WO2014206259A1

WO2014206259A1 - Use of prostate cancer gene marker in marking recurrence and metastasis of prostate cancer and method thereof

Info

Publication number: WO2014206259A1
Application number: PCT/CN2014/080539
Authority: WO
Inventors: 叶林; 叶定伟
Original assignee: 复旦大学附属肿瘤医院
Priority date: 2013-06-24
Filing date: 2014-06-23
Publication date: 2014-12-31
Also published as: CN103602720A; CN105861692A

Abstract

Provided in the present invention is the use of a prostate cancer gene marker MS4A8B gene in marking the recurrence and metastasis of prostate cancer, wherein the expression of the gene in a tissue sample to be detected is an indicator for marking the recurrence and metastasis of prostate cancer. The present invention also discloses the use of a reagent for detecting the expression of the MS4A8B gene in a sample in marking the recurrence and metastasis of prostate cancer and a method for marking the recurrence and metastasis of prostate cancer using the MS4A8B gene.

Description

Use and method of prostate cancer gene marker in labeling prostate cancer recurrence and metastasis

技术领域 Technical field

The present invention relates to compositions and methods for cancer diagnosis, research and treatment, including but not limited to cancer markers. More specifically, the present invention relates to a genetic marker for use as a marker for prostate cancer recurrence and metastasis.

发明背景 Background of the invention

Prostate cancer is the most common malignant tumor in men and women in Europe and the United States, and is the second most common cause of male cancer death in the United States, followed by lung cancer. According to statistics, the United States 2012 There were 24,1740 newly diagnosed prostate cancer patients, accounting for 29% of all new cancer patients; 2,8170 people died of prostate cancer, accounting for 9% of all tumor deaths. . In recent years, the incidence of prostate cancer in China has also increased.

The only clinical marker used for prostate cancer diagnosis and post-treatment recurrence monitoring is the serological marker prostate specific antigen (PSA), but About 1/3 of the patients found on PSA-based screening For the occult prostate cancer with slow progress, low invasiveness and no clinical symptoms, it leads to over-diagnosis of some cases, which increases unnecessary pain and injury, and screening has no substantial improvement on the overall mortality rate of prostate cancer, suggesting that many hidden Diagnosis and treatment of sexual subclinical tumors. This shifts the research focus from previous disease risk susceptibility studies to early diagnosis of invasive prostate cancer and discovery of molecular markers for lethal prostate cancer.

The discovery and rational application of tumor markers is the premise of early detection and early diagnosis of tumors. To date, global markers for predicting early recurrence and poor prognosis of prostate cancer are not yet mature, and for newly diagnosed prostate cancer patients, there are limited tools for understanding the risk of disease progression and guiding the course of treatment. Therefore, the detection of invasive prostate cancer markers has become a major scientific problem that prostate cancer, which has high molecular and clinical heterogeneity, is urgently needed to be solved. Several studies have attempted to identify high-risk markers of prostate cancer progression, but most have failed to achieve their intended goals.

At present, high-risk histological markers of prostate cancer recurrence and metastasis are mostly serological markers and histological markers.

1 . Serological marker

PSA derived parameters such as PSA Rate, doubling time and density can improve the sensitivity of invasive prostate cancer detection. An experimental study of active follow-up in the European Prostate Cancer Research International shows PSA Dynamic changes and density are the most appropriate predictors for assessing risk and individualized treatment (Bul et al., 2013); in the absence of secondary biopsy and Gleason In the case of other indicators of progression, such as elevated grades, pure PSA is not sufficient to initiate active treatment (Adamy et al., 2011).

Currently only PSA density enters 2012 NCCN The guidelines are recommended for predicting parameters for low-risk prostate cancer, but most doctors only test total PSA . Diagnosing bone metastasis is mainly based on bone imaging technology, which is insensitive and invasive at an early stage and is expensive. Factors derived from bone metabolism in plasma are associated with a high risk of bone metastasis in prostate cancer, reflecting an imbalance in the process of bone remodeling between tumor and bone microenvironment, including bone formation markers and bone resorption markers. In the diagnosis of bone metastases, it is not possible to predict the high risk of metastasis in advance.

2. Histological markers

Since the prostate gland is the center of a series of oncogenic polygenic events, the predictive value of histological markers for high-risk progression cannot be ignored. Several studies that attempt to identify high-risk markers of prostate cancer progression have failed to achieve the intended goals, and no histological markers with high risk of progression have been used clinically.

Recent evidence suggests that there is no key (no oncogene) in the acinar cells of the prostate. The activation event occurs or is inhibited so that it remains in a subclinical state for life. The difference between occult cancer and clinical cancer suggests that these events are initiated in prostate cancer / Early progression can lead to the development of cell senescence, which acts as a mechanism for the body to inhibit tumors, especially the down-regulation of the tumor suppressor gene NKX3.1 is an early persistent event and is associated with increased prostate epithelial cell proliferation and poor prognosis. (Lin et al., 2009). It is currently believed that the TMPRSS2-ERG translocation caused by loss of NKX3.1 represents the occurrence of early events in prostate cancer in situ, followed by PTEN, Loss and down-regulation of p27(Kip), RB, E-cadherin, and TP53 are thought to be the cause of early and late metastasis (Netto and Epstein, 2010).

A series of studies using immunohistochemistry and transgenic techniques confirmed the upstream molecule of PTEN, SMAD-4 (a TGF β/ Downregulation of the osteogenesis signal axis member and up-regulation of cyclin D1 and cell adhesion molecule pontin are associated with biochemical recurrence and metastasis of prostate cancer (Ding et al., 2011). Methylation of the glutathione-S-transferase promoter region associated with the clearance of foreign substances and carcinogens is considered an early event in the progression of prostate cancer, in some cases with TMPRSS2 Translocation is associated with early invasion (Ahmed, 2010). Histone methylation enzyme EZH2, which enables DNA methylation and histone modification to down-regulate the tumor suppressor gene RAS GTPase, thereby activating Up-regulation of RAS and NF-κB expression by DAB2IP proteins promotes tumor proliferation and metastasis, and disease states that are more deteriorating with prostate cancer, including metastasis (Min et al., 2010) .

The pathogenesis, progression, and metastasis of prostate cancer are associated with AR mutations, and current treatments are also around blocking this pathway. But because of AR It is a downstream transcriptional target gene. The diagnosis and castration treatment of prostate cancer is not completely dependent on the expression of AR. Some prostate cancer tissues and cells do not express AR; in addition, androgen-sensitive prostate cancer (HSPC) Transformation to castration-resistant prostate cancer (CRPC) involves multiple complex processes such as AR pathway and partial non-AR pathway activation, with multiple genes, signaling molecules, and phenotypic changes involved, which limit AR Value in prostate cancer progression prediction (Saraon et al., 2011).

Prostate-specific membrane antigen (PSMA) is a form secreted by prostate epithelial cells. A complete transmembrane glycoprotein is considered a prostate epithelial marker. There are reports of PSMA The level of patients with high-grade, staged prostate cancer and hormone-refractory prostate cancer is significantly elevated, which is associated with poor clinical outcomes. However, due to the high expression rate in the benign epithelium of the prostate, PSMA The value in prostate cancer progression prediction is greatly reduced.

However, unlike other tumors, prostate cancer often has multiple pathological histomorphies coexisting or even mixed with normal gland. This unique feature makes the study of prostate cancer progression and recurrence prediction of tissue markers difficult, although the investment is huge but little progress. Starting with the bioinformatics analysis method, starting from the disease progression model, studying the pathogenesis of prostate cancer from the overall level of the gene will make it possible to find a more suitable molecular marker for prostate cancer progression, which will be the diagnosis and prevention of prostate cancer. Treatment provides a key basis.

发明内容 Summary of the invention

One of the objects of the present invention is to provide a gene for prostate cancer-specific expression for labeling the development of prostate cancer, and providing a basis for formulating treatment decisions for prostate cancer.

The second object of the present invention is to provide a gene for prostate cancer-specific expression for labeling the recurrence of prostate cancer, thereby being used for identifying and judging the recurrence of prostate cancer, and providing a basis for formulating treatment decisions for prostate cancer.

The third object of the present invention is to provide a gene specifically expressed by prostate metastasis, which can be used for labeling prostate cancer metastasis, thereby being used for identifying and judging prostate cancer metastasis, and providing a basis for formulating treatment decisions for prostate cancer.

