CN116515988A - Markers for assessing T lymphocyte function after prolonged glucocorticoid use - Google Patents
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Abstract
The invention provides a methylation biomarker for evaluating T lymphocyte function after long-term use of glucocorticoid and application thereof. Use of mTORC1 pathway regulatory genes for the preparation of a test product for detecting T lymphocyte function after prolonged use of a glucocorticoid, the test product assessing T lymphocyte function by detecting the methylation level of one or more specific genes in the mTORC1 pathway regulatory genes in a test sample DNA, the specific genes comprising FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC1, PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3. Based on the elevated/reduced methylation levels of specific genes, T lymphocyte function in a subject can be assessed, facilitating monitoring and assessment of the immune system at any time, by taking countermeasures to reduce the risk of infection.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and particularly relates to a methylation biomarker for evaluating T lymphocyte function after long-term use of glucocorticoid and application thereof.
Background
Natural and synthetic glucocorticoids (also known as steroid hormones, abbreviated GC/GCs) are used to treat a variety of diseases and are widely used in anti-inflammatory, antitoxic, antishock and immunosuppressive therapies. Long-term use of glucocorticoids inhibits the activity and function of immune cells, e.g. affects the function of T lymphocytes, which are an important immune cell in the human immune system, and they recognize and destroy abnormal cells and pathogens, thus maintaining the immune state of the body; thus, long-term use of glucocorticoids can affect the body's recognition and clearance of pathogens by inhibiting T cell function, increasing the risk of infection.
The prior art has not yet been effective in determining the effect of glucocorticoid use on the immune system, as the response of the immune system may be different in different individuals after the same dose of glucocorticoid. This also makes it difficult for a physician to predict the response of the immune system and susceptibility to infection after glucocorticoid use. Thus, there is a need to closely monitor and evaluate the immune system response during long term use of glucocorticoids and to minimize the time and dosage of use of glucocorticoids to reduce damage to the immune system.
In view of the above, there is a need to develop a diagnostic tool for rapid clinical assessment of T lymphocyte function after prolonged use of glucocorticoids, which is useful for guiding the clinical use of glucocorticoids to reduce the occurrence of infections.
Disclosure of Invention
The invention aims to improve methylation markers for assessing T lymphocyte function after long-term use of a glucocorticoid, so as to monitor the condition of an immune system during long-term use of the glucocorticoid and timely reduce damage to the immune system.
To achieve the above object, the present invention provides a use of mTORC1 pathway regulatory genes for preparing a test product for detecting T lymphocyte function after long-term use of a glucocorticoid, the test product evaluating the function of T lymphocyte by detecting methylation level of one or more specific genes among the mTORC1 pathway regulatory genes in a test sample DNA, the specific genes including FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC1, PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3.
In a specific embodiment, the PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3 gene in the specific gene is a positive regulatory gene, and the FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC gene in the specific gene is a negative regulatory gene, when the methylation level of the positive regulatory gene is increased and/or the methylation level of the negative regulatory gene is decreased, the risk of assessing impaired T lymphocyte function in the subject is high.
In a specific embodiment, the glucocorticoid impairs the viability of cd4+ T cells by inhibiting the positive regulatory gene and promoting the inhibition of transcription of the mTORC1 pathway by the negative regulatory gene.
In a specific embodiment, the detection product is a kit.
In a specific embodiment, the kit contains primer sequence pairs and/or probe sequences for detecting a particular gene.
The invention also provides the use of a reagent for detecting the methylation level of one or more specific genes in a sample DNA, wherein the reagent is used for detecting the methylation level of the specific genes in the sample DNA, the specific genes comprise FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC1, PLD1, PIK3CD, PLD3, AKT3, PIK3R1, HRASLS, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, HRASLS5, HRASLS2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1 and RPS6KA3, in the preparation of a reagent for detecting the methylation level of DNA after long-term use of glucocorticoid.
In a specific embodiment, the specific gene comprises at least one CpG site and the reagent is used to detect the methylation level of any one of the plurality of CpG sites or an average of the methylation levels of the plurality of CpG sites of the specific gene.
