CN114350808A - Breast cancer adjuvant immunotherapy target gene SHISAL2A detection kit and application thereof - Google Patents
Breast cancer adjuvant immunotherapy target gene SHISAL2A detection kit and application thereof Download PDFInfo
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- CN114350808A CN114350808A CN202210056901.7A CN202210056901A CN114350808A CN 114350808 A CN114350808 A CN 114350808A CN 202210056901 A CN202210056901 A CN 202210056901A CN 114350808 A CN114350808 A CN 114350808A
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Abstract
The invention belongs to the technical field of biology, and relates to a breast cancer adjuvant immunotherapy target gene SHISAL2A detection kit and application thereof. The present study demonstrated a significant increase in the expression of SHISAL2A in breast cancer. The expression level of SHISAL2A is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMATESCORE, is positively correlated with various immune cell infiltrates and is positively correlated with the expression of immune checkpoint genes, and a SHISAL2A detection kit is designed.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting the expression level of SHISAL2A in preparation of a preparation for breast cancer detection and/or adjuvant immunotherapy, and a kit and application of a target gene SHISAL2A for breast cancer adjuvant immunotherapy.
Background
Breast cancer (BRCA) is one of the common malignant tumors in women worldwide, and its new incidence and mortality are the first of all types of cancer. Although the incidence of breast cancer is relatively low in China, the population is large, the total incidence is large, the incidence rate tends to rise year by year, and the incidence age tends to be younger. Immunotherapy is a promising new breast cancer treatment mode, but has the problems of low treatment response rate, side effects and the like, so that the development of a novel auxiliary immunotherapy target has important significance for clinical tumor immunotherapy.
Disclosure of Invention
The invention aims to provide a novel breast cancer immunotherapy target SHISAL2A, and provides application of a reagent for detecting SHISAL2A expression level in preparation of a preparation for breast cancer detection and/or adjuvant immunotherapy, and a breast cancer adjuvant immunotherapy target gene SHISAL2A detection kit and application.
The invention provides application of a reagent for detecting the expression level of SHISAL2A in preparing a preparation for breast cancer detection and/or adjuvant immunotherapy.
Further, the detecting the expression level of the SHISAL2A comprises detecting the expression level of mRNA of SHISAL2A and/or detecting the expression level of protein of SHISAL 2A.
Further, the method for detecting the expression level of SHISAL2A comprises the following steps: detecting the expression quantity of SHISAL2A mRNA in breast cancer and paracarcinoma tissues by an RT-qPCR method; detecting the expression quantity of SHISAL2A mRNA in breast cancer and paracarcinoma tissues by a molecular probe technology; the expression level of SHISAL2A protein in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of the SHISAL2A is an oligonucleotide probe, a PCR primer or an antibody targeting the SHISAL2A of the DNA sequence or mRNA sequence of the SHISAL2A gene.
According to the present invention, preferably, the reagent for detecting the expression level of SHISAL2A is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-CTTCCGCGACCACAAGTA-3’(SEQ ID NO：1)
5’-ATGAGAGCGCCAATGCTGA-3’(SEQ ID NO：2)
The second aspect of the present invention provides a breast cancer adjuvant immunotherapy target gene SHISAL2A detection kit, which comprises: reagents for detecting the expression level of SHISAL 2A.
Further, the reagent for detecting the expression level of the SHISAL2A is an oligonucleotide probe, a PCR primer or an antibody targeting the SHISAL2A of the DNA sequence or the mRNA sequence of the SHISAL2A gene.
Specifically, the reagent for detecting the expression level of SHISAL2A is a reagent with the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
According to the present invention, the kit may further contain other conventional reagents for real-time fluorescence quantification, and preferably, the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethanol, RNase-free water, random primers, 5 XM-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, a primer having the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and (4) ACTB real-time fluorescence quantitative PCR specific primers of the nucleotide sequence shown in the specification.
5’-GGCACCCAGCACAATGAAGA-3’(SEQ ID NO：3)
5’-ACTCCTGCTTGCTGATCCAC-3’(SEQ ID NO：4)
In a third aspect, the invention provides the use of the kit in the preparation of a preparation for breast cancer detection and/or adjuvant immunotherapy.
