CN110903997B - Fusobacterium nucleatum obtained from colorectal cancer tumor tissue and application thereof - Google Patents
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Abstract
The invention provides a Fusobacterium nucleatum THCT14E2 which is obtained from colorectal cancer tumor tissue and does not belong to known Fusobacterium nucleatum subspecies, and the gene sequence of 16SrRNA of the Fusobacterium nucleatum is shown as SEQ ID NO. 1. The classification of the THCT14E2 is named as Fusobaterium nucleatum, and the preservation number is CCTCC NO: m2019361, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection. The result of co-culture of the colorectal cancer cell line and the THCT14E2 shows that the THCT14E2 can obviously promote the proliferation of colorectal cancer cells, so that the THCT14E2 can promote the understanding of the microecological diversity of colorectal cancer and is beneficial to the functional research of fusobacterium nucleatum from the colorectal cancer.
Description
Technical Field
The invention relates to the field of fusobacterium nucleatum, and particularly relates to fusobacterium nucleatum obtained from colorectal cancer tumor tissues and application thereof.
Background
Colorectal cancer is a high-grade cancer in the world, and the disease burden caused by the colorectal cancer is a difficult problem for scientists to struggle. At present, China has become the country with the most new cases of colorectal cancer in the year. Previous epidemiological investigations and experimental studies have generally confirmed that only 5% -15% of colorectal cancer attacks are due to genetic factors, and the rest over 85% are mainly influenced by the environment, such as dietary structure changes and intestinal microecological imbalance. Colorectal cancer and intestinal micro-ecological disorder are being studied, in which fusobacterium nucleatum (fusobacterium nucleatum) in the intestinal tract is closely related to the occurrence and development of colorectal cancer.
Fusobacterium nucleatum is an oral colonization bacterium, and four subspecies are known, including nucleate, pleomorphic, venus, and animal subspecies. Clinical studies have demonstrated significantly elevated fusobacterium nucleatum levels in colorectal cancer patients, but the abundance thereof has not been determined accurately to sub-species levels. The laboratory studies were carried out using essentially the standard strain of fusobacterium nucleatum belonging to the subspecies nucleatum, but not from isolates of intestinal origin.
The intestinal flora has polymorphism, and bacteria have difference in strain and strain level. There is currently a lack of diverse fusobacterium nucleatum strains for colorectal cancer studies.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides fusobacterium nucleatum obtained from colorectal cancer tumor tissues and application of fusobacterium nucleatum in colorectal cancer microecological research.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a Fusobacterium nucleatum obtained from colorectal cancer tumor tissue, which is named THCT14E2, and the 16SrRNA gene sequence of the Fusobacterium nucleatum is shown as follows:
>16S_rRNA_gene THCT14E2[CCTCC M 2019361]
AGAGTTTGATCCTGGCTCAGGATGAACGCTGACAGAATGCTTAACACATGCAAGTCAACTTGAACTTCGGTTTGGGTGGCGGACGGGTGAGTAACGCG TAAAGAACTTGCCTCACAGTTAGGGACAACATTTGGAAACGAATGCTAATACCTGATATTATGATTTTAGGGCATCCTAGAATTATGAAAGCTATATGCGCTGTGAGAGAGCTTTGCGTCCCATTAGCTAGTTGGAGAGGTAACGGCTCACCAAGGCGATGATGGGTAGCCGGCCTGAGAGGGTGAACGGCCACAAGGGGACTGAGACACGGCCCTTACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGACCAAGAGTCTGATCCAGCAATTCTGTGTGCACGATGAAGTTTTTCGGAATGTAAAGTGCTTTCAGTTGGGAAGAAAAAAATGACGGTACCAACAGAAGAAGTGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCACAAGCGTTATCCGGATTTATTGGGCGTAAAGCGCGTCTAGGTGGTTATGTAAGTCTGATGTGAAAATGCAGGGCTCAACTCTGTATTGCGTTGGAAACTGTGTAACTAGAGTACTGGAGAGGTAAGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATGGGGAAGCCAGCTTACTGGACAGATACTGACGCTAAAGCGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTTGGGGGTCGAACCTCAGCGCCCAAGCAAACGCGATAAGTAATCCGCCTGGGGAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAGGAACCTTACCAGCGTTTGACATCTTAGGAATGAGACAGAGATGTTTCAGTGTCCCTTCGGGGAAACCTAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTTTCGTATGTTACCATCATTAAGTTGGGGACTCATGCGATACTGCCTGCGATGAGCAGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATACGCTGGGCTACACACGTGCTACAATGGGTAGTACAGAGAGTCGCAAAGCCGTGAGGTGGAGCTAATCTCAGAAAACTATTCTTAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGCGAATCAGCAATGTCGCGGTGAATACGTTCTCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTGGTTGCACCTGAAGTAGCAGGCCTAACCGCAAGGAGGGATGCTCCGAGGGTGTGATTAGCGATTGGGGTGAAGTCGTAACAAGGTATCCGTACGGGAACGTGCGGATGGATCACCT(SEQ ID NO:1)。
further, the strain is classified and named as Fusobaterium nucleatum, and the preservation number of the strain is CCTCC NO: m2019361, wherein the preservation date is 2019, 05 and 17, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university in Wuhan City.