In a specific embodiment, the PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3 gene in the specific gene is a positive regulatory gene, and the FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC gene in the specific gene is a negative regulatory gene, when the methylation level of the positive regulatory gene is increased and/or the methylation level of the negative regulatory gene is decreased, the risk of assessing impaired T lymphocyte function in the subject is high.
In a specific embodiment, the reagent is used to detect the methylation level of at least one positive regulator gene and at least one negative regulator gene in the sample DNA.
In a specific embodiment, the reagent further comprises specific primer pairs and probes for detecting methylation status of the specific gene, wherein the number of the specific primer pairs and the number of the probes are the same as the number of CpG sites of the specific gene, different specific primer pairs are used for amplifying to obtain different gene fragments of the specific gene, and each probe is used for detecting whether one CpG site of the specific gene is subjected to methylation change.
In a specific embodiment, the probes are biochip probes, each specific gene corresponds to one probe, and the sequence of the probes corresponding to the specific genes one by one is shown as SEQ ID NO. 1 to SEQ ID NO. 33 according to the arrangement sequence of the specific genes from front to back.
The invention also provides the use of a reagent for detecting the methylation level of DNA in the preparation of a reagent kit for detecting the methylation level of a target gene fragment of one or more specific genes in sample DNA after long-term use of glucocorticoid, wherein the target gene fragment of the specific genes comprises at least one CpG site, and the reagent kit is used for evaluating the function of T lymphocytes according to the methylation level.
The beneficial effects of the invention at least comprise:
1. the present invention provides an agent capable of detecting the methylation level of the specific gene, and evaluating the T lymphocyte function after long-term use of glucocorticoid according to the change of the methylation level to guide the application of glucocorticoid in clinic, and evaluating the risk of damaging the T lymphocyte function when the methylation level of the specific gene is abnormal is large, wherein the damage to the immune system is reduced by reducing the use time and the use dosage of glucocorticoid, so as to reduce the risk of infection.
2. The reagent for detecting the methylation level of one or more specific genes in the DNA of the test sample is applied to a T lymphocyte function kit for evaluating the glucocorticoid after long-term use; thus, during long-term use of glucocorticoids, T lymphocyte function can be rapidly assessed to be able to monitor and assess the immune system response at any time, reducing the risk of infection by taking countermeasures.
Drawings
FIG. 1 (A) is a graph showing changes in CD4+ and CD8+ caused by conventional GC treatment;
FIG. 1 (B) is a graph showing changes in CD3+CD4+ T cells caused by conventional GC treatment;
FIG. 1 (C) is a graph showing changes in CD3+CD8+ T cells caused by conventional GC treatment;
FIG. 1 (D) is a graph showing the change in CD4/CD8 ratio caused by conventional GC treatment;
FIG. 2 is a heat map of a real-time PCR array showing mTORC1 pathway genes with significant expression changes (P < 0.05) after 2 months of GC treatment;
FIG. 3 (A) is a graph showing the number of different methylated mTorrC 1 regulatory genes by time series analysis;
FIG. 3 (B) is a ratio graph showing different methylated mTorrC 1 regulatory genes by time series analysis;
FIG. 4 (A) is a time course analysis of individual CpG methylation changes in the promoter region of the negative mTorrC 1 pathway gene;
FIG. 4 (B) is a time course analysis of individual CpG methylation changes in the promoter region of the positive mTorrC 1 pathway gene;
FIG. 4 (C) time course mRNA expression profiles of DNMT1, DNMT3a, DNMT3b, TET1, TET2 and TET 3.
Detailed Description
The invention is described in detail below with reference to the drawings and examples, but the invention can be practiced in many different ways, which are limited and covered by the claims.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Definition: the term "methylation" refers to methylation of cytosine at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. The term "methylation level," which may also be referred to as methylation state, refers to the presence, absence, and/or amount of methylation at a nucleotide in a particular nucleotide or portion of DNA. Both elevated and reduced methylation levels are compared to normal methylation levels.
The methylation level of a specific gene provided by the invention can be used to assess T lymphocyte function, i.e., the methylation level of the specific gene is a DNA methylation marker that assesses T lymphocyte function after prolonged use of a glucocorticoid.