SHISAL2A is a protein coding gene, and the research of the invention proves that the expression of SHISAL2A in breast cancer is obviously increased. The expression level of SHISAL2A is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMATESCORE, is positively correlated with various immune cell infiltrates and is positively correlated with the expression of immune checkpoint genes, so that SHISAL2A can be used as a new target for breast cancer immunotherapy. And based on the result, a SHISAL2A detection kit is designed.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Figure 1 shows the change in expression of the target gene, SHISAL2A in breast cancer. The expression of SHISAL2A was significantly elevated in breast cancer tissues.
Figure 2 shows the correlation of SHISAL2A expression with the tumor immune microenvironment in breast cancer. SHISAL2A expression was significantly positively correlated with ESTIMATESCORE, StromalScore, and ImmuneScore.
Figure 3 shows the correlation of SHISAL2A expression with immune cell infiltration in breast cancer. (A) The EPIC method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (B) the Timer method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (C) the MCPcounter method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (D) the positive correlation between SHISAL2A expression and immune cell infiltration is detected by an xCELL method.
Figure 4 shows the correlation of SHISAL2A expression with immune checkpoint genes in breast cancer. The SHISAL2A expression was significantly positively correlated with the immune checkpoint gene.
Figure 5 shows the change in the expression of shal 2A in breast cancer.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
This example serves to demonstrate that the expression of the target gene SHISAL2A is significantly upregulated in breast cancer.
Analysis of SHISAL2A expression in breast cancer:
1. data downloading and sorting:
TCGA breast cancer expression profile data and clinical data are downloaded by using a UCSC XENA website (https:// xenoxybronower. net/datapages /), and SHISAL2A expression profile data are subjected to log2 transformation and then subjected to inter-group comparison.
2. Differential analysis of SHISAL2A expression in Breast cancer
The difference in the shi al2A mRNA levels between breast cancer tissue and normal tissue was analyzed using the R software package, and the ggplot2 package was used for visualization, as shown in fig. 1.
Figure 1 shows the change in expression of the target gene, SHISAL2A in breast cancer. The expression of SHISAL2A was significantly up-regulated in breast cancer tissues. P < 0.0001.
Example 2
This example demonstrates that SHISAL2A expression in breast cancer is significantly and positively correlated with tumor immune microenvironment markers.
Correlation of SHISAL2A expression with tumor immune microenvironment in breast cancer:
the R software package, ESTIMATE, was used to calculate the stromal score, ImmuneScore, and ESTIMATE scores for each breast cancer patient tissue, and the R software package, corr. As shown in fig. 2.
Figure 2 shows the correlation of SHISAL2A expression with the tumor immune microenvironment in breast cancer. SHISAL2A expression was significantly positively correlated with both (A) ImmuneScore, (B) ESTIMATESCORE, and (C) StromalScore.
Example 3
This example was used to verify that the expression of SHISAL2A was positively correlated with immune cell infiltration in breast cancer.
Correlation of SHISAL2A expression with immune cell infiltration in breast cancer:
the EPIC, Timer, MCPcounter and xCELL methods of the R software package IOBR are used for calculating the immune cell infiltration score of each colorectal cancer patient respectively, the corr.test function of the R software package psych is used for calculating the Pearson correlation coefficient of SHISAL2A and the immune cell infiltration score, and the ggplot2 is used for visualization. As shown in fig. 3.
Figure 3 shows the correlation of FAM129C expression with immune cell infiltration in colorectal cancer. (A) The EPIC method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (B) the Timer method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (C) the MCPcounter method detects that SHISAL2A expression is obviously and positively correlated with immune cell infiltration; (D) the positive correlation between SHISAL2A expression and immune cell infiltration is detected by an xCELL method.
Example 4
This example was used to verify that the expression of SHISAL2A was positively correlated with the expression of an immune checkpoint gene in breast cancer.
Correlation of SHISAL2A expression with immune checkpoint genes in breast cancer:
the Pearson correlation coefficient of SHISAL2A expression and immune checkpoint gene was calculated respectively by using the corr.test function of the R software package psych, and ggplot2 was used for visualization. As shown in fig. 4.
Figure 4 shows the correlation of SHISAL2A expression with immune checkpoint genes in breast cancer. The SHISAL2A expression was significantly positively correlated with the immune checkpoint gene.
Example 5
This example illustrates the preparation of a kit for detecting the expression level of SHISAL 2A.
1. Primer design
PCR primers were designed based on the CDS region sequence of the transcript of the human SHISAL2A Gene (Gene ID:348378), and the sequences of the primers are shown in Table 1.