A fourth object of the present invention is to provide a method for labeling the recurrence and metastasis of prostate cancer, which specifically marks the development of prostate cancer by a gene specifically expressed by prostate cancer, and provides a basis for formulating treatment decisions for prostate cancer. .

A fifth object of the present invention is to provide a method for labeling recurrence and metastasis of prostate cancer, which specifically marks a recurrence of prostate cancer by a gene specifically expressed by prostate primary cancer, thereby being capable of identifying and judging the prostate The recurrence of cancer provides a basis for making treatment decisions for prostate cancer.

A sixth object of the present invention is to provide a method for labeling recurrence and metastasis of prostate cancer, which specifically labels prostate cancer metastasis by a gene specifically expressed by prostate metastasis, thereby being used for identifying and judging prostate cancer The transfer provides a basis for making treatment decisions for prostate cancer.

A seventh object of the present invention is to provide an agent for detecting prostate cancer-specific expression in a sample for labeling prostate cancer recurrence and metastasis, for labeling the development of prostate cancer, and providing a basis for formulating treatment decisions for prostate cancer. .

An eighth object of the present invention is to provide an agent for detecting prostate cancer-specific expression in a sample for labeling prostate cancer recurrence and metastasis, for labeling prostate cancer recurrence, thereby being used for identifying and judging prostate cancer. Recurrence provides a basis for making treatment decisions for prostate cancer.

A ninth object of the present invention is to provide an agent for detecting prostate cancer-specific expression in a sample for labeling prostate cancer recurrence and metastasis for labeling prostate cancer metastasis, thereby being useful for identifying and judging prostate cancer. Transfer, provide a basis for the development of treatment decisions for prostate cancer.

To achieve the above object, the present invention discloses the use of the MS4A8B gene for the recurrence and metastasis of labeled prostate cancer.

Wherein, the expression of the MS4A8B gene in the tissue sample to be detected is an indicator for labeling prostate cancer.

Among them, the higher the expression of the MS4A8B gene in the tissue sample to be detected, the higher the probability of recurrence and metastasis of prostate cancer.

Among them, the MS4A8B gene expression was scored, and the MS4A8B gene expression score was given. The MS4A8B gene expression score was used to determine the recurrence and metastasis of prostate cancer.

Based on the use of the MS4A8B gene disclosed in the present invention for labeling the recurrence and metastasis of prostate cancer, the present invention further discloses a The MS4A8B gene-labeled method for recurrence and metastasis of prostate cancer, wherein the expression of the MS4A8B gene in a tissue sample to be examined is an indicator of prostate cancer.

Preferably, the MS4A8B gene-labeling method for recurrence and metastasis of prostate cancer comprises the following steps:

Detecting the expression of the MS4A8B gene in the tissue sample to be examined;

The higher the expression of the MS4A8B gene in the tissue sample to be examined, the higher the probability of prostate cancer recurrence and metastasis.

The MS4A8B gene expression was scored and the MS4A8B gene expression score was given;

According to the obtained MS4A8B gene expression score, the recurrence and metastasis of prostate cancer were judged.

Among them, the MS4A8B gene expression of the tissue sample to be examined can be passed, including but not limited to, using DNA Probes or fluorescent probes directly detect the MS4A8B gene in the DNA genome of prostate cancer tissues, and design PCR primers for MS4A8B using reverse transcriptase polymerase chain reaction (RT-PCR) The method was to detect the mRNA of MS4A8B, and detect the protein of MS4A8B gene by immunohistochemistry, immunofluorescence and flow cytometry, and stain the protein.

Among them, for the expression of the obtained MS4A8B gene, the MS4A8B gene expression score was given by the following criteria:

Percentage of positive cells expressing MS4A8B gene based on tissue samples to be tested:

The number of positive cells is <5 % for 0, 5 % to 25 % for 1 point, and 26 % to 50 % for 2 points. 51% to 75 % 3 points, 76% to 100% is 4 points;

Positive intensity factor based on MS4A8B gene expression of the tissue sample to be tested:

Negative (-) is 0, weak positive (+) is 0.25, positive (++) is 0.5, strong positive (+++) 1 ;

The score of the percentage of positive cells multiplied by the score of the positive intensity coefficient is the MS4A8B gene expression score.

Among them, MS4A8B gene expression score can be converted into MS4A8B gene expression positive grade: score multiplication <1 Divided into negative (-), score multiplication ≥ 1 is divided into weak positive (+), score multiplication ≥ 2 is divided into positive (++), score multiplication ≥ 3 is strongly positive (+++) .

Among them, when using immunohistochemistry, immunofluorescence, flow cytometry and other methods to detect transcription MS4A8B When the protein of the gene is stained with the protein, if immunohistochemical staining is used, the percentage of the positive cells is converted to the percentage of the number of stained cells, and the positive intensity coefficient is converted into the positive coloring intensity coefficient: colorless is 0. , light yellow is 0.25, brownish yellow is 0.5, and tan is 1.

Among them, the criteria for judging the recurrence of prostate cancer based on the obtained MS4A8B gene expression score are:

The higher the MS4A8B gene expression score, the higher the probability of prostate cancer recurrence and metastasis.

Specifically, for every 1 point increase in the MS4A8B gene expression score, the probability of prostate cancer recurrence increased by 110.6%. For every 1 point increase in MS4A8B gene expression score, the risk of distant metastasis increases by 31.7%.

Preferably, the criteria for determining the recurrence of prostate cancer based on the obtained MS4A8B gene expression score are:

MS4A8B gene expression score of not more than 1 is low expression, and its prostate cancer recurrence rate is low and the metastasis rate is low;

MS4A8B gene expression score greater than 1 but not greater than 2 is moderate expression, its prostate cancer recurrence rate and metastasis rate;

The MS4A8B gene expression score greater than 2 is highly expressed, and its prostate cancer recurrence rate is high and metastasis rate.

Specifically, the MS4A8B gene expression score is no more than 1 point, and the probability of recurrence in a year is 97.22%. The two-year recurrence-free survival probability was 91.00%, the three-year recurrence-free survival probability was 90.99%; the MS4A8B gene expression score was greater than 1 point but not more than 2 points, and the one-year recurrence-free survival probability was 96.39%, the two-year recurrence-free survival probability was 87.68%, the three-year recurrence-free survival probability was 67.84%; the MS4A8B gene expression score was greater than 2 points, and the one-year recurrence-free survival probability was 94.73%, the two-year recurrence-free survival probability was 72.25%, and the three-year recurrence-free survival probability was 53.552%.

Specifically, the MS4A8B gene expression score is less than or equal to 1 point, and the probability of distant metastasis is 15.625%; The MS4A8B gene expression score was greater than 1 point, and the probability of distant metastasis was 23.30%.

Based on the use of the MS4A8B gene disclosed in the present invention for labeling recurrence and metastasis of prostate cancer, the present invention further discloses a test sample The use of an agent for MS4A8B gene expression in the labeling of prostate cancer recurrence and metastasis, wherein the expression of the MS4A8B gene in a tissue sample to be examined is an indicator of prostate cancer.

Preferably, the higher the expression of the MS4A8B gene in the tissue sample to be detected, the higher the probability of prostate cancer recurrence and metastasis.

Preferably, the determining the recurrence and metastasis of prostate cancer is to score the obtained MS4A8B gene expression and give MS4A8B Gene expression scores were used to determine the recurrence and metastasis of prostate cancer based on the obtained MS4A8B gene expression score.

Wherein the reagent for detecting the expression of the MS4A8B gene in the sample is based on the test MS4A8B Gene expression specificity for MS4A8B gene, MS4A8B gene mRNA, MS4A8B gene transcription protein gene fragment, gene chimera, RNA Specific proteins, polypeptides, and reagents containing the same, including but not limited to direct detection of MS4A8B in the DNA genome of prostate cancer tissue using DNA probes or fluorescent probes Gene-configured reagent for the design of MS4A8B PCR primers using reverse transcription polymerase chain reaction (RT-PCR) to detect MS4A8B mRNA The reagent to be configured is a reagent configured by detecting a protein encoding the MS4A8B gene and staining the protein by immunohistochemistry, immunofluorescence, or flow cytometry.