In the embodiment of the invention, the methylation level of one site of a specific gene can be detected to be used as a marker for evaluating the function of T lymphocytes, the methylation level of a plurality of sites of the specific gene can be detected to be used as a marker for evaluating the function of T lymphocytes, the methylation level of a plurality of specific genes can be detected to be used as a marker for evaluating the function of T lymphocytes, and the methylation level of a plurality of specific genes can be the methylation level of one site or the methylation level of a plurality of sites can be the average value of the methylation levels of a plurality of sites.
Preferably, the methylation level of at least one positive regulatory gene and at least one negative regulatory gene is detected as a marker for assessing T lymphocyte function, which may provide the sensitivity of the kit.
Since it is possible to use a gene fragment containing a site to be tested as a marker for evaluating the function of T lymphocytes by detecting the methylation level of one site or two sites or three sites of a specific gene, the methylation level of a gene fragment of a specific gene can also be used as a DNA methylation marker for evaluating the function of T lymphocytes after long-term use of glucocorticoid.
Example 1
Screening of DNA methylation markers for assessing T lymphocyte function after prolonged glucocorticoid use
1. Prospective time series study
10 patients diagnosed with minimal change in glomerular disease (MCD) and with onset of Nephrotic Syndrome (NS) were selected for 8 months of follow-up. In this time series study, all patients were adequately informed and received a standard conventional glucocorticoid regimen starting with either high doses of oral prednisone (1 mg/kg/day, up to 80 mg/day) or comparable doses of methylprednisolone (0.8 mg/kg/day), for 6 to 8 weeks, with a 5mg decrease every two weeks after complete remission. Patients who were included in the study did not receive any immunosuppressive agent other than glucocorticoid. Patients who failed to achieve complete remission of renal proteinuria or who developed early relapse within 8 weeks were excluded from the study and received additional immunosuppressive agent treatment.
2. Human CD4+ T cell isolation and stimulation experiments
Cd4+ T cells were purified from Peripheral Blood Mononuclear Cells (PBMCs) using a magnetic bead isolation kit (Miltenyi Biotec). Cd4+ T cells were activated in 10% FBS supplemented RPMI-1640 medium for 72 hours using a T cell expansion kit (Miltenyi Biotec) comprising biotinylated antibodies to human CD2, CD3 and CD 28. Activated CD4+ T cells were cultured with TGF-beta (2 ng/ml) and IL-2 (200U/ml), inducing the production of tregs. For pathogen stimulation experiments, phytohemagglutinin (PHA, sigma-Aldrich,5 g/ml) was used as a mitotic stimulator, PHA was used in cultured T cells, followed by T cell isolation. Experiments on mTORC1 were performed using commercial non-targeting siRNA, PTEN siRNA and PS6K siRNA, following manufacturer's instructions (Cell Signaling, #6568, #6251 and # 6566).
3. Flow cytometry analysis
The flow cytometry comprises the following steps: single cell suspensions were stained at 4 ℃ for 30 min with the following directly bound antibodies: CD3-PerCP, CD4-PE, CD8-PE/Cy7, CD25-APC/Cy7, FOXP3-FITC, IFNG-Pacificblue, IL10-Pacificblue, ki67-Pacificblue, rabbit isotype control IgG (BioLegend), bcl-xL-FITC (Invitrogen), and Phospho-S6-APC (Cell Signaling); performing isotype control and a control of fluorescence reduction in a multiplex color immunofluorescence experiment to ensure proper threshold setting; for intracellular staining of cytokines, single cell suspensions were fixed and permeabilized using Foxp 3/transcription factor staining buffer set (eBioscience), operating according to the manufacturer's protocol; apoptosis assays were performed using the FITC annexin v/PI apoptosis detection kit (Biolegend); cell sorting was collected and analyzed using BD FACSCalibur flow cytometer and FlowJo software (Tree Star).
