CN115011710B - Intragastric flora marker related to prognosis of gastric cancer and application thereof - Google Patents
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Abstract
The invention discloses an intragastric flora marker related to prognosis of gastric cancer and application thereof. Application of methyl bacillus as a stomach flora marker in preparing a reagent for predicting prognosis of gastric cancer. The application of a reagent for detecting the abundance of the bacillus methylotrophicus in preparing a reagent for predicting the prognosis of gastric cancer. According to the invention, the bacillus methylobacterium is firstly found to be related to prognosis of gastric cancer, the abundance of the bacillus methylobacterium shows a significant difference between patients with longer survival time and patients with shorter survival time, and the abundance of the bacillus methylobacterium is significantly and negatively related to TRM cells related to prognosis of tumor, so that the bacillus methylobacterium can be used as a detection marker for predicting prognosis of gastric cancer patients.
Description
Technical Field
The invention belongs to the field of biological detection, and relates to a gastric flora marker related to prognosis of gastric cancer and application thereof.
Background
Gastric cancer, the fifth most common malignancy in the world, occurs in about one million new cases each year, of which about 738,000 die, severely threatening human life health and quality of life. Gastric cancer progression is generally from Atrophic Gastritis (AG) to Intestinal Metaplasia (IM) further to gastric cancer, and is closely related to host-related factors such as eating habits, genetic susceptibility, and environmental factors, including microbial infection. Most stomach cancer belongs to adenocarcinoma, has no obvious symptoms in early stage, is often similar to the symptoms of chronic gastric diseases such as gastritis, gastric ulcer and the like, is easy to ignore, has the characteristics of strong invasiveness, high recurrence rate and the like, and brings great challenges to clinical treatment. Currently, endoscopic resection is the main treatment method for early gastric cancer, and non-early surgically resectable gastric cancer can be treated by surgery to resect lesions. The advanced gastric cancer mainly comprises radiotherapy and chemotherapy, has poorer prognosis and median survival time of less than one year.
Recently, it has been found that the flora in tumor tissue is closely related to the development of tumors. There is a correlation between the predicted function of the intratumoral bacterial type, flora in a particular tumor type and tumor subtype and the smoking status of the patient, tumor immunotherapy sensitivity. However, at present, the related flora in tissues of other diseases has not been reported yet.
Disclosure of Invention
One of the purposes of the invention is to provide an application of the intragastric flora marker related to prognosis of gastric cancer. The invention provides that the abundance of the bacillus methylobacterium in the stomach has negative correlation with prognosis of gastric cancer patients for the first time, the abundance of the bacillus methylobacterium can be used as a stomach adenocarcinoma biomarker, and the promotion effect of the bacillus methylobacterium in stomach adenocarcinoma diseases is revealed, thereby providing a basis for treating gastric cancer.
It is a second object of the present invention to provide a product for predicting prognosis of gastric cancer.
The aim of the invention can be achieved by the following technical scheme:
application of methyl bacillus as a stomach flora marker in preparing a reagent for predicting prognosis of gastric cancer.
The application of a reagent for detecting the abundance of the bacillus methylotrophicus in preparing a reagent for predicting the prognosis of gastric cancer.
Preferably, the reagent for detecting the abundance of the methylobacterium is selected from a probe, a primer or an antibody for detecting the abundance of the methylobacterium.
As a preferred embodiment of the present invention, the primer is a primer for detecting 16SrRNA of Methylobacillus bacterium.
A reagent for predicting prognosis of gastric cancer, comprising a reagent for detecting abundance of methylobacterium.
Preferably, the reagent of the present invention further comprises a reagent for extracting genomic DNA of a microorganism.
The application of the bacillus methylobacterium serving as the intragastric flora marker in screening gastric cancer therapeutic drugs.
A system for predicting prognosis of gastric cancer using intragastric flora markers, comprising:
(1) A nucleic acid sample separation unit for separating a nucleic acid sample of the intragastric flora from a subject;
(2) A sequencing unit for sequencing the isolated intragastric flora nucleic acid sample to obtain a sequencing result;
(3) The data processing unit is used for detecting the relative abundance of the microbial markers in the gastric flora according to the sequencing result, and analyzing the obtained relative abundance value to obtain the critical value of the microbial markers;
(4) And the result judging unit is used for comparing the critical value of the microbial marker obtained by the data processing unit with a set diagnostic value.
With this system, the relative abundance of the markers of the invention in the intragastric flora can be detected. Thus, it is possible to determine whether the subject individual is a gastric cancer patient and a gastric cancer prognosis by comparing the obtained relative abundance with a predetermined threshold value.