Experimental results show that MS4A8B The gene has specific characteristics for prostate cancer due to its biological characteristics, and can be used as a genetic marker for identifying and diagnosing recurrence and metastasis of prostate cancer:

1, MS4A8B The gene has obvious expression differences in normal tissues and cancer tissues, and there are obvious expression differences in different cancer tissues. Therefore, the MS4A8B gene is a gene specifically expressed in prostate cancer;

2, MS4A8B gene in benign prostate tissue, adjacent tissues, HPIN There were significant differences in expression between prostate cancer and metastatic cancer samples. The expression of MS4A8B gene in prostate cancer and metastatic cancer samples was much higher than that in benign prostate tissue, adjacent tissues, and HPIN. therefore The MS4A8B gene is a prostate cancer metastasis-specific expression gene;

3, MS4A8B There are significant differences in the expression of genes in normal prostate tissue cells, prostate cancer cells and prostate cancer metastasis cells. MS4A8B in prostate cancer metastatic cancer samples. The expression of the gene was also higher than that of the MS4A8B gene in the primary prostate cancer. Therefore, the MS4A8B gene is a prostatic primary cancer, especially a metastatic cancer-specific expression gene;

4, MS4A8B gene acts on G1-S Cell cycle regulatory points promote cell proliferation and therefore play an important role in prostate cancer progression.

The experimental results further indicate that MS4A8B The gene has specific expression for prostate cancer, especially for primary and metastatic cancers of prostate cancer, among which:

1. The higher the MS4A8B gene expression score, the higher the risk of biochemical recurrence and metastasis.

MS4A8B gene expression score greater than 2 is highly expressed, and its prostate cancer recurrence rate is high and metastasis rate;

2, MS4A8B gene highly expressed patients showed the characteristics of easy recurrence, and this feature became more apparent over time;

3, MS1A8B gene expression score increased by 1 point, the risk of biochemical recurrence increased by 110.6%;

4. For every 1 point increase in MS4A8B gene expression score, the risk of distant metastasis increased by 31.7%.

Therefore, MS4A8B Genes can be used as genetic markers to identify and diagnose postoperative recurrence and metastasis of prostate cancer, thereby predicting prostate cancer recurrence and metastasis and making treatment decisions.

MS4A8B gene expression is low-grade or moderately expressed in early stage prostate cancer patients, 15.625% in radical prostatectomy The probability of metastasis, while patients with radical prostatectomy, the high expression of MS4A8B gene, the probability of distant metastasis was 23.3%. If according to MS4A8B The gene expression was given to patients with prostate cancer lymph node area adjuvant radiotherapy, NNT ( number need to treat) = 13.29. That is, according to MS4A8B Gene expression, 13 patients per lymph node, one patient benefited.

附图说明 DRAWINGS

Figure 1 shows the expression levels of the mRNA level of the MS4A8B gene in prostate cancer and various tumors and corresponding normal tissues.

Figure 2A-2B shows differential expression of the MS4A8B gene in prostate cell lines and MS4A8B in 4 prostate cancer cell lines. Schematic diagram of cell cycle assay results after down-regulation.

Figure 3A-3F shows down-regulation of MS4A8B in LNCaP cell line Schematic diagram of the proliferative capacity and cell cycle changes after the gene.

Figure 4 is a graphical representation of changes in cell cycle-associated proteins after down-regulation of the MS4A8B gene in the LNCaP cell line.

Figure 5 is a graphical representation of changes in metastatic capacity after down-regulation of the MS4A8B gene in the PC3 cell line.

Figure 6 is a graphical representation of changes in EMT markers after down-regulation of the MS4A8B gene in the PC3 cell line.

Figure 7A-7E shows overexpression of the MS4A8B gene in normal prostate epithelial RWPE-1 Schematic diagram of the effects of cell cycle and cell proliferation.

Figures 8A-8F are schematic representations of immunohistochemical analysis of MS4A8B protein in prostate cancer tissue microarray samples.

Figure 9 shows the MS4A8B gene in benign prostate tissue, adjacent tissues, HPIN Expression profiles in primary prostate cancer metastatic tumors and metastatic cancer samples.

Figure 10A-10C shows MS4A8B gene expression and Gleason Schematic diagram of correlation analysis of score, proliferation index, and apoptotic index.

Figure 11 is a graphical representation of the correlation between MS4A8B gene expression and recurrence-free survival time (RFS).

具体实施方式 detailed description

In view of the fact that prostate cancer is different from other tumors, it often has multiple pathological histomorphies coexisting or even mixed with normal glands, making it difficult to study the tissue markers of prostate cancer progression, recurrence and metastasis prediction. Through the bioinformatics analysis method, from the prostate cancer disease progression model, the pathogenesis of prostate cancer is studied from the overall level of the gene, and a more suitable molecular marker for prostate cancer progression is found, which provides for prostate cancer recurrence and metastasis prediction. Gene markers and criteria for judgment.

基因标志物的筛选： MS4A8B 基因 Screening of gene markers: MS4A8B gene

Based on this, the bioinformatics analysis software independently developed by the research group DataAnalysorOne 1.0 Multi-class, multi-level, multi-angle, multi-series screening of massive high-throughput data on prostate cancer progression models, respectively, and multiple comparisons between prostate cancer tissue and benign prostate tissue, matched prostate cancer and adjacent cancer Screening for a transmembrane protein family member between different groups of tissues, between prostate cancer primary metastases and metastases The MS4A8B gene, which is specifically elevated in prostate cancer tissues and associated with prostate cancer progression, recurrence, and metastasis.

In the follow-up study, Kaplan-Meier Survival curve analysis showed that patients with high expression of this protein in prostate cancer tissues had significantly lower biochemical recurrence-free survival time (RFS) in patients with lower expression of this protein (p<0.05). Further studies found that the protein was positively correlated with Gleason score and proliferation index (Spearman analysis) (p<0.001).

In an in vitro cell model study, specific knockout of the gene is expressed in a prostate cancer cell line that highly expresses the protein, resulting in decreased proliferative and metastatic ability of prostate cancer; and, in normal prostate epithelial cells that underexpress the protein The expression of this gene is up-regulated in the line, resulting in enhanced proliferative and metastatic ability of prostate epithelial cells.

MS4A8B 基因简介 MS4A8B gene introduction

MS4A8B, a human transmembrane protein, full name membrane-spanning 4-domains, Subfamily A, member 8B, official name MS4A8, alias MS4A4; 4SPAN4; CD20L5. NCBI Gene ID: 83661. MS4A8B is a hydrophobic protein MS4A containing four transmembrane domains (Membrane-spanning 4-domains, subfamily A) One of the family members.

For further study of MS4A8B Differential expression of genes in different tissues of humans. We collected common human cancer tissues and corresponding adjacent tissues, and detected mRNA of MS4A8B gene by PCR. The expression of the MS4A8B gene in various normal and tumor tissues of humans was obtained and expressed in various normal and tumor tissues, and the corresponding expression profiles were analyzed.

All statistical analyses were processed using SPSS 17.0 statistical software. Independent t test (constant variable) for comparison between different groups The t-test was used to compare the means between the two groups, the 分类 2 test was used to compare the categorical variables, and the one-way ANOVA was used to compare the multiple groups. Values are expressed as mean ± SD. P<0.05 It is considered that there is a significant difference and is statistically significant.

MS4A8B 基因对前列腺癌的特异性表达 Specific expression of MS4A8B gene in prostate cancer

We collected 13 common tumor samples, including prostate cancer samples, which A common tumor sample was a surgically resected frozen tissue sample, which was stored at the Fudan University Affiliated Tumor Hospital Tissue Bank at -70 °C (except for all tumor samples except small cell lung cancer) and the Second Affiliated Hospital of Dalian Medical University. (Non-small cell lung cancer samples), Medical Ethics Committee ethics number 050432-4-1212B All patients signed informed consent. All samples included paired paracancerous tissues, in which frozen sections of prostate cancer samples were stained by pathologists to determine more than 80% of the glandular glands, and laser microdissection (LCM) was performed. Obtain cancer and adjacent normal tissue samples.

RT-PCR was used to detect the mRNA of MS4A8B in 13 normal and tumor tissues of the human. Level, and using the housekeeping gene β-actin to correct the mRNA expression intensity of MS4A8B in each sample, the MS4A8B of 13 human tissues shown in Table 1 was obtained. Expression profiling.

