit, or reagent kit for inhibiting Helicobacter pylori-infected gastric diseases, said composition comprising the targeted inhibitor of claim 4; or, said kit or reagent kit comprising said composition.

8. A method for inhibiting Helicobacter pylori, comprising: The target inhibitor of claim 4 or the composition of claim 7 is administered to the target that needs to be inhibited.

9. A method for screening potential substances that inhibit Helicobacter pylori or infectious gastric diseases induced by it, the method comprising: (1) Treatment of a system expressing or containing a polynucleotide with a candidate substance, said polynucleotide comprising: a polynucleotide derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; and (2) Detect the transcription, expression, or activity of the polynucleotide in the system; If the candidate substance can reduce the transcription, expression, or activity of the polynucleotide, it indicates that the candidate substance is a potential substance for inhibiting Helicobacter pylori or infectious gastric diseases caused by it.

10. Use of polynucleotides in the preparation of diagnostic reagents or kits for detecting Helicobacter pylori-infected gastric diseases; said polynucleotides include: Polynucleotides derived from or homologous to Helicobacter pylori having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO:

5.

11. The use of a detection reagent that specifically recognizes or amplifies polynucleotides for the preparation of a kit for detecting Helicobacter pylori-infected gastric diseases; said polynucleotides include: A polynucleotide having or homologous to the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5; preferably, the detection reagent comprises: primers for specifically amplifying the polynucleotide, probes for specifically recognizing the polynucleotide, or a chip for specifically recognizing the polynucleotide.

12. A method for preparing a diagnostic reagent for detecting Helicobacter pylori-infected gastric diseases, comprising: Using polynucleotides as the target, prepare reagents that specifically recognize or amplify the polynucleotides; the polynucleotides include: polynucleotides having the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 and / or SEQ ID NO: 5 or polynucleotides homologous to them; Preferably, the detection reagent includes: primers for specifically amplifying the polynucleotide, probes for specifically recognizing the polynucleotide, or chips for specifically recognizing the polynucleotide.

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