Further, the strain was isolated from a fresh surgically excised specimen using a selection medium containing neomycin sulfate, vancomycin, and crystal violet. The separation method comprises the following steps: mechanically breaking the specimen, diluting for 2-4 times according to gradient of 10 times, laying on the selective culture medium, anaerobic culturing at 37 ℃, selecting single colony, purifying and culturing, and identifying the strain.
Furthermore, the strain is cultured under anaerobic condition at 37 deg.C in a Columbia blood plate or thioglycollate fluid medium.
Furthermore, the strain is red and slender rod-shaped under a mirror after gram staining.
Further, the strain is rough, round, irregular in edge, white in middle, translucent in edge, and colony on a columbia blood plate.
In a second aspect of the invention, there is provided the use of fusobacterium nucleatum isolated from colon cancer tumor tissue in the microecological study of colorectal cancer.
Further, the colorectal cancer cell is the HCT116 cell line or the LoVo cell line.
The present invention provides a Fusobacterium nucleatum isolated from colon cancer, which is not a known subspecies, and demonstrated to promote proliferation of colorectal cancer cell lines. The fusobacterium nucleatum is helpful for understanding the microecological diversity of colorectal cancer and is also helpful for functional research of fusobacterium nucleatum from colorectal cancer.
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The invention discloses Fusobacterium nucleatum THCT14E2 obtained from colorectal cancer tumor tissue, which is preserved and is classified and named as Fusobacterium tuberculosis, and the preservation number is CCTCC NO: m2019361, the preservation date is 2019, 05 and 17 months, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university in Wuhan City.
FIG. 1 is a graph showing the proliferation of the HCT116 cell line after co-culture with THCT14E2 in one embodiment of the present invention (in the left graph, the growth curve of HCT116 cells; in the right graph, the proliferation is compared for 72 hours of co-culture (two-tailed t test p ═ 0.03));
FIG. 2 is a graph showing the proliferation of a LoVo cell line after co-culture with THCT14E2 in one embodiment of the present invention (in which the left graph is a LoVo cell growth curve; and the right graph is a comparison of proliferation at 72 hours of co-culture (two-tailed t-test p < 0.01)).
Detailed Description
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
The Fusobacterium nucleatum THCT14E2 obtained from colorectal cancer tumor tissue is preserved and named as Fusobacterium nucleatum with the preservation number of CCTCC NO: m2019361, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection.
The sequence of the 16SrRNA gene of the strain is shown as SEQ ID NO. 1, and the strain has 99.59% consistency with the sequence of the 16SrRNA gene of F.nuclear subsp.polymorphhum strain ATCC 10953 (NCBI accession number NR _113141.1), and has 97.84% consistency with the sequence of the 16SrRNA gene of F.nuclear subsp.nuclear strain ATCC25586 (NCBI accession number NR _ 114702.1). 97.83% identity with the 16s rrna gene sequence of f.nuclear subsp.vincentii strain ATCC 51190 (NCBI accession No. NR — 113040.1). 97.63% identity with the 16s rrna gene sequence of f.nuclear subsp.animalis strain JCM 11025 (NCBI accession No. NR _ 113378.1).