Table 1. Analysis of MS4A8B gene expression profile in 13 human tissues

	例数 Number of cases	恶性 (mean±SD) Malignant (mean±SD)	良性 (mean±SD) Benign (mean±SD)	P 值 P value
乳腺 Mammary gland	6 6	4.69E-11±4.65E-11 4.69E-11±4.65E-11	5.63E-12±4.38E-12 5.63E-12±4.38E-12	0.1250 0.1250
膀胱 bladder	6 6	1.38E-11±8.22E-12 1.38E-11±8.22E-12	1E-11±8.54E-12 1E-11±8.54E-12	0.8125 0.8125
胰腺 pancreas	6 6	3.45E-11±3.12E-11 3.45E-11±3.12E-11	1.9E-11±1.8E-11 1.9E-11±1.8E-11	0.4762 0.4762
肾 kidney	6 6	1.14E-05±2.31E-05 1.14E-05±2.31E-05	6.87E-06±9.35E-06 6.87E-06±9.35E-06	0.4698 0.4698
肝脏 liver	6 6	5.35E-11±8.75E-11 5.35E-11±8.75E-11	2.85E-11±2.53E-11 2.85E-11±2.53E-11	0.9372 0.9372
甲状腺 thyroid	6 6	5.09E-11±3.2E-11 5.09E-11±3.2E-11	6.1E-11±7.61E-11 6.1E-11±7.61E-11	0.9143 0.9143
卵巢 Ovary	6 6	2.17E-10±2.82E-10 2.17E-10±2.82E-10	4.14E-11±3.53E-11 4.14E-11±3.53E-11	0.1320 0.1320
结肠 colon	6 6	2.37E-07±3.45E-07 2.37E-07±3.45E-07	1.41E-05±1.33E-05 1.41E-05±1.33E-05	0.0043 0.0043
胃 stomach	6 6	4.05E-05±7.42E-05 4.05E-05±7.42E-05	5.67E-06±4.3E-06 5.67E-06±4.3E-06	1.0000 1.0000
前列腺 prostate	6 6	0.000206±0.000221 0.000206±0.000221	3.44E-05±7.28E-05 3.44E-05±7.28E-05	0.0128* 0.0128*
肺 lung	6 6	2.06E-05±2.04E-05 2.06E-05±2.04E-05	0.000728±0.001331 0.000728±0.001331	0.0303* 0.0303*
宫颈 cervix	6 6	1.47E-06±3.89E-06 1.47E-06±3.89E-06	2.41E-05±4.25E-05 2.41E-05±4.25E-05	0.0177* 0.0177*
软组织 Soft tissue	6 6	7.18E-12±1.78E-12 7.18E-12±1.78E-12	2E-11±2.35E-11 2E-11±2.35E-11	0.0952 0.0952

According to the MS4A8B expression profiling of 13 human tissues shown in Table 1, the results are shown in Figure 1, by RT-PCR. The mRNA levels of MS4A8B in 13 normal and tumor tissues were detected, and the Y-axis was the intensity of MS4A8B after correction by the housekeeping gene β-actin. Figure 1 As shown, the mRNA level of the MS4A8B gene is in prostate cancer (p<0.05) and several normal tissues including lung (p<0.05), colon Significantly elevated (p < 0.01) and cervical (p < 0.05).

The results of Table 1 and Figure 1 indicate that the mRNA of the MS4A8B gene Not expressed in most tissues, only expressed in normal lung, colon and cervical tissues. MS4A8B gene is overexpressed in prostate cancer in tumor tissues, which is 3 times higher than normal control prostate tissue (p<0.05), while the magnitude of elevation in renal and gastric cancer was small and not statistically significant (p>0.05). This shows the MS4A8B Genes have significant expression differences in normal tissues and cancer tissues, and there are significant expression differences in different cancer tissues, so MS4A8B The gene is a prostate cancer-specific gene that is of great value for the specific recognition of prostate cancer in prostate cancer tissues in which various pathological histologies are present or even mixed with normal glands.

So far, the most widely used histological marker for the differential diagnosis of benign and malignant prostate cancer is α-formyl-CoA racemase (alpha). methylacyl-CoA racemase, AMACR, also known as P504S), which is highly expressed in prostate cancer tissues, but AMACR Poor tissue specificity, overexpressed in various tumor tissues such as renal papillary tumor, colorectal tumor, ovarian tumor, breast tumor, bladder tumor, lung tumor, lymphoma and melanoma, and with prostate cancer staging, Gleason scores, margins, biochemical recurrence, and other indicators that reflect the prognosis of patients. In contrast, MS4A8B The high specificity in prostate cancer tissue suggests that we can specifically identify prostate tissue derived from prostate tissue, especially ectopic metastatic prostate cancer tissue.

MS4A8B 基因对前列腺原发癌和转移癌的特异性表达 Specific expression of MS4A8B gene in primary prostate cancer and metastatic carcinoma

We used Western blot analysis to detect MS4A8B gene in prostate cell lines. Expression in RWPE-1, 22RV1, LNCaP, PC3 and DU145. As shown in Figure 2A, the results indicate that the MS4A8B gene is highly expressed in 4 The prostate cancer cell lines 22RV1, LNCaP, PC3 and DU145, but the MS4A8B gene was not expressed in the normal prostate immortalized epithelial RWPE-1 cell line.

This result indicates that the MS4A8B gene is derived from the prostate cancer cell line 22RV1 derived from prostate cancer. The prostate cell line derived from prostate cancer metastases is highly expressed in LNCaP, PC3 and DU145, but not in prostate cell line derived from normal prostate immortalized epithelium RWPE-1 Medium. It can be seen that the MS4A8B gene has obvious expression differences in normal prostate tissue cells, prostate primary cancer cells and prostate cancer metastasis cells, so MS4A8B The gene is a prostate cancer-specific gene, so MS4A8B The gene is a primary cancer and metastasis-specific expression gene of prostate cancer, which is of great value for the identification and diagnosis of recurrence and metastasis of prostate cancer.

MS4A8B 基因对前列腺癌细胞发展的特异性表达 Specific expression of MS4A8B gene in the development of prostate cancer cells

For the above four MS4A8B gene high expression of prostate cancer cell lines 22RV1, LNCaP, PC3 and DU145, we designed three siRNA#1-3 for the transcriptional protein of MS4A8B gene, and transfected the above four prostate cancer cell lines for flow detection. As shown in Figure 3B As shown, flow cytometry showed that siRNA #2 of the MS4A8B gene showed a significant increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the corresponding S phase. Figure 2B The results suggest that MS4A8B silencing leads to G1-S blockade in multiple prostate cancer cell lines, MS4A8B gene siRNA#2 in four prostate cancer cells 22RV1, LNCaP, PC3, DU145 caused the most pronounced down-regulation of the MS4A8B gene and G1-S blockade.

Different expression levels of MS4A8B gene in different prostate cancer cell lines and 3 different siRNA pairs For the interference efficiency of the MS4A8B gene, we selected LNCaP cells as a cell model and MS4A8B gene siRNA#2 for further study.

As shown in Figure 3A, siRNA#2 of the MS4A8B gene was transfected into LNCaP cells, MS4A8B gene expression was effectively inhibited (immunoblotted), followed by down-regulation of the MS4A8B gene protein (Western blot). As shown in Figure 3B and Figure 3C As shown, the flow-through assay was used to compare the siRNA #2 of MS4A8B gene with untransfected cells. The results showed that down-regulation of the MS4A8B gene protein resulted in prostate cancer cells LNCaP. G1-S block in the cells, the proportion of G0/G1 cells decreased, and the proportion of cells in the S phase increased. As shown in Figure 3D, using EdU assay, the results indicate that down-regulation of the MS4A8B gene protein results in S phase cells were significantly reduced in LNCaP cells. As shown in Figure 3E, the CCK8 experiment was repeated three times. The results showed that down-regulation of the MS4A8B gene protein led to LNCaP. The cell activity of S phase cells was significantly attenuated and the survival rate was significantly decreased (P<0.05). As shown in Figure 3F, a clone formation experiment was performed and the results showed that MS4A8B Downregulation of the gene protein resulted in a significant reduction in clone formation.

These results indicate that downregulation of the MS4A8B gene results in decreased prostate cancer proliferation.