4. Whole genome DNA methylation analysis
For analysis of the methylation profile of the whole genome, a DNA sample was extracted from a human whole blood sample using DNeasy Blood and Tissue kit (Qiagen), and then genomic DNA was subjected to sodium sulfite treatment and Infinium Human Methylation 850,850, 850BeadChip (Illumina) treatment according to the manufacturer's standard protocol. The methylation fraction of each CpG site is expressed as a beta value in terms of fluorescence intensity. The methylation fraction of CpG sites is expressed as a beta value (0 to 1) and the methylation level is expressed as a percentage. Two techniques and biological replicates were processed by chip technology to ensure that the differential methylation observed between study samples represents a true biological differential. For all replicates, the pearson correlation coefficient was >0.99, confirming good reproducibility of the chip processing.
5. mRNA sequencing and real-time PCR
To determine gene expression, total RNA was isolated from PBMC or cd4+ T cells using the RNeasy mini kit (Qiagen). The amount and quality of RNA was evaluated using NanoDrop to ensure that the 260/280 and 260/230 ratios were greater than 2.0 for all samples. Paired-end libraries were synthesized using TruSeq RNA Sample Preparation Kit (Illumina). Clusters were generated on cBot and sequenced on Illumina HiSeq X-ten (Illumina) as per manufacturer's instructions. Real-time PCR assays were performed using PCR stock and pre-designed primers to confirm the expression of specific genes. Gene expression (fold change for each gene of interest) was normalized to glycolytic product-3-phosphate dehydrogenase (GAPDH) using the 2-DeltaCt method. All assays were performed in triplicate.
6. Statistical analysis
Statistical analysis was performed using the SPSS 22 software package (IBM). Data are presented as mean ± SEM. FDR analysis of multiplex assays was performed and FDR <5% (q < 0.05) was considered to be a significant difference. The comparison of the differences between the groups was performed using a two-tailed Student t-test, paired t-test, wilcoxon rank sum test or chi-square test (χ2 test), as required. For three or more sets of comparisons, one-way anova was performed followed by Tukey test. Double sided P values <0.05 were considered statistically significant.
7. Experimental results
1) Chronic glucocorticoid treatment constantly alters the composition of circulating T cells
The results of the flow cytometry analysis are shown in fig. 1A to 1D, and fig. 1A reveals the ratio change of T cell subsets at 0, 2, 4, 8 months, and as can be seen from fig. 1B to 1D, the conventional GC protocol reduces the ratio of cd3+cd4 but promotes the ratio of cd3+cd8+t cells, resulting in a significant decrease in the CD4/CD8 ratio, and as can be seen from fig. 1, the GC drops to physiological equivalent at 8 months, these changes still exist, indicating that the conventional GC treatment has a continuous effect on cd4+t cells.
In fig. 1 (B) to 1 (D), the respective curves correspond to the following schemes:
MCD Individuals
MCD Average
Healthy Average
2) GC modulates CD4+ T cell biology by modulating mTorrC 1 pathway
GC was shown to significantly alter mRNA expression of multiple mTORC1 regulated genes in cd4+ T cells by a real-time qPCR array. The real-time PCR array showed a heat map of mTORC1 pathway genes with significant expression changes (P < 0.05) after 2 months of GC treatment, and the gene expression heat map indicated that GC exerted an inhibitory effect on transcription of mTORC1 pathway by mainly inhibiting positive regulatory genes and promoting negative regulatory genes (FIG. 2). Flow cytometry analysis of cd4+ T cells showed that GC significantly inhibited global expression of effector PS6 downstream of mTORC1 pathway. Using the siRNA approach, we specifically silenced the expression of PS6K and PTEN genes. Consistently, PS6KsiRNA further reduced proliferation of GC-exposed cd4+ T cells and increased apoptosis by inhibiting mTORC1 pathway. In contrast, activation of the mTORC1 pathway by PTENsiRNA counteracts the interfering effect of GC on cd4+ T cell viability. From the above experimental data, GC compromised the viability of cd4+ T cells by inhibiting the effects of mTORC1 signaling pathway.