The strain is separated from a fresh surgical excision specimen, the screening culture medium contains fusobacterium nucleatum screening culture medium containing neomycin sulfate, vancomycin and crystal violet, the fusobacterium nucleatum screening culture medium is rough, round, irregular in edge, white in the middle, semitransparent in edge and colony on a Columbia blood plate, and the fusobacterium nucleatum screening culture medium is red, slender and rod-shaped under a mirror after gram staining.
The genome of this strain was analyzed by Jspeces software, and had a genome wide ANIb value of 90.67 for F.nuclear subsp.nuclear strain ATCC25586 (NCBI genome accession number: GCA _003019295.1), of 89.13 for F.nuclear subsp.analimus strain 7_1(NCBI genome accession number: GCA _000158275.2), of 89.75 for F.nuclear subsp.vingia strain 3_1_36A2(NCBI genome accession number: GCA _000162235.2), and of 92.75 for F.nuclear subsp.polymorphum strain NCTC10562(NCBI genome accession number: GCA _ 001457555.1). ANIb, average nucleotide identity using BLAST (based on the average nucleotide sequence identity of BLAST), is an index for evaluating the affinity of strains, and generally is regarded as being equal to or greater than 95, i.e., a genus (species). Although the 16s rrna gene of THCT14E2 exhibited high identity with polymorphum subspecies, it did not belong to the known fusobacterium nucleatum subspecies from the genome-wide level.
The following demonstrates that F.nucleatum THCT14E2 obtained from colorectal cancer tumor tissue can promote the proliferation of colorectal cancer cell lines by co-culturing the colorectal cancer cell lines with THCT14E 2.
Example one
The THCT14E2 and the colorectal cancer HCT116 cell line are cultured together, and the specific operation steps are as follows:
collecting cultured HCT116 cells, digesting with pancreatin to obtain HCT116 cell suspension, countingInoculation into 24-well plates (1X 10)5One cell/well), cultured for 12-24 hours until the cells adhere. After attachment, wash with PBS and replace non-resistant medium. The experimental groups were added freshly prepared THCT14E2 bacteria and suspended with PBS such that each well was 1 × 108A CFU; an equal volume of PBS was added to the control group. Each set was prepared with 3 replicate wells. Culturing under conventional conditions. The cell proliferation activity was measured by the CCK8 method at 6 hours, 24 hours, 48 hours, and 72 hours of culture, respectively. Wherein the bacterial suspension was replaced every 24 hours.
As can be seen from fig. 1, the proliferation activity of HCT116 cells at 72 hours is significantly higher than that of the control group (p ═ 0.03), which indicates that THCT14E2 can significantly promote the proliferation of HCT116 cells.
Example two
THCT14E2 and colorectal cancer LoVo cell line were cultured together, the specific procedures were as follows:
taking cultured LoVo cells, digesting with pancreatin to prepare LoVo cell suspension, counting, inoculating to 24-well plate (1 × 10)5One cell/well), cultured for 12-24 hours until the cells adhere. After attachment, wash with PBS and replace non-resistant medium. THCT14E2 bacteria were freshly prepared and suspended in PBS and the bacterial suspension was added to the experimental group (1X 10)8CFU/well); an equal volume of PBS was added to the control group. Each set was prepared with 3 replicate wells. Culturing under conventional conditions. The cell proliferation activity was measured by the CCK8 method at 6 hours, 24 hours, 48 hours, and 72 hours of culture, respectively. Wherein the bacterial suspension was replaced every 24 hours.
From fig. 2, it can be seen that the proliferation activity of the LoVo cells at 72 hours is significantly higher than that of the control group (p <0.01), indicating that THCT14E2 can significantly promote the proliferation of the LoVo cells.
