CN115011710A - Gastric flora marker related to gastric cancer prognosis and application thereof - Google Patents
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Abstract
The invention discloses an intragastric flora marker related to gastric cancer prognosis and application thereof. Application of methylobacterium as a marker of gastric flora in preparation of a reagent for predicting gastric cancer prognosis. Application of the reagent for detecting the abundance of the methylobacterium in preparing the reagent for predicting gastric cancer prognosis. The invention discovers that the methylobacterium is related to the prognosis of the gastric cancer for the first time, the abundance of the methylobacterium is obviously different between patients with longer life time and patients with shorter life time, and the abundance of the methylobacterium is obviously and negatively related to TRM cells related to the prognosis of tumors, so that the methylobacterium can be used as a detection marker to predict the prognosis of the gastric cancer patients.
Description
Technical Field
The invention belongs to the field of biological detection, and relates to an intragastric flora marker related to gastric cancer prognosis and application thereof.
Background
Gastric cancer, the fifth most common malignancy in the world, occurs in about one million new cases each year, with about 738,000 people dying, and is a serious threat to human health and quality of life. Gastric cancer progression generally ranges from Atrophic Gastritis (AG) to Intestinal Metaplasia (IM) to gastric cancer, and is closely related to host-related factors such as dietary habits, genetic predisposition, and environmental factors, including microbial infection. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, are similar to the symptoms of chronic gastric diseases such as gastritis and gastric ulcer, are easy to ignore, have the characteristics of strong invasiveness, high recurrence rate and the like, and bring great challenges to clinical treatment. At present, endoscopic resection is the main treatment method for early gastric cancer, and non-early gastric cancer which can be resected by operation can be treated by operation to remove focus. The advanced gastric cancer mainly takes radiotherapy and chemotherapy as main components, the prognosis is poor, and the median survival time is 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 bacterial type, flora in a particular tumor type and tumor subtype within a tumor and the smoking status, tumor immunotherapy sensitivity of the patient. However, no report has been made on the relevant flora in tissues of other diseases.
Disclosure of Invention
The invention aims to provide an application of an intragastric flora marker related to gastric cancer prognosis. The invention firstly provides that the abundance of the intragastric flora methylobacterium has negative correlation with the prognosis of gastric cancer patients, the abundance of the methylobacterium can be used as a biomarker of gastric adenocarcinoma, and the promotion effect of the methylobacterium in the gastric adenocarcinoma diseases is disclosed, so that a basis is provided for the treatment of the gastric cancer.
The invention also aims to provide a product for predicting the prognosis of gastric cancer.
The purpose of the invention can be realized by the following technical scheme:
application of methylobacterium as a marker of gastric flora in preparation of a reagent for predicting gastric cancer prognosis.
Application of a reagent for detecting the abundance of methylobacterium in preparing a reagent for predicting gastric cancer prognosis.
Preferably, the reagent for detecting the abundance of methylobacterium is selected from a probe, a primer or an antibody for detecting the abundance of methylobacterium.
In a preferred embodiment of the present invention, the primer is a primer for detecting 16S rRNA of Methylobacterium.
An agent for predicting gastric cancer prognosis, comprising an agent for detecting methylobacterium abundance.
Preferably, the reagent further comprises a reagent for extracting genomic DNA of the microorganism.
Application of methylobacterium as a marker of gastric flora in screening gastric cancer treatment drugs.
A system for predicting gastric cancer prognosis using markers of the gastric flora, comprising:
(1) a nucleic acid sample separation unit for separating a nucleic acid sample of the gastric 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 a result judging unit for comparing the critical value of the microorganism 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 gastric flora can be detected. Therefore, whether the subject individual is a gastric cancer patient and the prognosis of gastric cancer can be determined by comparing the obtained relative abundance with a predetermined threshold value.
Further, the product also comprises a reagent for extracting microbial genome DNA and the like.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers that the methylobacterium is related to the prognosis of the gastric cancer for the first time, the abundance of the methylobacterium is obviously different between patients with longer life time and patients with shorter life time, and the abundance of the methylobacterium is obviously and negatively related to TRM cells related to the prognosis of tumors, so that the methylobacterium can be used as a detection marker to predict the prognosis of the gastric cancer patients.