3) GC has different effects on the DNA methylation and gene expression relationship of the mTorrC 1 pathway
Time series analysis of whole genome DNA methylation was performed on individuals 2 months, 4 months and 8 months before and after GC treatment, a list of 340 mTORC1 regulatory genes was selected and classified into negative (green), positive (red) and potential (orange) regulatory role groups based on PubMed and KEGG data. In the promoter regions of these genes (TSS 1500, TSS200 and 5' untranslated regions), 4642 CpG sites were examined in total and separated into different quadrants (Q1 to Q5, reflecting the change from hypomethylation to hypermethylation) according to their methylation level changes. The number of nucleotides with significant methylation changes increases during the GC taper. Quantitative analysis showed that the hypermethylated nucleotide (Q4) was significantly changed relative to the high dose GC exposure time points of 2 months (GCs/2 m) and 4 months (GCs/4 m), 8 months (GCs/8 m) (FIG. 3A). Further proportional analysis showed that during the high dose GC exposure period (GCs/2 m), the hypomethylated group (Q1) consisted mainly of mTORC1 negative regulator genes, while the hypermethylated group (Q4) consisted mainly of positive regulator genes (fig. 3B). The above epigenetic changes explain to some extent the global transcriptional inhibition of the mTORC1 pathway by GC at 2 months (fig. 2). However, at 8 months, in the genes responsible for modulating the mTORC1 pathway, the positive regulator was highly increased in the hypomethylated group (Q1) and decreased in the hypermethylated group (Q4), whereas the proportion of negative regulator was changed. Given the inhibition of gene expression by cytosine methylation, these data suggest that GC may dynamically affect mTORC1 pathway activity through dose-dependent epigenetic regulation.
The same samples were then subjected to high throughput RNA sequencing to further determine the relationship between the mTORC1 pathway of hypomethylation-promoting gene expression (Q1) and hypermethylation-inhibiting gene expression (Q3). Only a few genes can be identified as having altered transcription associated with changes in DNA methylation patterns for 2 months and 4 months. Whereas at 8 months of GC reduced to physiologically equivalent levels, the number of genes associated with Q1 and Q3 increased significantly. According to the results, a total of 33 mTORC1 regulatory genes may be affected by cytosine methylation, including 9 negative regulatory genes and 24 positive regulatory genes, wherein 9 negative regulatory genes include GSKIP, TSC1, PRKACB, CAB39L, PRKAR1A, FKBP, PTEN, PPKAG2, and 24 positive regulatory genes include hrals 2, AKT3, hrals, RAG1, RPS6KA3, IGF2, RPS6KA5, EIF4A2, hrals 5, IRS2, TELO2, RPS6KA1, PIK3R6, PIK3R3, PIK3CA, SGK1, PIK3R1, PLD3, AKT2, PLD1, EIF4G3, PIK3CD, PIK3R5.
The single cytosine methylation profile in the promoter regions of 33 identified mTORC1 regulatory genes was further evaluated. Time series analysis showed that the changes in average methylation levels in negative regulator genes tended to be modest, with the majority gradually reverting to baseline levels of 8 months [ fig. 4 (a) ]. In contrast, cytosine methylation changes in some upregulated genes were significant, particularly at 8 months [ fig. 4 (B) ]. Time series analysis of DNA methyltransferases (DNMTs) showed that mRNA expression of DNMT1 and DNMT3a was significantly inhibited at 2 months, but restored to normal levels at 8 months. The mRNA expression levels of undecamed methyl cytosine deoxyenzymes (TETs) 2 and 3 decreased significantly at 8 months, as shown in fig. 4 (C), from which it can be seen that GC had time-dependent differences in DNMTs and TETs regulation.
Taken together, the dynamic relationship between cytosine methylation levels and gene expression highlights the key role of epigenetic regulation in mTORC1 signaling dynamics, leading in a dose-independent manner to the long-term impact of conventional GC treatment on cd4+ T cell biology [ fig. 4 (C) ].
Based on the experimental study, the coordinate information of the DNA methylation markers obtained by screening is as follows:
the coordinate information of a specific gene (DNA methylation marker) based on the sequence of Hg19 of the human reference genome is as follows:
example 2
Design of specific primer pairs and probes
The 33 genes provided by the invention comprise a plurality of CpG sites, each CpG site is respectively corresponding to a specific primer pair (comprising an upstream primer and a downstream primer) and a probe, wherein the specific primer pair is used for amplification, and the probe pair is used for detecting whether methylation changes occur in the CpG sites of the genes.
