Fusobacterium sp. lscC04 and application thereof

Abstract

The application discloses a new fusobacterium sp. LSCC04 and application thereof, the preservation number of the fusobacterium sp. LSCC04 is CCTCC NO:M 2025587, the fusobacterium sp. LSCC04 is successfully separated from a laryngeal carcinoma tissue, the strain has key technical properties such as stable growth, repeatable culture and clear storage condition, and solves the problem of the lack of related strains in the current laryngeal carcinoma microecological research field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tumor microbiology, and particularly relates to a new Fusobacterium sp. LSCC04 and application thereof. BACKGROUND

[0002] Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors in the upper respiratory tract of the human body. Recent studies have shown that the microbiome associated with LSCC is closely related to tumor occurrence, development, metastasis, drug resistance, and the like. Among the numerous microorganisms in the human larynx, the role of Fusobacterium in tumor research has attracted the attention of researchers. Previous studies have shown that the abundance of Fusobacterium in the larynx of patients with LSCC is significantly higher than that in normal people. Fusobacterium promotes the proliferation, migration, invasion, and metastasis of LSCC through various mechanisms.

[0003] According to existing literature reports, Fusobacterium is divided into Fusobacterium nucleatum, animal Fusobacterium, Fusobacterium polymorpha, and periodontal Fusobacterium, and the type strains thereof are isolated and cultured from the oral cavity, intestinal tract, and the like. There is no literature or database report indicating that Fusobacterium is isolated from the throat tissue. The type strains of Fusobacterium nucleatum ATCC 25586 and Fusobacterium polymorpha ATCC 10953 are preserved in microbial preservation centers around the world and are used to construct in vivo and in vitro tumor experimental models to study the mechanism of tumor occurrence and development and to find specific targets for anti-tumor treatment. However, the niche characteristics of Fusobacterium derived from the oral cavity and intestinal tract are significantly different from those of the symbiotic Fusobacterium associated with LSCC. Although it can simulate part of the carcinogenic effect of Fusobacterium, it still has differences with the microenvironment of LSCC microorganisms, and cannot well reflect the carcinogenic effect of LSCC Fusobacterium, which limits the study of the carcinogenic mechanism of LSCC.

[0004] Currently, there is no research report on the isolation of active Fusobacterium specific strains from LSCC, which limits the study of Fusobacterium in the mechanism of LSCC occurrence and development, and cannot construct a precise and effective LSCC Fusobacterium carcinogenic cell and animal model. SUMMARY

[0005] In order to overcome the shortcomings of the above prior art solutions, we need to solve the following problems:

[0006] 1. The present application provides a new strain of Fusobacterium isolated and cultured from LSCC tissue, which ensures stable activity and can be passaged, and solves the problem of mismatched ecological niche and insufficient clinical relevance of existing type strains.

[0007] 2. Construct a co-culture experiment of the new Fusobacterium strain and LSCC cell line to verify the carcinogenic effect of the new Fusobacterium strain in the LSCC cell experiment.

[0008] The core technical solution of the present application is as follows:

[0009] (I) A strain of Fusobacterium sp. LSCC04 derived from laryngeal squamous carcinoma is provided, which is classified as Fusobacterium sp. LSCC04, and has a preservation number of CCTCC NO: M 2025587 and is preserved in the China Center for Type Culture Collection on March 25, 2025, and the address of the preservation unit is Wuhan University, Wuhan, China.

[0010] (II) The strain is isolated from a fresh surgical resection specimen of a patient with laryngeal squamous carcinoma, and is amplified by a Columbia blood agar plate or a thioethanolate liquid culture bottle.

[0011] (III) The application of Fusobacterium sp. LSCC04 in the research on the mechanism of laryngeal squamous carcinoma development is provided, and the strain is co-cultured with a laryngeal carcinoma cell line to verify that the strain can promote the malignant phenotype of the laryngeal carcinoma cell.

[0012] To solve the above technical problems, the first aspect of the present application provides a Fusobacterium sp. LSCC04, which has a preservation number of CCTCC NO: M 2025587.

[0013] In a preferred embodiment, the sequence of 16S rRNA of the Fusobacterium sp. LSCC04 is shown in SEQ ID NO: 1.

[0014] In a preferred embodiment, the full-length genome sequence of the Fusobacterium sp. LSCC04 is 2245556 bp.

[0015] In a preferred embodiment, the Fusobacterium sp. LSCC04 is red and fusiform under a microscope after Gram staining, and forms irregular round colonies that are semi-transparent grayish white, smooth and moist on the surface, and have a central microconvex after culture.

[0016] The second aspect of the present application provides a Fusobacterium colony, which is obtained by culturing the Fusobacterium sp. LSCC04 according to the first aspect of the present application.