Further, the product also includes an agent for extracting genomic DNA of a microorganism or the like.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the bacillus methylobacterium is firstly found to be related to prognosis of gastric cancer, the abundance of the bacillus methylobacterium shows a significant difference between patients with longer survival time and patients with shorter survival time, and the abundance of the bacillus methylobacterium is significantly and negatively related to TRM cells related to prognosis of tumor, so that the bacillus methylobacterium can be used as a detection marker for predicting prognosis of gastric cancer patients.
The inventor adopts 16s RNA sequencing technology in the early stage to screen out 10 genus level differential bacteria which are obviously enriched in gastric cancer from 53 gastric cancer tissues and 30 chronic gastritis tissues. Then, we count the prognosis survival time of gastric cancer patients, correlate the survival result with the abundance of differential bacteria, find that there is significant correlation between the methyl bacillus and the survival time of patients, and the abundance of the methyl bacillus in the stomach of patients with shorter average survival time is high. The combination of clinical pathological feature analysis shows that the high abundance of the methyl bacillus is obviously related to vascular cancer embolism. Suggesting that the methylobacterium is closely related to the development, prognosis and the like of gastric cancer.
To clarify the role of methylobacterium in gastric cancer: (1) first we found that methylobacterium is associated with peripheral blood lymphocytes from gastric cancer patients. (2) Further studies have found that the number of tissue-dominant memory T cells (TRMs) in tumors of a methylobacterium-enriched patient is small, suggesting that methylobacterium may inhibit the formation and residence of TRM cells, resulting in poor prognosis for the patient. (3) Furthermore, methylobacterium is inversely related to the expression amount of TGF- β in the tumor microenvironment, and TGF- β is a key cytokine required for TRM cell formation. The above results suggest that methylobacterium is a dominant flora in the gastric tumor microenvironment, and that it may inhibit anti-tumor TRM cell formation, a potential flora marker associated with prognosis. Thus, based on the above-described research work, the present invention proposes that methylobacterium can be used as a marker of the gastric flora for predicting prognosis of gastric cancer. Use of methylobacterium as a marker of intragastric flora in the preparation of a product for predicting prognosis of gastric cancer. Methylobacillus is used as a stomach flora marker to predict gastric cancer prognosis. Methylobacillus as a marker of intragastric flora provides a kit for predicting prognosis of gastric cancer.
Drawings
FIG. 1 comparison of the abundance of Methylobacillus bacteria in gastric tissues in gastric cancer patients and chronic gastritis patients
FIG. 2 ROC Curve analysis of relative abundance of Methylobacillus as a predictor of gastric cancer
FIG. 3 shows the probability of survival of clinical gastric cancer patients based on the abundance level of Methylobacillus in gastric cancer patients. Right graph, total survival probability; left graph, median survival probability
FIG. 4 comparison of clinical vascular carcinoma suppository characteristics of gastric cancer patients with Methylobacillus abundance in gastric tissue
FIG. 5 correlation heat map between Methylobacillus abundance in stomach tissue and peripheral blood immune cells of gastric cancer patient
FIG. 6 shows a typical example of co-expression of CD103 and CD8 by multicolor immunofluorescent staining of primary gastric cancer. CD8+CD103+ cell quantification in 53 independent cases, including representative cases
FIG. 7 Total survival probability of gastric cancer patients based on gastric cancer TRM cell infiltration level in ACRG database
FIG. 8 relationship between CD8+ TRM cells of gastric cancer patients and clinically characterized vascular cancer plugs
FIG. 9 flow cytometry analysis of the relationship between Methylobacillus abundance and TRM cells. Expression of CD103+ on CD8+ cells in 19 independent cases (flow cytometry data)
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
EXAMPLE 1 intragastric flora screening associated with gastric cancer
1. Sample acquisition:
30 chronic gastritis tissues were collected and designated as G group and 53 gastric cancer tissues were collected and designated as GC group. All samples were collected under sterile conditions, cryopreserved at-80 ℃ and ready for subsequent sequencing. All samples were taken with the ethical committee agreeing that data collection followed the principle outlined in the declaration of helsinki.
2. 16S rRNA sequencing
We delegated 16S rRNA sequencing by Tianjin North Bioinformation technologies Co.