The inventor previously applied 16s RNA sequencing technology to screen 10 genus-level differential bacteria which are obviously enriched in gastric cancer from 53 gastric cancer tissues and 30 chronic gastritis tissues. Then, the prognosis survival time of the gastric cancer patient is counted, the survival result of the gastric cancer patient is correlated with the abundance of the differential bacteria, the significant correlation between the methylobacterium and the survival time of the patient is found, and the intragastric methylobacterium of the patient with short average survival time is high in abundance. The high abundance of the methylobacterium is found to be obviously related to the vascular cancer embolus by combining with clinical pathological feature analysis. 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: firstly, the methylobacterium is found to be related to peripheral blood lymphocytes of patients with gastric cancer. Secondly, the further research shows that the quantity of the tissue mainstream memory T cells (TRM) in the tumor of the patient enriched with the methylobacterium is very small, which indicates that the methylobacterium can inhibit the formation and the retention of the TRM cells, and the prognosis of the patient is poor. And thirdly, the methylobacterium is in negative correlation with the expression quantity of TGF-beta in a tumor microenvironment, and the TGF-beta is a key cell factor required by TRM cell formation. The above results suggest that methylobacterium is the dominant flora in the gastric tumor microenvironment and that it may inhibit anti-tumor TRM cell formation, a potential flora marker relevant for prognosis. Therefore, based on the above research work, the present invention proposes that methylobacterium can be used as a marker of gastric flora for predicting gastric cancer prognosis. Application of methylobacterium as a marker of gastric flora in preparation of products for predicting gastric cancer prognosis. A system for predicting gastric cancer prognosis using Methylobacterium as a marker of the gastric flora. The methylobacterium as a marker of the gastric flora provides a kit for predicting gastric cancer prognosis.
Drawings
FIG. 1 comparison of the abundance of Methylobacterium in stomach tissues of patients with gastric cancer and chronic gastritis
FIG. 2 ROC curve analysis of relative abundance of Methylobacterium as prediction for gastric cancer
FIG. 3 clinical gastric cancer patient survival probability based on the abundance level of Methylobacterium in gastric cancer patients. Right graph, total survival probability; left graph, median survival probability
FIG. 4 comparison of clinical vascular cancer thrombus characteristics of gastric cancer patients with methylobacterium abundance in gastric tissues
FIG. 5 heatmap of correlation between Methylobacterium abundance in stomach tissue and peripheral blood immune cells in gastric carcinoma patients
Figure 6 shows a typical example of co-expression of CD103 and CD8 by multicolor immunofluorescent stained primary gastric cancer. CD8+ CD103+ cell quantification in 53 independent cases, including representative cases
FIG. 7 Total survival probability of clinical gastric cancer patients based on the TRM cell infiltration level of gastric cancer in ACRG database
FIG. 8 relationship between CD8+ TRM cells and clinical characteristic vascular cancer emboli of gastric cancer patients
Figure 9 flow cytometry analyses the relationship between methylobacterium 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 gastric 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 and designated as GC group. All samples were taken aseptically, stored frozen at-80 ℃ and prepared for subsequent sequencing. All the above samples were obtained with the consent of the ethics committee and the data collection followed the principles outlined in the declaration of helsinki.
2. 16S rRNA sequencing
We entrusted Tianjin Nowa Gene bioinformatics technology, Inc. with 16S rRNA sequencing.
3. Data analysis
Data pre-processing
Exporting the IonS5TMXL offline data to a fastq file. The data for each sample is distinguished according to the barcode sequence. The original 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 original Data is firstly spliced and filtered to obtain effective Data (clear Data). To investigate the species composition of each sample, OTUs (operational taxomic units) clustering was performed with 97% Identity (Identity) on the Effective Tags of all samples, followed by species annotation on representative sequences of OTUs. According to the OTUs clustering result, on one hand, species annotation is carried out on the sequence of each OTU, and corresponding species information and the abundance distribution condition based on the species are obtained. And simultaneously, analyzing abundance, Alpha diversity calculation, Venn diagram, petal diagram and the like of the OTUs to obtain abundance and uniformity information of species in the sample, common and specific OTUs information among different samples or components and the like.