[0017] The third aspect of the present application provides a Fusobacterium bacterial solution, which is obtained by culturing the Fusobacterium sp. LSCC04 according to the first aspect of the present application.

[0018] The fourth aspect of the present application provides a culture method of the fusobacterium colony as described in the second aspect of the present application or the fusobacterium liquid as described in the third aspect of the present application, comprising culturing the fusobacterium Fusobacterium sp. LSCC04 on a liquid culture medium or a solid culture medium to obtain.

[0019] The present application also provides an application of the fusobacterium Fusobacterium sp. LSCC04 as described in the first aspect of the present application in preparing the fusobacterium colony or the fusobacterium liquid.

[0020] The present application also provides an application of the fusobacterium Fusobacterium sp. LSCC04 as described in the first aspect of the present application, the fusobacterium colony as described in the second aspect of the present application or the fusobacterium liquid as described in the third aspect of the present application in promoting the proliferation or cloning of TU686 cells and / or AMC-HN-8 cells.

[0021] The present application also provides an application of the inhibitor of the fusobacterium Fusobacterium sp. LSCC04 as described in the first aspect of the present application, the fusobacterium colony as described in the second aspect of the present application or the fusobacterium liquid as described in the third aspect of the present application in preparing a drug for treating laryngeal squamous cell carcinoma.

[0022] In a preferable embodiment, the inhibitor comprises metronidazole and the like.

[0023] The present application also provides an application of the fusobacterium Fusobacterium sp. LSCC04 as described in the first aspect of the present application, the fusobacterium colony as described in the second aspect of the present application or the fusobacterium liquid as described in the third aspect of the present application in the research of laryngeal squamous cell carcinoma.

[0024] The present application also provides an application of the fusobacterium Fusobacterium sp. LSCC04 as described in the first aspect of the present application in constructing an in vitro verification model, wherein the in vitro verification model further comprises laryngeal squamous cell carcinoma cells, such as TU686 cells and / or AMC-HN-8 cells.

[0025] On the basis of common knowledge in the art, the above-mentioned preferable conditions can be combined in any manner, thereby obtaining preferable examples of the present application.

[0026] The reagents and raw materials used in the present application are commercially available.

[0027] The positive progress effect of the present application is that:

[0028] The present application successfully isolates and identifies a new species of Fusobacterium LSCC04 from laryngeal cancer tissue, combined with in vitro functional verification, which increases the scientificity and rigor of the study of laryngeal cancer microecology.

[0029] 1. Fusobacterium LSCC04 originating from laryngeal cancer tissue is successfully isolated, and the strain has stable growth, repeatable culture, and clear preservation conditions, solving the problem of the lack of related strains in the field of laryngeal cancer microecology research.

[0030] 2. Fusobacterium LSCC04 is identified as a new species of Fusobacterium, filling the gap in the specific strain resources of laryngeal cancer tumor niche.

[0031] 3. An in vitro verification model (Fusobacterium-laryngeal cancer cell co-culture model) of the effect of Fusobacterium LSCC04 on laryngeal cancer is established, providing a reproducible culture mode.

[0032] 4. The promoting effect of Fusobacterium LSCC04 on laryngeal cancer cell line proliferation and clone formation is preliminarily verified, confirming the influence of Fusobacterium LSCC04 on laryngeal cancer, providing experimental basis and strain resources for further mechanism research, and providing a new idea for the prevention and treatment of laryngeal squamous carcinoma.

[0033] 5. A reproducible operation process is provided for isolating anaerobic bacteria from laryngeal cancer tissue, providing reference in strain isolation, identification, preservation, and functional verification, and having promotional value.

[0034] 6. The candidate strain resource is provided for the transformation research of tumor-related bacteria (such as the development of live bacteria drugs, targeted microecological treatment, etc.), which is expected to become an important supplementary means for future precise treatment of laryngeal cancer.

[0035] Biological material preservation information

[0036] The Fusobacterium sp. LSCC04 of the present application has been preserved in the China Center for Type Culture Collection (CCTCC) on March 25, 2025, the preservation address is Wuhan University, Wuhan, China, the postcode is 510070, the preservation number is CCTCC NO: M 2025587, the culture name is Fusobacterium sp. LSCC04, and the classification name is Fusobacterium sp. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1The results of CCK8 experiment proved that Fusobacterium LSCC04 could promote the proliferation of TU686 and AMC-HN-8 cells.

[0038] Figure 2 The results of cell cloning experiment proved that Fusobacterium LSCC04 could promote the cloning of laryngeal cancer cells AMC-HN-8 and TU686.