In this embodiment, the gene detection is chip detection, and the designed probe is a biochip probe, specifically, a biochip of Illumina, which is composed of 2 parts: the 1 st is a glass substrate, and the 2 nd is a bead. The microbeads are the core part of the chip and have small volume, only micron-sized. The surface of each microbead is coupled with a DNA fragment of a sequence. There are hundreds of thousands of fragments per bead, and the fragments on one bead are all of the same species. These DNA fragments are 73 bases in length, and the 73 bases are divided into 2 functional regions. The sequence of 23 bases near this end of the bead, called the Address sequence, is also the 5' end of the DNA fragment. It is a tag sequence that identifies the microbead. The tag sequence, through the arrangement and combination of bases, obtains a plurality of possibilities, and each sequence is the identification card number (ID number) of the corresponding microbead. The sequence of the 50 bases of the DNA fragment at the end remote from the bead, i.e.the 3' end, is called Probe sequence, and functions to complementarily hybridize with the target DNA. An Address sequence corresponds to a Probe sequence. There is a one-to-one correspondence between them.
Specifically, the sequence of the biochip probe is as follows:
it should be noted that the target sites hereinafter refer to CpG sites.
The sequence of the probe for detecting the FKBP5 gene target site is shown as SEQ ID NO:1, wherein the coordinates of the target site of the FKBP5 gene are as follows: chr6:35655607-35656856;
the sequence of the probe for detecting the target site of the PRKAG2 gene is shown as SEQ ID NO:2, wherein the coordinates of the target site of the PRKAG2 gene are as follows: chr7, 151572761-151575106;
the sequence of the probe for detecting the target site of the CAB39 gene is shown as SEQ ID NO:3, wherein the coordinates of the target site of the CAB39 gene are: chr2, 231577309-231578504;
the sequence of the probe for detecting the target site of the CAB39L gene is shown as SEQ ID NO:4, wherein the coordinates of the target site of the CAB39L gene are: chr13, 50018121-50018738;
the sequence of the probe for detecting the GSKIP gene target site is shown as SEQ ID NO:5, wherein the coordinates of the target site of the GSKIP gene are as follows: chr14, 96829340-96830334;
the sequence of the probe for detecting the target site of the PRKACB gene is shown as SEQ ID NO:6, wherein the coordinates of the target site of the PRKACB gene are as follows: chr1, 84543146-84544261;
the sequence of the probe for detecting the PTEN gene target site is shown as SEQ ID NO:7, wherein the coordinates of the PTEN gene target site are as follows: chr10, 89621772-89624128;
the sequence of the probe for detecting the target site of the PRKAR1A gene is shown as SEQ ID NO:8, wherein the coordinates of the target site of the PRKAR1A gene are as follows: chr17, 66507790-66508956;
the sequence of the probe for detecting the target site of the TSC1 gene is shown as SEQ ID NO:9, wherein the coordinates of the target site of the TSC1 gene are as follows: chr9, 135819526-135820529;
the sequence of the probe for detecting the PLD1 gene target site is shown as SEQ ID NO:10, wherein the coordinates of the target site of the PLD1 gene are: chr3, 171527617-171528458;
the sequence of the probe for detecting the target site of the PIK3CD gene is shown as SEQ ID NO:11, wherein the coordinates of the target site of the PIK3CD gene are: chr1, 9711780-9713001;
the sequence of the probe for detecting the PLD3 gene target site is shown as SEQ ID NO:12, wherein the coordinates of the target site of the PLD3 gene are: chr19:40854180-40854733;
the sequence of the probe for detecting the AKT3 gene target site is shown as SEQ ID NO:13, wherein the coordinates of the target site of the AKT3 gene are: chr1, 243876720-243877450;
the sequence of the probe for detecting the target site of the PIK3R1 gene is shown as SEQ ID NO:14, wherein the coordinates of the target site of the PIK3R1 gene are: chr5:67510872-67512386;
the sequence of the probe for detecting the target site of the HRASLS gene is shown as SEQ ID NO:15, wherein the coordinates of the target site of the hrals gene are: chr17, 66507790-66508956;
the sequence of the probe for detecting the target site of the PIK3R5 gene is shown as SEQ ID NO:16, wherein the coordinates of the target site of the PIK3R5 gene are: chr17, 8868469-8869372;