2 flora species differential analysis
According to species annotation and abundance information of all samples (groups) at the genus level, a maximum value sorting method is adopted to select the genus with the top abundance ranking 35 (the abundance of the group 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 group in each sample, and a heatmap is drawn by using a R software pheatmap package, so that the fact that which species are more or less aggregated in which samples can be found is facilitated. In order to avoid the influence caused by too low abundance, T-test among groups is carried out in the species with the top ten abundance levels, and the species with obvious difference (p value <0.05) is found out.
3 results of
In combination with clinical data, we find that there is a high correlation between methylobacterium in the differential bacteria and gastric cancer, and the species difference analysis results show that the abundance of methylobacterium in the tissues of gastric cancer patients is significantly higher than that of chronic gastritis patients (fig. 1). The suggestion is that the methylobacterium can be used as a detection marker for the diagnosis or prognosis of gastric cancer.
Example 2 correlation with gastric cancer
1. The clinical information of the patients collected and the results of the sequencing of example 1 were analyzed
2. Results
Using Methylobacterium as a detection variable, the analysis yielded a ROC curve (see FIG. 2). The results of the ROC curve show that the AUC value is 0.7732, which indicates that the methylobacterium applied to the diagnosis of gastric cancer has higher diagnosis efficiency. In addition, the results obtained by correlating the abundance of methylobacterium with the mean survival time of patients show that the mean survival time of the methylobacterium-enriched patients is short (fig. 3), and the methylobacterium is positively correlated with the clinical characteristic vascular cancer embolus of the patients (fig. 4), which suggests that the methylobacterium can be used as the gastric flora marker for gastric cancer prognosis.
Example 3 investigation of the Effect of Methylobacterium on TRM cells
1. Methylobacterium and peripheral blood lymphocyte relationship
Blood routine information was obtained clinically for 53 patients with gastric cancer, including: total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, NK cells, B cells, NKT cells, and activated T cell numbers. The correlation between the abundance of the methylobacterium and the number of the peripheral blood lymphocytes is further analyzed, and the influence of the methylobacterium on the peripheral blood lymphocytes is researched.
2. Multicolor immunofluorescence
Staining was performed using primary antibodies for markers CD103(TRM cell marker), CD8 and CD3 for immunohistochemical analysis. Antibody detection was performed using Opal polymer HRP Ms + Rb immunohistochemical detection reagent (PelkinElmer, Boston, MA). Staining was performed sequentially using the same protocol as immunohistochemistry and detection of each marker was done before the next antibody was applied. The staining channel includes: PPD520, PPD570, PPD650 and DAPI. After staining was complete, sections were imaged using a Vectra 3.0 spectral imaging system (PerkinElmer). CD8 and TRM cell infiltration were observed and counted.
3. And (3) carrying out correlation analysis on methylobacterium and TRM cells:
the methylobacterium are divided into a high group and a low group (taking a median as a reference), and the high group and the low group are correlated with fluorescence data for analysis, so that the influence of the methylobacterium on the TRM cell content in a gastric tumor microenvironment is researched.
4. Asian Cancer Research Group (ACRG) survival data analysis
We obtained survival data from 300 patients with gastric adenocarcinoma from the Asian Cancer Research Group (ACRG), analyzed and collated these data to explore the relationship between TRM cell infiltration number and prognosis of evidence-changing survival.
5. Flow cytometry
The TRM cell number of CD8+ CD103+ is detected by flow cytometry, the sample is divided into a methylobacterium high group and a methylobacterium low group, and the influence of the methylobacterium on the TRM cell number in the multicolor fluorescence result is verified.