Among them, the G1-S cell cycle regulatory point controls the eukaryotic cells from the G1 phase into the S phase, at this regulatory point, Cyclin D1, CyclinE1 and several other cell cycle related proteins p21, p27/Kip1 play a role. Based on this, we further tested cell cycle-associated proteins and expressed cell proliferation activities. Expression of PCNA protein. As shown in Figure 4, the results showed that after down-regulation of the MS4A8B gene, LNCaP cells showed lower Cyclin D1 than negative control cells. Cyclin D1 , Cyclin E1 , Cyclin D1 , Cyclin E1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin D1 , Cyclin The p21 and PCNA proteins were significantly down-regulated, indicating significant changes in markers associated with decreased proliferation of LNCaP cells.

The most common metastatic site in patients with metastatic prostate cancer is bone metastasis, one of the most commonly used cell line models in PC3 cell line, which is highly expressed. The MS4A8B gene, and thus we selected the PC3 cell line as a cell model for the transfer function of the MS4A8B gene. As shown in Figure 5, we performed MS4A8B on PC3 cells. The RNAi was subjected to a scratch test 48 hours after transfection, and it was found that the migration ability of these prostate cancer cells was significantly attenuated after down-regulating the MS4A8B protein.

Based on this, we further examined the expression of metastasis-associated proteins, as shown in Figure 6, we found that the transfer ability is strong, EMT After the phenotype of PC3 cells was down-regulated by MS4A8B, Vimentin also showed a significant down-regulation, indicating that EMT with decreased PC3 cell transfer ability Markers also changed significantly.

Upregulate the protein of MS4A8B gene in RWPE-1 cells to promote cell proliferation to further elucidate MS4A8B The role of genes in prostate cancer. We transfected an overexpression vector with the MS4A8B open reading frame (ORF) into an immortalized normal prostate epithelium that does not express the MS4A8B gene. RWPE-1 cells were compared to the control group. As shown in Figure 7A, after RWPE-1 cells were transfected into the MS4A8B gene overexpression vector, the MS4A8B gene was effectively overexpressed and transfected. The expression level of MS4A8B gene in RWPE-1 cells with MS4A8B gene overexpression vector was significantly increased. As shown in Figure 7B and Figure 7C, flow detection cell cycle, RWPE-1 After transfection of the MS4A8B gene overexpression vector, the proportion of G1/G0 phase cells decreased and the proportion of S phase cells increased compared with the transfection negative control vector. As shown in Figure 7D, using the CCK8 experiment, When RWPE-1 cells were transfected with the MS4A8B gene overexpression vector, cell viability was increased compared to the transfection negative control vector. As shown in Figure 7E, the clone formation experiment was repeated three times. When RWPE-1 cells were transfected into the MS4A8B gene overexpression vector, the clone formation was significantly increased compared with the transfection negative control vector, p<0.05.

The above results indicate that the MS4A8B gene acts on G1-S Cell cycle regulation points promote cell proliferation and therefore play an important role in prostate cancer progression, which is of great value for the identification and diagnosis of prostate cancer recurrence and metastasis.

MS4A8B 基因作为前列腺癌的基因标志物 MS4A8B gene as a genetic marker for prostate cancer

In summary, MS4A8B The gene has specific characteristics for prostate cancer due to its biological characteristics, and can be used as a genetic marker for identifying and diagnosing recurrence and metastasis of prostate cancer:

Thus, the present invention discloses the use of the MS4A8B gene for the recurrence and metastasis of labeled prostate cancer. Wherein the MS4A8B The expression of the gene in the tissue sample to be tested is an indicator of prostate cancer. Wherein the MS4A8B The higher the expression of the gene in the tissue sample to be tested, the higher the probability of prostate cancer recurrence and metastasis. Among them, the MS4A8B gene expression was scored and given to MS4A8B. Gene expression scores were used to determine the recurrence and metastasis of prostate cancer based on the obtained MS4A8B gene expression score.

MS4A8B according to the present invention The use of a gene for labeling recurrence and metastasis of prostate cancer, the present invention further discloses an application of an agent for detecting expression of a MS4A8B gene in a sample for labeling prostate cancer recurrence and metastasis, wherein The expression of the MS4A8B gene in the tissue sample to be tested is an indicator of prostate cancer. Wherein the MS4A8B The higher the expression of the gene in the tissue sample to be tested, the higher the probability of prostate cancer recurrence and metastasis. Wherein, the determination of prostate cancer recurrence and metastasis is to score the obtained MS4A8B gene expression, and The MS4A8B gene expression score was used to judge the recurrence and metastasis of prostate cancer based on the obtained MS4A8B gene expression score.

The reagent for detecting the expression of the MS4A8B gene in the sample is based on the test MS4A8B Gene expression specificity for MS4A8B gene, MS4A8B gene mRNA, MS4A8B gene transcription protein gene fragment, gene chimera, RNA Specific proteins, polypeptides, and reagents containing the same, including but not limited to direct detection of MS4A8B in the DNA genome of prostate cancer tissue using DNA probes or fluorescent probes Gene-configured reagent for the design of MS4A8B PCR primers using reverse transcription polymerase chain reaction (RT-PCR) to detect MS4A8B mRNA The reagent to be configured is a reagent configured by detecting a protein encoding the MS4A8B gene and staining the protein by immunohistochemistry, immunofluorescence, or flow cytometry.

Application of MS4A8B gene disclosed in the present invention for labeling prostate cancer recurrence and metastasis and detecting sample MS4A8B The use of an agent for gene expression in the labeling of prostate cancer recurrence and metastasis, the present invention further discloses a method for recurring and metastasizing prostate cancer of the MS4A8B gene, wherein the MS4A8B The expression of the gene in the tissue sample to be tested is an indicator of prostate cancer, and the higher the expression of the MS4A8B gene in the tissue sample to be examined, the higher the probability of prostate cancer recurrence and metastasis.

The MS4A8B gene-labeled prostate cancer recurrence and metastasis method comprises the following steps:

The number of positive cells is <5 % for 0, 5% to 25 % for 1 point, and 26% to 50% for 2 Points, 51% to 75 % 3 points, 76% to 100% are 4 points;

Negative (-) is 0, weak positive (+) is 0.25, positive (++) is 0.5, strong positive (+++) is 1;

Among them, the criteria for judging the recurrence of prostate cancer based on the obtained MS4A8B gene expression score are: MS4A8B The higher the gene expression score, the higher the probability of prostate cancer recurrence and metastasis. Specifically, for every 1 point increase in MS4A8B gene expression score, the probability of prostate cancer recurrence increased by 110.6%, MS4A8B For every 1 point increase in gene expression score, the risk of distant metastasis increases by 31.7%.

Specifically, we used the above steps to detect 140 cases of paraffin-embedded prostate cancer tissue samples. The MS4A8B gene expression of paraffin-embedded prostate cancer tissue samples was used to label prostate cancer recurrence and metastasis in 140 paraffin-embedded prostate cancer tissue samples.

1 、取样 1, sampling

Paraffin-embedded tissue samples containing prostate tissue from the Fudan University Affiliated Tumor Hospital from January 2009 to 2010 A total of 140 patients with clinically localized prostate cancer who underwent endocrine therapy and radiotherapy before surgery, and a tissue sample of radical prostatectomy and as a control A prostate specimen undergoing radical cystectomy for bladder cancer was embedded in 10% formalin fixed paraffin, aged 60-89 years, with an average of 72.3 years.

According to the 2002 AJCC TNM staging, the pathological grade was based on the Gleason score. All HE Slices were screened by a pathologist and labeled for benign prostate tissue, high-grade prostatic intraepithelial neoplasia (HGPIN) Representative tumor regions such as prostate cancer and metastatic lymph node tissues serve as templates for tissue microarray chips.

HE staining, also known as hematoxylin-eosin staining One of the commonly used staining methods in paraffin sectioning technology. Hematoxylin is sensitive to the chromatin in the nucleus and the ribosome in the cytoplasm is purple-blue; eosin is an acid dye, mainly cytoplasm and extracellular matrix. The ingredients in it are red. HE staining is the most basic and widely used technical method in histology, embryology, pathology teaching and research, and clinical pathology practice.

In addition, clinical and pathological trait data were collected for all patients.