The above examples illustrate that the fusobacterium nucleatum subspecies thetaiotaomicron isolate THCT14E2 significantly promotes the proliferation of colorectal cancer tumor cell lines, contributes to the understanding of the diversity of colorectal cancer microecology, and will also contribute to the functional study of fusobacterium nucleatum from colorectal cancer.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> tenth people hospital in Shanghai City
<120> Fusobacterium nucleatum obtained from colorectal cancer tumor tissue and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1494
<212> DNA
<213> 16S_rRNA_gene THCT14E2 [CCTCC M 2019361]
<400> 1
agagtttgat cctggctcag gatgaacgct gacagaatgc ttaacacatg caagtcaact 60
tgaacttcgg tttgggtggc ggacgggtga gtaacgcgta aagaacttgc ctcacagtta 120
gggacaacat ttggaaacga atgctaatac ctgatattat gattttaggg catcctagaa 180
ttatgaaagc tatatgcgct gtgagagagc tttgcgtccc attagctagt tggagaggta 240
acggctcacc aaggcgatga tgggtagccg gcctgagagg gtgaacggcc acaaggggac 300
tgagacacgg cccttactcc tacgggaggc agcagtgggg aatattggac aatggaccaa 360
gagtctgatc cagcaattct gtgtgcacga tgaagttttt cggaatgtaa agtgctttca 420
gttgggaaga aaaaaatgac ggtaccaaca gaagaagtga cggctaaata cgtgccagca 480
gccgcggtaa tacgtatgtc acaagcgtta tccggattta ttgggcgtaa agcgcgtcta 540
ggtggttatg taagtctgat gtgaaaatgc agggctcaac tctgtattgc gttggaaact 600
gtgtaactag agtactggag aggtaagcgg aactacaagt gtagaggtga aattcgtaga 660
tatttgtagg aatgccgatg gggaagccag cttactggac agatactgac gctaaagcgc 720
gaaagcgtgg gtagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgatt 780
actaggtgtt gggggtcgaa cctcagcgcc caagcaaacg cgataagtaa tccgcctggg 840
gagtacgtac gcaagtatga aactcaaagg aattgacggg gacccgcaca agcggtggag 900
catgtggttt aattcgacgc aacgcgagga accttaccag cgtttgacat cttaggaatg 960
agacagagat gtttcagtgt cccttcgggg aaacctaaag acaggtggtg catggctgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cctttcgtat 1080
gttaccatca ttaagttggg gactcatgcg atactgcctg cgatgagcag gaggaaggtg 1140
gggatgacgt caagtcatca tgccccttat acgctgggct acacacgtgc tacaatgggt 1200
agtacagaga gtcgcaaagc cgtgaggtgg agctaatctc agaaaactat tcttagttcg 1260
gattgtactc tgcaactcga gtacatgaag ttggaatcgc tagtaatcgc gaatcagcaa 1320
tgtcgcggtg aatacgttct cgggtcttgt acacaccgcc cgtcacacca cgagagttgg 1380
ttgcacctga agtagcaggc ctaaccgcaa ggagggatgc tccgagggtg tgattagcga 1440
ttggggtgaa gtcgtaacaa ggtatccgta cgggaacgtg cggatggatc acct 1494
Claims (2)
1. A Fusobacterium nucleatum separated from colon cancer tumor tissue is characterized in that the gene sequence of 16SrRNA of the Fusobacterium nucleatum is shown as SEQ ID NO. 1; the Fusobacterium nucleatum is THCT14E2, and is named by classificationFusobacterium nucleatumThe preservation number is CCTCC NO: m2019361, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection.
2. Use of fusobacterium nucleatum isolated from colon cancer tumor tissue as claimed in claim 1 for the study of colorectal cancer microecology for non-disease diagnosis and treatment.
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| CN111004738B (en) * | 2019-10-23 | 2021-09-21 | 上海市第十人民医院 | Fusobacterium nucleatum subspecies pleomorphus isolate and application thereof |
| CN111205994B (en) * | 2019-10-23 | 2021-09-21 | 上海市第十人民医院 | Fusobacterium nucleatum subspecies animal strain and application thereof |
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