the sequence of the probe for detecting the target site of the EIF4G3 gene is shown as SEQ ID NO:17, wherein the coordinates of the target site of the EIF4G3 gene are: chr1, 21503115-21503836;
the sequence of the probe for detecting the target site of the TELO2 gene is shown as SEQ ID NO:18, wherein the coordinates of the target site of the TELO2 gene are: chr16:1550144-1550829;
the sequence of the probe for detecting the PIK3CA gene target site is shown as SEQ ID NO:19, wherein the coordinates of the target site of the PIK3CA gene are: chr3, 178866068-178866908;
the sequence of the probe for detecting the AKT2 gene target site is shown as SEQ ID NO:20, wherein the coordinates of the target site of the AKT2 gene are: chr19:40790737-40791607;
the probe for detecting the IGF2 gene target site has a sequence shown in SEQ ID NO:21, wherein the coordinates of the target site of IGF2 gene are: chr11, 2158951-2162484;
the sequence of the probe for detecting the target site of the HRASLS5 gene is shown as SEQ ID NO:22, wherein the coordinates of the target site of the hrals 5 gene are: chr11, 63258378-63258804;
the sequence of the probe for detecting the target site of the HRASLS2 gene is shown as SEQ ID NO:23, wherein the coordinates of the target site of the hrals 2 gene are: chr11, 63330425-63330670;
the sequence of the probe for detecting the target site of the EIF4A2 gene is shown as SEQ ID NO:24, wherein the coordinates of the target site of the EIF4A2 gene are: chr3, 186500738-186502048;
the sequence of the probe for detecting the target site of the PIK3R3 gene is shown as SEQ ID NO:25, wherein the coordinates of the target site of the PIK3R3 gene are: chr1, 46598126-46599129;
the sequence of the probe for detecting the target site of the RPS6KA1 gene is shown as SEQ ID NO:26, wherein the coordinates of the target site of the RPS6KA1 gene are: chr1, 26856191-26856684;
the sequence of the probe for detecting the target site of the RAG1 gene is shown as SEQ ID NO:27, wherein the coordinates of the target site of the RAG1 gene are: chr11, 36588220-36588490;
the sequence of the probe for detecting the target site of the PIK3R6 gene is shown as SEQ ID NO:28, wherein the coordinates of the target site of the PIK3R6 gene are: chr17, 8702342-8702824;
the sequence of the probe for detecting the target site of the IRS-2 gene is shown as SEQ ID NO:29, wherein the coordinates of the target site of the IRS2 gene are: chr13, 110434466-110440180;
the sequence of the probe for detecting the target site of the RPS6KA5 gene is shown as SEQ ID NO:30, wherein the coordinates of the target site of the RPS6KA5 gene are: chr14, 91526233-91527552;
the sequence of the probe for detecting the target site of the EIF4G1 gene is shown as SEQ ID NO:31, wherein the coordinates of the target site of the EIF4G1 gene are: chr3, 184314606-184335358;
the sequence of the probe for detecting the target site of the SGK1 gene is shown as SEQ ID NO:32, wherein coordinates of the target site of the SGK1 gene are: chr6:134638797-134639021;
the sequence of the probe for detecting the target site of the RPS6KA3 gene is shown as SEQ ID NO:33, wherein the coordinates of the target site of the RPS6KA3 gene are: chrysX 20284127-20286811.
TABLE 1 nucleotide sequences of the respective probes
It should be noted that, a person skilled in the art can design a primer pair for amplification based on the coordinate information of a specific gene and the coordinates of a disclosed target site, and the design of the primer pair belongs to the prior art and is not described herein.
In addition, the kit of the invention can also comprise other conventional reagents required by methylation quantitative PCR, such as one or more of DNA extraction reagents, sulfite, deionized water, taq mix buffer and the like. Because the common reagents for methylation quantitative PCR can be purchased independently or prepared by self through a market way, the reagents are particularly required to be assembled into the kit, can be prepared according to the actual needs of customers, and can be assembled into the kit for convenience.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments, and is not intended to limit the practice of the invention to such description. It will be apparent to those skilled in the art that several simple deductions and substitutions can be made without departing from the spirit of the invention, and these are considered to be within the scope of the invention.