2 、组织切片和组织微阵列芯片制作 2, tissue section and tissue microarray chip production

All samples of HE in advance The stained sections and corresponding wax blocks were placed under a microscope to find the intended sampling area and marked on the wax block, and the position in the microarray chip was planned to make an Excel sheet. Use Quick-Ray The drill bit removes the marked area from the wax block, the wax block is placed horizontally on the table top, grips the Quick-Ray, the Quick-Ray probe is perpendicular to the marked area, and the Quick-Ray probe is inserted into the wax block and slowly removed. 5mm depth wax column. Use the Quick-Ray probe to remove the wax column (organization block) into the recipient block designed by UNITMA . The prefabricated wax block is placed on a horizontal tabletop, and the tissue extracted from the donor wax block is vertically inserted into the hole of the prefabricated wax block, and the Quick-Ray drill bit is slowly used to inject approximately 4 mm vertically. Use a smoothed tool or hand to adjust the height of all extracted tissue. Place the prefabricated wax block (the piece to be cut down) in the embedding mold and place it in the oven at 60 ° C 30 Minutes (the part that needs to be cut is the smoothed side down). After the wax block is taken out in a transparent form, the wax is embedded and the pre-made wax block is embedded. The pre-made wax block is placed in a cold dish to allow the wax block to be solidified, and the slicer is sliced (about 4 microns). .

All pathological indicators of the sections were determined by a senior doctor in the pathology department.

Tissue microarrays (TMAs), also known as tissue microarrays (tissue) Chip), an important branch of biochip technology, is to arrange many different individual tissue specimens on the same slide in a regular array to perform in situ histological studies of the same index. The technology since 1998 Since its inception in the year, it has been widely promoted and applied with its advantages of large-scale, high-throughput, and standardization. The tissue chip together with the gene chip and protein chip constitutes a series of biochips, enabling humans to effectively use hundreds of tissue samples for the first time, and research at the three levels of genome, transcriptome and proteome, known as medicine A revolution in the field of biology. Tissue microarray technology can be combined with many other conventional techniques such as immunohistochemistry (IHC), nucleic acid in situ hybridization (ISH), fluorescence in situ hybridization (FISH), in situ PCR In combination with applications, its application areas are constantly expanding. As an emerging biological research technology, it is demonstrating its potential with its absolute superiority.

3 、 MS4A8B 基因表达、 MS4A8B 基因表达评分和 MS4A8B 基因表达阳性等级 3, MS4A8B gene expression, MS4A8B gene expression score and MS4A8B Positive expression of gene expression

In the above steps, immunohistochemical staining of microarray chips of paraffin-embedded sections of prostate cancer tissue samples was performed for MS4A8B. Expression studies of protein levels of genes.

Immunohistochemical staining using MaxVisionTMplus Immunohistochemistry Kit (KIT-5020) Purchased from Fuzhou Maixin Biotechnology Co., Ltd., refer to the instruction manual of the kit for operation. After 10% formalin-fixed, paraffin-embedded sections were dewaxed by xylene, the gradient ethanol was hydrated and then placed in double distilled water. 0.01 mM Tris Buffer (pH 6.0) Heated at 121 °C for 20 minutes to repair the antigen, allowed to cool at room temperature and then treated with Tris buffer containing 1% BSA at room temperature. The endogenous immunogen is blocked in minutes. Anti-MS4A8B rabbit polyclonal antibody (dilution 1:50) and PCNA mouse monoclonal antibody (dilution 1:500) overnight at 4 °C. HRP-mer goat anti-rabbit/mouse IgG secondary antibody was incubated and DAB was developed. Universal biotinylated goat anti-mouse IgG was incubated at 37 °C for 20 minutes, DAB was developed, Hematoxylin counterstained, neutral gum seal.

Immunohistochemical staining is the basic principle of applied immunology -- The antigen-antibody reaction, that is, the principle of specific binding of an antigen to an antibody, determines the antigen (polypeptide and protein) in the tissue cell by chemically reacting the color developing agent (fluorescein, enzyme, metal ion, or isotope) of the labeled antibody. Positioning, qualitative and quantitative research, called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).

The positive expression of MS4A8B gene is brownish yellow particles on the cell membrane and/or cytoplasm. Using semi-quantitative results, the percentages of the positive cells and the staining intensity of the above samples were scored according to the following criteria:

1. Number of stained cells: 5 high power fields ( × 200) were observed on each slice, the percentage of positive cells was counted, and the number of positive cells was counted. <5 % is 0 points, 5 % to 25 % is 1 point, 26 % to 50 % is 2 points, 51 % to 75 % 3 points, 76 % to 100 % is 4 Minute;

2, positive coloring intensity coefficient: colorless is 0, light yellow is 0.25, brownish yellow is 0.5, tan is 1 ;

3, the score of the number of colored cells and the score of the positive coloring intensity coefficient, the score of the two is the MS4A8B gene expression score, which can be converted into MS4A8B gene expression positive grade: score multiplication <1 is divided into negative (-), score multiplication ≥ 1 is divided into weak positive (+), score multiplication ≥ 2 is positive (++) , the score multiplication ≥ 3 is divided into strong positive (+++).

The MS4A8B gene expression score and MS4A8B of 140 paraffin-embedded prostate cancer tissue samples were obtained. Positive expression of gene expression.

4, PCNA positive reaction

In the above steps, the TUNEL apoptosis experiment was performed on a microarray chip of a slice of a prostate cancer tissue sample embedded in paraffin, and PCNA was performed. Expression studies of positive reactions.

The Roche TUNEL Apoptosis Detection Kit (No. 11684817910) was used. Dewaxing of xylene 10 Minutes, change fresh xylene and dewax for 5-10 minutes. Anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Add 20g/ml without DNase protease K, 20-37 °C for 15-30 minutes, washed 3 times with PBS buffer solution. 3% hydrogen peroxide solution (3% H2O2) in PBS In PBS) Incubate for 10 min at room temperature to inactivate the endogenous peroxidase in sections and wash 3 times with PBS. Biotin labeling, incubation at 37 °C for 60 minutes. PBS washing 1 The reaction stop solution was added dropwise, incubated for 10 minutes at room temperature, and washed three times with PBS. The DAB of the sample was developed and counterstained with hematoxylin.

Proliferating Cell Nuclear Antigen (PCNA) Miyachi was first discovered and named in the serum of patients with SLE (systemic lupus erythematosus) in 1978, and was named for its presence only in normal proliferating cells and tumor cells. Later studies found that PCNA is closely related to cell DNA synthesis, plays an important role in the initiation of cell proliferation, and is a good indicator reflecting the state of cell proliferation. Therefore, PCNA has been set up in recent years. The research boom, especially in oncology.

Nuclear brown staining was used as positive apoptotic cells, and the percentage of positive apoptotic cells was counted as the apoptotic index (AI). The specific calculation is to count at least 5 fields of view per sample, and count 1000 cells per field, and take the average value as the apoptotic index.

The positive PCNA reaction is brown-yellow uniform particles located in the nucleus, and each sample slice is observed at least 5 Representative high-power fields were observed, and positive staining cells were observed in not less than 500 cells. The expression of PCNA was calculated as the percentage of total cells in the cell-positive cells:

Based on the obtained 140 paraffin-embedded prostate cancer tissue samples of MS4A8B Gene expression scores were used to determine the probability of recurrence and metastasis of prostate cancer in each sample.

MS4A8B 基因表达与前列腺癌的复发转移关系的统计学分析 Statistical analysis of the relationship between MS4A8B gene expression and recurrence and metastasis of prostate cancer

Since the MS4A8B gene is a prostate cancer-specific expression gene, particularly prostate primary cancer and metastatic cancer-specific expression genes, The MS4AS8B gene can be used as an independent predictor of biochemical recurrence of prostate cancer, and its expression can be used to predict the recurrence rate and metastasis rate of prostate cancer, that is, when representing the expression of MS4A8B gene. When the MS4A8B gene expression score is low, the prostate cancer recurrence rate and metastasis rate are low, when MS4A8B represents MS4A8B gene expression. When the gene expression score is high, the prostate cancer recurrence rate and metastasis rate are high.

Therefore, the MS4A8B gene expression score was divided into three groups:

For the criteria for determining the probability of recurrence and metastasis of prostate cancer by MS4A8B gene expression score, we Patients with paraffin-embedded prostate cancer tissue samples were followed up for 3-4 years, followed by statistical analysis of the above-mentioned 140 paraffin-embedded prostate cancer tissue samples of MS4A8B. Gene expression scores and actual recurrence and metastasis of the corresponding patients.