3. Data analysis
Data preprocessing
The fastq file is exported from the IonS5TMXL down machine data. The data of each sample is distinguished according to the barcode sequence. The Raw Data (Raw Data) obtained by sequencing has a certain proportion of interference Data (Dirty Data), and in order to make the result of information analysis more accurate and reliable, the Raw Data is spliced and filtered to obtain effective Data (Clean Data). To study the species composition of each sample, the Effective Tags of all samples were OTUs (Operational Taxonomic Units) clustered with 97% Identity, and then species annotated for the sequences representative of OTUs. And according to the OTUs clustering result, performing species annotation on the sequence of each OTU on one hand to obtain corresponding species information and abundance distribution conditions based on the species. Meanwhile, the OTUs are analyzed in terms of abundance, alpha diversity calculation, venn diagrams, petal diagrams and the like, so that information on the abundance and uniformity of species in the samples, common and specific OTUs among different samples or groups and the like are obtained.
2 flora species differential analysis
According to species annotation and abundance information of all samples (groups) at the genus level, a maximum ranking method is adopted, the genus with the top 35 abundance ranks (the abundance of the groups is the average abundance of all samples in the group), clustering is carried out from the species level according to the abundance information of the genus in each sample, and an R software pheeatmap package is used for drawing a heat map so as to be convenient for finding which species are more aggregated or have lower content in which samples. To avoid the effects of too low abundance, T-test tests between groups were performed in the top ten abundance ranked species to find species with significant differences (p-value < 0.05).
3 results
In combination with clinical data, we found that there was a higher correlation between methylobacterium in the differential bacteria and gastric cancer, and the results of species differential analysis showed that the tissue abundance of methylobacterium in patients with gastric cancer was significantly higher than that in patients with chronic gastritis (figure 1). It is suggested that the methylobacterium can be used as a detection marker for diagnosis or prognosis of gastric cancer.
Example 2 correlation with gastric cancer
1. Analysis of the collected patient clinical information and sequencing results of example 1
2. Results
The ROC curve was analyzed using methylobacterium as a detection variable (see fig. 2). The ROC curve result shows that the AUC value is 0.7732, which indicates that the application of the methylobacterium to the diagnosis of gastric cancer has higher diagnosis efficiency. Furthermore, the results after correlating the abundance of methylobacterium with the average survival of the patient showed that the average survival of the methylobacterium-enriched patient was short (fig. 3), and that methylobacterium was positively correlated with the patient's clinical signature vascular cancer plug (fig. 4), suggesting that methylobacterium could be an intragastric flora marker for prognosis of gastric cancer.
EXAMPLE 3 investigation of the effect of Methylobacillus on TRM cells
1. Methylobacillus and peripheral blood lymphocyte relationship
Blood routine information of 53 gastric cancer patients is clinically obtained, including: total lymphocytes, cd3+ T cells, cd4+ T cells, cd8+ T cells, NK cells, B cells, NKT cells, and activated T cell numbers. And (3) carrying out correlation analysis on the abundance of the methylobacterium and the quantity of the peripheral blood lymphocytes, and exploring the influence of the methylobacterium on the peripheral blood lymphocytes.
2. Multicolor immunofluorescence
Staining was performed using primary antibodies to markers CD103 (TRM cell markers), CD8 and CD3 for immunohistochemical analysis. Antibody detection uses Opal polymer HRP ms+rb immunohistochemical detection reagent (PelkinElmer, boston, MA). Staining was performed consecutively using the same protocol as immunohistochemistry and detection of each label was completed before the next antibody was applied. The staining channel includes: four types of PPD520, PPD570, PPD650 and DAPI are used. After staining was completed, the sections were imaged using a Vectra 3.0 spectral imaging system (PerkinElmer). Infiltration of CD8 and TRM cells was observed and counted.
3. Association analysis of methylobacterium with TRM cells:
methylobacillus were grouped into high and low groups (based on median) and analyzed in association with fluorescence data to investigate the effect of Methylobacillus on TRM cell content in gastric tumor microenvironment.
4. Survival data analysis of Asian Cancer Research Group (ACRG)
We obtained survival data for 300 gastric adenocarcinoma patients from Asian Cancer Research Group (ACRG), analyzed and collated these data, and explored the relationship between TRM cell infiltration number and survival prognosis.
5. Flow cytometry
The number of TRM cells of CD8+CD103+ was examined by flow cytometry, and the samples were classified into a high group of Methylobacillus bacteria and a low group of Methylobacillus bacteria, and the effect of Methylobacillus bacteria on the number of TRM cells in the above polychromatic fluorescence results was examined.