[0039] Figure 3 The schematic diagram of Fusobacterium LSCC04 genome sequence.

[0040] Figure 4 The schematic diagram of Fusobacterium LSCC04 under light microscope after Gram staining.

[0041] Figure 5 The schematic diagram of Fusobacterium LSCC04 colony on Columbia medium. DETAILED DESCRIPTION

[0042] The present application will be further described in the following examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to conventional methods and conditions, or according to the instructions of the products.

[0043] Example 1: Preservation of Fusobacterium LSCC04, characteristics and identification of new species

[0044] A strain of Fusobacterium sp. LSCC04 derived from laryngeal squamous cell carcinoma is provided, with the preservation number CCTCC NO: M 2025587, preserved in the China Center for Type Culture Collection on March 25, 2025, and the address of the preservation unit is China·Wuhan·Wuhan University.

[0045] The light microscope morphology is red, fusiform after Gram staining, as shown in Figure 4 After 7 days of culture on Columbia medium, relatively large, irregular round edge, flat, translucent grayish white colonies were formed Figure 5 The full length of Fusobacterium LSCC04 genome sequence is 2245556 bp, which consists of a circular chromosome (see Figure 3 ). The number of coding genes is 2198. The total length of all coding genes is 2,048,661 bp, the average length is 932 bp, and the coding region accounts for 91.23% of the total length of the genome.

[0046] The 16S rRNA sequence is (SEQ ID NO: 1):

[0047] AGAGTTTGATCCTGGCTCAGGATGAACGCTGACAGAATGCTTAACACATGCAAGTCTACTTGAATTTGGGTCTTTG

[0048] ACTTAAATTTGGGTGGCGGACGGGTGAGTAACGCGTAAAGAACTTGCCTCACAGCTAGGGACAACATTTGGAAACG

[0049] AATGCTAATACCTGATATTATGATTTTAGGGCATCCTAAGATTATGAAAGCTATATGCGCTGTGAGAGAGCTTTGC

[0050] GTCCCATTAGCTAGTTGGAGAGGTAACGGCTCACCAAGGCGATGATGGGTAGCCGGCCTGAGAGGGTGAACGGCCA

[0051] CAAGGGGACTGAGACACGGCCCTTACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGACCAAAAGTCTG

[0052] ATCCAGCAATTCTGTGTGCACGATGACGTTTTTCGGAATGTAAAGTGCTTTCAGTTGGGAAGAAAAAAATGACGGT

[0053] ACCAACAGAAGAAGTGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCACAAGCGTTATCCGGATTT

[0054] ATTGGGCGTAAAGCGCGTCTAGGTGGTTATGTAAGTCTGATGTGAAAATGCAGGGCTCAACTCTGTATTGCGTTGG

[0055] AAACTGTGTAACTAGAGTACTGGAGAGGTAAGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGA

[0056] ATGCCGATGGGGAAGCCAGCTTACTGGACAGATACTGACGCTAAAGCGCGAAAGCGTGGGTAGCAAACAGGATTAG

[0057] ATACCCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTTGGGGGTCGAACCTCAGCGCCCAAGCAAACGCGT

[0058] AAGTAATCCGCCTGGGGAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAG

[0059] CATGTGGTTTAATTCGACGCAACGCGAGGAACCTTACCAGCGTTTGACATCTTAGGAATGAGACAGAGATGTTTCA

[0060] GTGTCCCTTCGGGGAAACCTAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT

[0061] CCCGCAACGAGCGCAACCCCTTTCGTATGTTACCATCATTAAGTTGGGGACTCATGCGATACTGCCTACGATGAGT

[0062] AGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATACGCTGGGCTACACACGTGCTACAATGGGTAGTA

[0063] CAGAGAGTCGCAAAGCCGCGAGGTGGAGCTAATCTCAGAAAACTATTCTTAGTTCGGATTGTACTCTGCAACTCGA

[0064] GTACATGAAGTTGGAATCGCTAGTAATCGCGAATCAGCAATGTCGCGGTGAATACGTTCTCGGGTCTTGTACACAC

[0065] CGCCCGTCACACCACGAGAGTTGGTTGCACCTGAAGTAGCAGGCCTAACCGTAAGGAGGGATGCTCCGAGGGTGTG

[0066] ATTAGCGATTGGGGTGAAGTCGTAACAAGGTATCCGTACGGGAACGTGCGGATGGATCACCT

[0067] ANI comparison was performed on all reference strains of Fusobacterium species in the NCBI database, among which the similarity between the periodontal Fusobacterium ATCC 33693 (GCF_000160475.1) and Fusobacterium LSCC04 was the highest. The average nucleotide identity (ANI) value of the whole genome comparison between Fusobacterium LSCC04 and the most similar periodontal Fusobacterium ATCC 33693 (GCF_000160475.1) was 91.45. It is generally believed that ANI≥95 is the same species, indicating that the strain belongs to a new species of Fusobacterium.