All statistical analyses were processed using SPSS 17.0 statistical software. Independent t test (constant variable) for comparison between different groups The t-test was used to compare the means between the two groups, the 分类 2 test was used to compare the categorical variables, and the one-way ANOVA was used to compare the multiple groups. Values are expressed as mean ± SD. P<0.05 It is considered that there is a significant difference and is statistically significant. The expression level of MS4A8B gene in benign, paracancerous, precancerous lesions, prostate cancer and metastatic cancer is not normally distributed. Therefore, nonparametric Wilcoxon is used. Symbol rank sum test or Mann-Whitney U test. Spearman rank correlation coefficient analysis for analysis of MS4A8B gene expression with Gleason Correlation between score, proliferation index, and apoptotic index. Univariate and multivariate analyses were used to assess the value of the MS4A8B gene for predicting clinical parameters. Kaplan-Meier method for plotting recurrence-free survival time (RFS) curve, two-sided log-rank method to assess the statistical correlation between MS4A8B and RFS.

Follow-up was calculated to the last clinical follow-up of tumor-free recurrence. At p<0.05 Differences are considered significant and statistically significant. There are at least three results for each analysis. Values are expressed as mean ± SD.

1, as shown in Figure 8A-8F is the prostate cancer tissue microarray chip sample MS4A8B Immunohistochemical analysis of the protein, in which brown represents the positive expression of the MS4A8B gene. Among them, Fig. 8A shows the case where benign prostate tissue does not express the MS4A8 gene. Figure 8B In the middle, the left side expresses the MS4A8B gene in prostate cancer, the right side of the prostate adjacent gland does not express the MS4A8B gene, the white arrow shows PIN, and the MS4A8B gene is slightly expressed. The Gleason score is , 3+3=6.

Figure 8C ~ 8E, different Gleason The primary prostate cancer scores were graded from medium to high in patients with clinically localized prostate cancer. The sample in Figure 8C was Gleason score, 3+3=6, and the sample in Figure 8D was Gleason score, 4+3=7, the sample of Figure 8E is Gleason score, 4+4=8. Prostate metastatic cancer sample (Lymph node metastasis) in Figure 8F , showing the strongest staining of the MS4A8B gene.

Immunohistochemical staining of MS4A8B for the above 140 paraffin-embedded prostate cancer tissue samples Gene expression was statistically analyzed to obtain statistical results of the expression of MS4A8B gene in benign prostate tissue, paracancerous tissues, HPIN, primary prostate cancer, and metastatic cancer samples. All data are averaged + Standard error shows that all five groups showed significant differences (Wilcoxon signed rank sum test, p value <.001).

2. According to the statistical results of the above expression levels, the MS4A8B gene shown in Figure 9 is obtained in benign prostate tissue, adjacent tissues, Expression profiles in HPIN, primary prostate cancer, and metastatic cancer samples. Figure 9 shows that the MS4A8B gene is highly expressed in prostate cancer and metastatic carcinoma, so MS4A8B The gene is a primary cancer and metastasis-specific expression gene of prostate cancer, using MS4A8B As a marker, the gene can accurately and effectively identify and determine primary and metastatic cancers of prostate cancer, which plays an important role in the recognition and diagnosis of prostate cancer recurrence and metastasis.

3. Based on the above results, the relationship between MS4A8B gene expression and clinicopathological features of prostate cancer was studied. Analysis 140 Clinical characteristics of patients with paraffin-embedded prostate cancer tissue samples, in which the rate of biochemical recurrence (bRFR) in all prostate cancer patients was 75%, PSA and Gleason The scores are related to biochemical recurrence. The sample sampling, statistics and follow-up data of this group are more objective.

As shown in Figures 10A-10C, MS4A8B gene expression and Gleason Correlation analysis of score, proliferation index, and apoptotic index.

As shown in Figure 10A, MS4A8B gene expression was positively correlated with Gleason score (Spearman Level correlation analysis, correlation coefficient 0.206, p<.001). As shown in Figure 10B, MS4A8B gene expression was positively correlated with proliferation index (Spearman) Grade correlation analysis, correlation coefficient 0.411, p<.001). As shown in Figure 10C, MS4A8B gene expression was positively correlated with apoptotic index (Spearman) Grade correlation analysis, correlation coefficient 0.213, p<.001).

4. Further analysis of MS4A8B gene expression and clinicopathological features of prostate cancer, as shown in Table 4.

Table 4 Single-factor and multi-factor correlation analysis of MS4A8B protein expression and clinicopathological parameters

Clinical Parameters Clinical Parameters	Crude OR(95%CI) Crude OR (95% CI)	p-value * P-value *	adjusted OR(95%CI) Adjusted OR (95% CI)	p-value^§ P-value ^§
血管侵犯 Vascular invasion	1.376(1.025,1.846) 1.376 (1.025, 1.846)	0.033* 0.033*	1.282(0.874,1.880) 1.282 (0.874, 1.880)	0.204 0.204
包膜侵犯 Envelope invasion	1.100(0.861,1.406) 1.100 (0.861, 1.406)	0.446 0.446	0.997(0.704,1.410) 0.997 (0.704, 1.410)	0.985 0.985
神经侵犯 Neurological invasion	1.206(0.876,1.661) 1.206 (0.876, 1.661)	0.25 0.25	1.137(0.814,1.589) 1.137 (0.814, 1.589)	0.451 0.451
切缘阳性 Positive margin	1.260(0.333,4.762) 1.260 (0.333, 4.762)	0.733 0.733	0.922(0.132,6.441) 0.922 (0.132, 6.441)	0.935 0.935
远隔转移 Remote transfer	1.481(0.721,3.044) 1.481 (0.721, 3.044)	0.285 0.285	1.586(0.748,3.362) 1.586 (0.748, 3.362)	0.229 0.229
盆腔淋巴结转移 Pelvic lymph node metastasis	1.407(1.048,1.890) 1.407 (1.048, 1.890)	0.023* 0.023*	1.462(0.864,2.473) 1.462 (0.864, 2.473)	0.157 0.157
生化复发 Biochemical recurrence	1.478(1.068,2.046) 1.478 (1.068, 2.046)	0.018* 0.018*	1.730(1.196,2.503) 1.730 (1.196, 2.503)	0.004^§ 0.004 ^§

* Crude OR, p < 0.05

^§ adjusted OR, adjusted OR for age, GS, PSA at diagnosis, T stage, p < 0.05

Univariate analysis indicated that the MS4A8B gene was vascularized (OR=1.376, 95% CI, 1.025 to 1.846, p<.05), pelvic lymph node metastasis (OR=1.407, 95% CI, 1.048 to 1.890, p<.05) and biochemical recurrence Predictor for (OR=1.478, 95% CI, 1.068 to 2.046, p<.05). Age, Gleason score, PSA level at diagnosis and T Multivariate analysis after stage correction showed that the MS4A8B gene was an independent predictor of biochemical recurrence (OR=1.730, 95% CI, 1.196 to 2.503, p<.01).

5. According to the above follow-up results and clinical features, MS4A8B gene expression and recurrence-free survival time (RFS) Correlation analysis.

The MS4A8B gene expression scores were divided into 3 groups as described above:

A total of 140 patients were divided into 3 groups according to the above MS4A8B gene expression score, with an average follow-up of 38.2. Months, low and high expression of MS4A8B showed statistical differences between the two groups (bilateral log-rank method, p <0.05), the other two comparisons did not show statistical differences (low expression) Vs. Moderate expression, p=0.306; moderate expression vs. high expression, p=0.460). Using Kaplan-Meier The survival curve was used to treat the relationship between different MS4A8B gene expression levels in patients with prostate cancer after radical surgery, and Figure 11 was obtained.

As shown in Figure 11, the above results suggest that although the median follow-up time is only 3.61 years, MS4A8B can still be obtained. Genes are the independent predictors of biochemical recurrence after radical surgery in patients with prostate cancer.