(1) Cell resuscitation: taking tumor-infiltrating lymphocytes of a gastric cancer patient frozen in a laboratory (-80 ℃) and resuscitating in a constant-temperature water bath kettle at 37 ℃ (note that the cells are damaged due to the rapid resuscitating and long time);
(2) Diluting the resuscitated cells with PBS, mixing well, centrifuging (1500 r,5 min), centrifuging, and pouring out supernatant;
(3) Adding 1ml of culture medium into the tube after discarding the supernatant, repeatedly blowing with a pipetting gun to resuspend the cells, transferring the resuspended cells into well plates (plates), adding 2 μl of stimulant into each well plate, shaking uniformly, and culturing in a constant temperature incubator at 37deg.C for 4h;
(4) After 4h of culture, the well plate was removed and the supernatant was aspirated into 1.5ml EP tubes (macrophage adherent growth, T cell suspension growth);
(5) After centrifugation of EP tube at 4℃for 500g for 5min, the supernatant was pipetted off (note: to prevent the bottom cells from being inadvertently discarded when the supernatant was discarded, it is preferable to reserve 100. Mu.l of supernatant);
(6) Washing: washing the cells with 1ml of PBS solution, mixing well and centrifuging (4 ℃,500g,5 min);
(7) Closing: centrifuging, removing supernatant, adding 1 μl of blocking solution, mixing, and incubating at 4deg.C for 20min;
(8) Cell surface staining: adding surface antibody into the tube (the surface antibody is prepared in advance), mixing uniformly, and incubating at 4 ℃ for 30min;
(9) Washing: same (6)
(10) Cell fixation and membrane rupture: add 250. Mu.l of fixative to the tube and incubate at 4℃for 20min (note: mix cells before addition);
(11) Washing: washing the cells with 1 XBD Perm/Wash buffer (membrane rupture liquid) 2 times (1 ml each time), mixing, and centrifuging (4deg.C, 1000g,5 min);
(12) Intracellular factor staining: centrifuging, removing supernatant, adding intracellular antibody (prepared with intracellular antibody) into the tube, mixing, and incubating at 4deg.C for 30min;
(13) Washing: same (11)
(14) And (3) filtering: after discarding the supernatant, 250. Mu.l PBS was added to the tube, which was filtered into a new EP tube after mixing (note: no filtration is needed if the cell content is low);
(15) And detecting the sample by a flow cytometer.
6. Results
Methylobacillus is associated with total lymphocytes in patient peripheral blood lymphocytes, suggesting that Methylobacillus may affect prognosis by acting on lymphocytes (FIG. 5). Multicolor immunofluorescence and flow cytometry results showed that methylobacterium was significantly correlated with TRM cells in the tumor microenvironment, with a high population of methylobacterium having a low TRM cell content and a low population of TRM cells having a high TRM cell content (fig. 6 and 9). TRM cells play an important role in anti-tumor, so we found by analysis of gastric cancer sample data in the ACRG database that samples with high numbers of TRM cells survived significantly longer than samples with low numbers (fig. 7). Furthermore, TRM cells were significantly inversely related to vascular cancer plugs, suggesting that TRM cells may be a factor in preventing the occurrence of vascular cancer plugs (fig. 8). Overall, methylobacterium may result in poor prognosis for gastric cancer patients by inhibiting TRM cells.
Claims (3)
1. Application of methyl bacillus as a stomach flora marker in preparing a reagent for predicting prognosis of gastric cancer.
2. Application of a reagent for detecting the abundance of the bacillus methylotrophicus in preparing a reagent for predicting the prognosis of gastric cancer; the reagent for detecting the abundance of the methyl bacillus is selected from a probe, a primer or an antibody for detecting the methyl bacillus.
3. The use according to claim 2, wherein the primer is a primer for detecting 16SrRNA of methylobacterium.
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| CN108220410A (en) * | 2016-12-16 | 2018-06-29 | Md保健株式会社 | For by the macro genome analysis of bacterium come the kit and method of diagnosis of gastric cancer |
| CN112980977A (en) * | 2021-03-19 | 2021-06-18 | 天水师范学院 | Pathogenic bacteria identification primer for apple tree branch rot and disease degree grading method |
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| CN112980977A (en) * | 2021-03-19 | 2021-06-18 | 天水师范学院 | Pathogenic bacteria identification primer for apple tree branch rot and disease degree grading method |
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| Dysbiosis of the microbiome in gastric carcinogenesis;Natalia Castano-Rodriguez等;SCIENTIFIC REPORTS;第7卷;15957第4页、图3 * |
| 胃炎癌转变过程中胃内菌群构成变化的研究;董雨;中国优秀硕士学位论文全文数据库医药卫生科技辑;摘要、第8-9、17、22页 * |
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