(1) Cell recovery: taking tumor infiltrating lymphocytes of gastric cancer patients cryopreserved in a laboratory at (-80 ℃), and resuscitating the tumor infiltrating lymphocytes in a constant-temperature water bath at 37 ℃ (the cells are damaged if rapid resuscitation is needed and the time is too long);
(2) diluting the recovered cells with PBS, mixing uniformly, centrifuging (1500r, 5min), and pouring out the supernatant after centrifuging;
(3) removing supernatant, adding 1ml of culture medium into the tube, repeatedly blowing by using a pipette to resuspend cells, transferring the resuspended cells into a pore plate (plate paving), adding 2 mul of stimulant into each pore plate, shaking uniformly, and culturing in a constant-temperature incubator at 37 ℃ for 4 h;
(4) after 4h of culture, the well plate was removed and the supernatant was aspirated into a 1.5ml EP tube (macrophage adherent growth, T cell suspension growth);
(5) after centrifuging the EP tube at 4 ℃ for 5min at 500g, the supernatant was discarded by pipetting (note: 100. mu.l of supernatant is preferably reserved in order to prevent discarding the supernatant by carelessness);
(6) washing: adding 1ml PBS solution to wash the cells, mixing uniformly and centrifuging (4 ℃, 500g, 5 min);
(7) and (3) sealing: centrifuging, removing supernatant, adding 1 μ l of blocking solution, mixing, and incubating at 4 deg.C for 20 min;
(8) cell surface staining: adding a surface antibody (the surface antibody is prepared in advance) into the tube, uniformly mixing, and incubating at 4 ℃ for 30 min;
(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 well before fixative addition);
(11) washing: washing the cells with 1 XBD Perm/Wash buffer (1 ml each time), mixing and centrifuging (4 deg.C, 1000g, 5 min);
(12) intracellular factor staining: centrifuging, removing supernatant, adding intracellular antibody (prepared intracellular antibody), mixing, and incubating at 4 deg.C for 30 min;
(13) washing: in (11)
(14) And (3) filtering: after discarding the supernatant, 250. mu.l PBS was added to the tube, mixed well and filtered into a new EP tube (note: no filtration is needed if the cell content is low);
(15) the samples were examined on a flow cytometer.
6. Results
Methylobacterium were associated with total lymphocytes in the patient's peripheral blood lymphocytes, suggesting that methylobacterium might affect prognosis by acting on lymphocytes (fig. 5). The results of multicolor immunofluorescence and flow cytometry indicate that the methylobacterium is obviously related to TRM cells in a tumor microenvironment, the high group of the methylobacterium has low TRM cell content, and the low group of the methylobacterium has high TRM cell content (fig. 6 and fig. 9). TRM cells play an important role in anti-tumor, so we found that the survival time of the samples with high TRM cell number is significantly higher than that of the samples with low TRM cell number by analyzing the gastric cancer sample data in the ACRG database (fig. 7). In addition, TRM cells were significantly negatively associated with vascular cancer emboli, suggesting that TRM cells may be a factor in preventing the occurrence of vascular cancer emboli (fig. 8). In general, methylobacterium may lead to poor prognosis of gastric cancer patients by inhibiting TRM cells.
Claims (7)
1. Application of methylobacterium as a marker of gastric flora in preparation of a reagent for predicting gastric cancer prognosis.
2. Application of a reagent for detecting the abundance of methylobacterium in preparing a reagent for predicting gastric cancer prognosis.
3. The use of claim 2, wherein the reagent for detecting the abundance of Methylobacterium is selected from a probe, a primer or an antibody for detecting the abundance of Methylobacterium.
4. The use according to claim 2, wherein the primer is a primer for detecting 16S rRNA of Methylobacterium.
5. An agent for predicting gastric cancer prognosis, which comprises an agent for detecting the abundance of Methylobacterium.
6. The reagent according to claim 5, wherein the reagent further comprises a reagent for extracting genomic DNA of the microorganism.
7. Application of methylobacterium as a marker of gastric flora in screening gastric cancer treatment drugs.
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| NATALIA CASTANO-RODRIGUEZ等: "Dysbiosis of the microbiome in gastric carcinogenesis", SCIENTIFIC REPORTS, vol. 7, pages 15957 * |
| 董雨: "胃炎癌转变过程中胃内菌群构成变化的研究", 中国优秀硕士学位论文全文数据库医药卫生科技辑, pages 8 - 9 * |
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