Analysis of MS4A8B gene expression in 140 cases of paraffin-embedded prostate cancer tissue samples

Correlation analysis between MS4A8B gene expression and recurrence-free survival time (RFS) was performed based on follow-up results and clinical features. Total 140 patients were divided into 3 groups according to the MS4A8B gene expression level (0 or 1 for low expression; 2 for moderate expression; 3 or 4 for high expression), with an average follow-up of 38.2. Months. Statistical analysis The data of MS4A8B gene expression in each sample of 140 paraffin-embedded prostate cancer tissue samples and the follow-up data of prostate cancer recurrence in the corresponding patients of the sample were obtained. The statistics shown.

Table 5 MS4A8B gene expression and recurrence and metastasis

	MS4A8B 基因表达情况 MS4A8B gene expression
	表达评分 Expression score	不大于 1 Not more than 1	大于 1 但不大于 2 Greater than 1 but not greater than 2	大于 2 Greater than 2
		低度表达患者 Low expression patient	中度表达患者 Moderately expressed patient	高度表达患者 Highly expressed patient
一年无复发生存概率 One year recurrence-free survival probability		97.22% 97.22%	96.39% 96.39%	94.73% 94.73%
两年无复发生存概率 Two-year recurrence-free survival probability		91.00% 91.00%	87.68% 87.68%	72.25% 72.25%
三年无复发生存概率 Three-year recurrence-free survival probability		90.99% 90.99%	67.84% 67.84%	53.552% 53.552%
前列腺癌复发概率 Prostate cancer recurrence probability		低 low	中 in	高 high
发生远处转移的概率 Probability of a distant transfer		15.625% 15.625%	23.30% 23.30%	23.30% 23.30%
前列腺癌转移概率 Prostate cancer metastasis probability		低 low	中 in	中 in

Among them, the MS4A8B gene showed low statistical expression and high expression between the two groups (two-sided log-rank method, p<0.05), the other two comparisons did not show statistical differences (low expression vs. moderate expression, p=0.306; moderate expression vs. high expression, p=0.460 ). Kaplan-Meier survival curve was used to treat the relationship between different MS4A8B gene expression levels in patients with prostate cancer after radical surgery, and Figure 11 was obtained.

The statistics in Table 5 further validate MS4A8B The relationship between gene expression score and prostate cancer recurrence rate and metastasis rate, namely:

1. Patients with low expression of MS4A8B gene expression score of not more than 1 have a clinical recurrence-free survival probability of 97.22%. The two-year recurrence-free survival probability was 91.00%, and the three-year recurrence-free survival probability was 90.99%, indicating that the prostate cancer has a low recurrence rate and a low metastasis rate;

2, MS4A8B gene expression score greater than 1 but not greater than 2 moderately expressed patients, the clinical one-year recurrence-free survival probability 96.39%, two-year recurrence-free survival probability of 87.68%, and three-year recurrence-free survival probability of 67.84%, indicating that the prostate cancer recurrence rate is moderate and metastatic;

3, MS4A8B gene expression score greater than 2 high-expression patients, the clinical one-year recurrence-free survival probability 94.73% The two-year recurrence-free survival probability was 72.25%, and the three-year recurrence-free survival probability was 53.552%, indicating a high recurrence rate and metastasis rate of prostate cancer.

In summary, combined with Table 5 and Figure 11, based on 140 cases of paraffin-embedded prostate cancer tissue samples of MS4A8B The gene expression situation further proves that the MS4A8B gene has specific expression for prostate cancer, especially for primary and metastatic cancers of prostate cancer, among which:

2, MS4A8B Patients with high gene expression show signs of recurrence, and this feature becomes more apparent over time;

Claims

1. Application of MS4A8B gene in the recurrence and metastasis of prostate cancer.

2. The use of the MS4A8B gene according to claim 1 for labeling recurrence and metastasis of prostate cancer, characterized in that said MS4A8B The expression of the gene in the tissue sample to be tested is an indicator of the recurrence and metastasis of prostate cancer.

3. The use of the MS4A8B gene according to claim 2 for labeling recurrence and metastasis of prostate cancer, characterized in that said MS4A8B The higher the expression of the gene in the tissue sample to be tested, the higher the probability of prostate cancer recurrence and metastasis.

4. The use of the MS4A8B gene according to claim 3 for labeling recurrence and metastasis of prostate cancer, characterized in that the obtained MS4A8B The gene expression was scored, the MS4A8B gene expression score was given, and the recurrence and metastasis of prostate cancer were judged based on the obtained MS4A8B gene expression score.

5. A method for labeling recurrence and metastasis of prostate cancer with a MS4A8B gene, characterized in that said MS4A8B The expression of the gene in the tissue sample to be tested is an indicator of prostate cancer.

6. The MS4A8B of claim 5 A method of genetically labeling recurrence and metastasis of prostate cancer, characterized in that the method comprises the steps of:

Detecting the expression of the MS4A8B gene in the tissue sample to be examined;

The higher the expression of the MS4A8B gene in the tissue sample to be examined, the higher the probability of prostate cancer recurrence and metastasis.

7. The MS4A8B of claim 6 A method of genetically labeling recurrence and metastasis of prostate cancer, characterized in that the method comprises the steps of:

Detecting the expression of the MS4A8B gene in the tissue sample to be examined;

The MS4A8B gene expression was scored and the MS4A8B gene expression score was given;

According to the obtained MS4A8B gene expression score, the recurrence and metastasis of prostate cancer were judged.

The method for recurring and metastasizing prostate cancer of the MS4A8B gene according to claim 7, wherein the obtained MS4A8B is obtained. For gene expression, the MS4A8B gene expression score was given using the following criteria:

Percentage of positive cells expressing MS4A8B gene based on tissue samples to be tested: positive cells <5 % is 0, 5% to 25 % is 1 point, 26%~ 50% is 2 points, 51%~75% 3 points, 76%~100% is 4 points;

The positive intensity coefficient of MS4A8B gene expression based on the tissue sample to be tested: negative (-) is 0, weak positive (+) is 0.25, positive (++) is 0.5 and strong positive (+++) is 1;

The score of the percentage of positive cells multiplied by the score of the positive intensity coefficient is the MS4A8B gene expression score.

9. The MS4A8B gene-tagged prostate cancer recurrence and metastasis method according to claim 8, wherein the obtained MS4A8B is obtained according to claim 8. Gene expression scores to determine the recurrence and metastasis of prostate cancer are:

The higher the MS4A8B gene expression score, the higher the probability of prostate cancer recurrence and metastasis.

The method for recurring and metastasizing prostate cancer of the MS4A8B gene according to claim 8, wherein the obtained MS4A8B is obtained according to claim 8. Gene expression scores to determine the recurrence and metastasis of prostate cancer are:

MS4A8B gene expression score of not more than 1 is low expression, and its prostate cancer recurrence rate is low and the metastasis rate is low;

MS4A8B gene expression score greater than 1 but not greater than 2 is moderate expression, its prostate cancer recurrence rate and metastasis rate;

The MS4A8B gene expression score greater than 2 is highly expressed, and its prostate cancer recurrence rate is high and metastasis rate.

The method for recurring and metastasizing prostate cancer of the MS4A8B gene according to claim 5, wherein the tissue sample to be detected is The MS4A8B gene expression can be designed by directly detecting the MS4A8B gene in the DNA genome of prostate cancer tissue using DNA probes or fluorescent probes. The primers were detected by reverse transcriptase polymerase chain reaction (RT-PCR) to detect the mRNA of MS4A8B. Immunohistochemistry, immunofluorescence and flow cytometry were used to detect the transcription of MS4A8B. The protein of the gene and the protein are stained and labeled.

12. Use of an agent for detecting expression of a MS4A8B gene in a sample for labeling prostate cancer recurrence and metastasis, characterized in that said MS4A8B The expression of the gene in the tissue sample to be tested is an indicator of prostate cancer.

13. The test sample according to claim 12, MS4A8B Use of an agent for gene expression in the labeling of prostate cancer recurrence and metastasis, characterized in that said MS4A8B The higher the expression of the gene in the tissue sample to be tested, the higher the probability of prostate cancer recurrence and metastasis.

14. The test sample according to claim 13 in the sample MS4A8B The application of the agent for gene expression in labeling prostate cancer recurrence and metastasis is characterized in that the expression of the obtained MS4A8B gene is scored, and the MS4A8B gene expression score is given, according to the The MS4A8B gene expression score was used to determine the recurrence and metastasis of prostate cancer.