[0068] Example Two: In vitro functional verification of the carcinogenic effect of Fusobacterium LSCC04

[0069] CCK8 specific experimental steps: Take freshly prepared TU686 (purchased from the Chinese Academy of Sciences) and AMC-HN-8 (purchased from the Chinese Academy of Sciences) cell suspension, inoculate into 96-well plates (1×10 3 cells / well). After 24 hours of culture until the cells adhere, wash with PBS and replace the anti-culture medium. Take freshly prepared Fusobacterium LSCC04 bacterial suspension, centrifuge and resuspend the bacteria with PBS, and co-culture with TU686 and AMC-HN-8 cells (with a multiplicity of infection MOI of 100, 200, 400, and 800, respectively); use the same volume of PBS as a blank control, each with 7 repeated wells. Culture under normal conditions, and after 4 days, detect the cell proliferation activity by CCK8 method (follow the steps given in the instruction manual). Replace the freshly prepared bacterial suspension every 24 hours. The results are shown in Figure 1 Fusobacterium LSCC04 can significantly promote the proliferation of TU686 and AMC-HN-8 cells, and the proliferation activity at MOI=200 is significantly higher than that of the control group (P<0.001).

[0070] Cell clone experiment steps: cells in logarithmic growth phase were trypsinized, resuspended into cell suspension with complete culture medium (basic medium + 10% fetal bovine serum) and counted, 500 cells per well were inoculated in 6-well plates, fresh Fusobacterium LSCC04 bacterial suspension was prepared, centrifuged, resuspended with PBS, and co-cultured with TU686 and AMC-HN-8 cells respectively, MOI was 200, the optimal concentration of CCK8 experiment, and the culture was continued until the cell number in most single clones was greater than 50, the medium was changed every 3 days during the process and the cell state was observed; PBS was washed once, 1 mL of 4% paraformaldehyde was added per well for fixation for 30-60 min, PBS was washed once; crystal violet staining was performed for 15 min, PBS was washed for 3 times; and observation was performed by taking pictures. The results are shown in Figure 2 Fusobacterium LSCC04 can significantly promote the clone formation of laryngeal cancer cells AMC-HN-8 and TU686.

Claims

1. A type of fusobacterium Fusobacterium sp.LSCC04 Its accession number is CCTCC NO: M 2025587.

2. The Clostridium as described in claim 1 Fusobacterium sp.LSCC04 Its 16S rRNA sequence is shown in SEQ ID NO:

1.

3. The Clostridium as described in claim 1 Fusobacterium sp.LSCC04 Its full-length genome sequence is 2245556 bp.

4. The Clostridium as described in claim 1 Fusobacterium sp.LSCC04 Its characteristics are, The Fusobacterium Fusobacterium sp.LSCC04 After Gram staining, it appears red and spindle-shaped under a microscope. After culturing, it forms irregular round colonies with neat edges, smooth and moist surfaces, and a slightly convex center, appearing as translucent grayish-white.

5. A Fusobacterium colony, characterized in that, The Fusobacterium colonies are composed of Fusobacterium as described in any one of claims 1-4. Fusobacterium sp.LSCC04 Acquired through training.

6. A Fusobacterium bacterial suspension, characterized in that, The fusobacterium bacterial suspension is composed of fusobacterium as described in any one of claims 1-4. Fusobacterium sp.LSCC04 Acquired through training.

7. The method for culturing Fusobacterium colonies as described in claim 5 or Fusobacterium bacterial suspension as described in claim 6, characterized in that, Including the Clostridium Fusobacterium sp.LSCC04 It is obtained by culturing on liquid or solid culture media.

8. The Clostridium as described in any one of claims 1-4 Fusobacterium sp.LSCC04 Application in the preparation of Fusobacterium colonies or Fusobacterium bacterial suspensions.

9. The Clostridium as described in any one of claims 1-4 Fusobacterium sp.LSCC04 The use of the Fusobacterium colonies as described in claim 5 or the Fusobacterium bacterial suspension as described in claim 6 in promoting the proliferation or cloning of TU686 cells and / or AMC-HN-8 cells.

10. The Clostridium as described in any one of claims 1-4 Fusobacterium sp. LSCC04 Application in constructing an in vitro validation model, wherein the in vitro validation model is laryngeal squamous cell carcinoma.

11. The application as described in claim 10, characterized in that, The laryngeal squamous cell carcinoma cells were TU686 cells and / or AMC-HN-8 cells.

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