Claims (10)
- Use of mTORC1 pathway regulatory genes, for the preparation of a test product for detecting T lymphocyte function after prolonged use of a glucocorticoid, the test product assessing T lymphocyte function by detecting the methylation level of one or more specific genes in the mTORC1 pathway regulatory genes in a test sample DNA, the specific genes comprising FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC, PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, tele 2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3.
- 2. The use of mTORC1 pathway regulatory genes according to claim 1, wherein the PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3 genes in the specific genes are positive regulatory genes, and the FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR A, TSC1 genes in the specific genes are negative regulatory genes, and the risk of impaired T lymphocyte function in the subject is assessed when the methylation level of the positive regulatory genes is increased and/or the methylation level of the negative regulatory genes is decreased.
- 3. The use of mTORC1 pathway regulatory gene according to claim 2, wherein the glucocorticoid impairs the viability of cd4+ T cells by inhibiting the positive regulatory gene and promoting the transcription of the mTORC1 pathway by the negative regulatory gene.
- 4. Use of a reagent for detecting the methylation level of a DNA for the preparation of a kit for detecting the methylation level of one or more specific genes in a sample DNA, including FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR1A, TSC1, PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, tele 2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3, for the detection of the methylation level of a DNA after prolonged use of a glucocorticoid.
- 5. The use according to claim 4, wherein the specific gene comprises at least one CpG site and the reagent is for detecting the methylation level of any one of the plurality of CpG sites of the specific gene or an average of the methylation levels of the plurality of CpG sites.
- 6. The use according to claim 4, wherein the PLD1, PIK3CD, PLD3, AKT3, PIK3R1, hrals, PIK3R5, EIF4G3, TELO2, PIK3CA, AKT2, IGF2, hrals 5, hrals 2, EIF4A2, PIK3R3, RPS6KA1, RAG1, PIK3R6, IRS2, RPS6KA5, EIF4G1, SGK1, RPS6KA3 genes in the specific gene are positive regulatory genes, the FKBP5, PPKAG2, CAB39L, GSKIP, PRKACB, PTEN, PRKAR A, TSC1 genes in the specific gene are negative regulatory genes, and the subject is assessed for a large risk of impaired T lymphocyte function when the methylation level of the positive regulatory genes is increased and/or the methylation level of the negative regulatory genes is decreased.
- 7. The use according to claim 6, wherein the reagent is used for detecting the methylation level of at least one positive regulatory gene and at least one negative regulatory gene in the sample DNA.
- 8. The use according to claim 4, wherein the reagent further comprises specific primer pairs and probes for detecting methylation status of the specific gene, the number of the specific primer pairs and the number of the probes are the same as the number of CpG sites of the specific gene, different specific primer pairs are used for amplifying different gene fragments of the specific gene, and each probe is used for detecting whether methylation change occurs in one CpG site of the specific gene.
- 9. The use according to claim 8, wherein the probes are biochip probes, each of the specific genes corresponds to one of the probes, and the sequence of the probes corresponding to the specific genes one by one is shown in SEQ ID NO. 1 to SEQ ID NO. 33 in the order of the specific genes arranged from front to rear.
- 10. Use of a reagent for detecting the methylation level of DNA for the preparation of a kit for T lymphocyte function after prolonged use of a glucocorticoid, wherein the reagent is used for detecting the methylation level of a target gene fragment of one or more specific genes according to claim 4 in a test sample DNA, said target gene fragment of a specific gene comprising at least one CpG site, said kit being used for assessing the function of T lymphocytes on the basis of said methylation level.
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| CN104363914A (en) * | 2011-11-23 | 2015-02-18 | 因特利凯有限责任公司 | Enhanced treatment regimens using mtor inhibitors |
| CN106659758A (en) * | 2014-06-02 | 2017-05-10 | 儿童医疗中心有限公司 | Methods and compositions for immunomodulation |
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