2. Kit Components
(1)Trizol 50.0mL；
(2) 50.0mL of isopropanol;
(3) 50.0mL of chloroform;
(4) 50.0mL of absolute ethyl alcohol;
(5) 5.0mL of RNase-free water;
(6) 1.0. mu.M Random primers (Random primers) 50.0. mu.L;
(7) 2.0mL of 5 XM-MLV buffer;
(8)10.0mM base triphosphate deoxynucleotides (dNTPs) 100.0. mu.L;
(9) 40U/. mu.L RNase inhibitor 50.0. mu.L;
(10) 200U/. mu. L M-MLV reverse transcriptase 50.0. mu.L;
(11)ABI 2×PCR Mix 2.0mL；
(12) 10.0. mu.M SHISAL2A real-time fluorescent quantitative PCR specific primer 30.0. mu.L, the primer sequence is shown in Table 1;
(13) 10.0. mu.M ACTB real-time fluorescent quantitative PCR specific primers 30.0. mu.L, and the primer sequences are shown in Table 1.
TABLE 1 fluorescent quantitative RT-PCR primer sequences
3. Identification of changes in SHISAL2A expression in Breast cancer by RT-PCR
(1) Extraction of total RNA from tissues: the experiment was performed in an ice bath. Placing 30-50 mg of tissue (fresh or tissue preserved at-70 ℃ in liquid nitrogen) into a 1.5mL centrifuge tube, adding 1mL Trizol, homogenizing fully, and standing at room temperature for 5 min; adding 200 μ L chloroform into each tube, mixing vigorously for 30sec, standing for 15min, and centrifuging at 4 deg.C and 12000rpm for 15 min; gently sucking 400 μ L of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 12000rpm at 4 deg.C for 10 min; discarding the supernatant, adding 1ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 12000rpm for 10 min; the supernatant was discarded as much as possible, air-dried at room temperature for 10min, and dissolved by adding 10. mu.L of RNase-free water to each tube (10-15 min. lysis-promoting at 65 ℃). Determination of OD260And calculating the RNA concentration.
RNA(mg/mL)＝40×OD260X dilution multiple (n)/1000
(2) Reverse transcription: each 25. mu.L reverse transcription system contained 100pmol of random primer, 2. mu.g of total RNA, 1. mu.L of M-MLV reverse transcriptase, 0.625. mu.L of RNase inhibitor, 1.25. mu.L of dNTPs (10mM), 5. mu.L of 5 XM-MLV buffer, and 25. mu.L of RNase-free water. The reaction conditions are as follows: 1h at 37 ℃ and 5min at 95 ℃.
(3) Quantitative PCR: each 20. mu.L reaction system contained 2 XPCR Mix 10. mu.L, upstream and downstream primers 0.4. mu.L each, cDNA 1. mu.L, ddH2O8.2. mu.L. The reaction conditions are as follows: 94 ℃ for 2min, 94 ℃ for 15s, 60 ℃ for 40s, 40 cycles.
(4)2-ΔΔCTThe method calculates the relative expression quantity of SHISAL 2A: ACTB as reference gene, target gene C determined by qPCRTC of reference gene ACTB with the same value as that of the tissue sourceTSubtracting the values to obtain Δ CTThen, Δ C is addedTAnd control group Δ CTSubtracting to obtain Δ Δ CT(taking a paracarcinoma sample Δ CTHas an average value of Δ CTControl), the relative expression of SHISAL2A in each group was calculated using the Power function in Excell tables. Expression difference between 17 breast cancer samples (BRCA) and 17 paracarcinoma samples (normal), P-test, was analyzed using software GraphPad Prism 6.0 mapping, T-test<A difference of 0.05 is statistically significant. The results are shown in FIG. 5.
Figure 5 shows the change in the expression of shal 2A in breast cancer. It can be seen that the expression of SHISAL2A was significantly increased in breast cancer.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
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Claims (10)
1. Use of an agent for detecting the expression level of SHISAL2A in the preparation of a formulation for breast cancer detection and/or adjuvant immunotherapy.
2. The use of claim 1, wherein said detecting the level of SHISAL2A expression comprises detecting the level of mRNA expression of SHISAL2A and/or detecting the level of protein expression of SHISAL 2A.
3. The use of claim 1, wherein the method of detecting the expression level of SHISAL2A comprises: detecting the expression quantity of SHISAL2AmRNA in breast cancer and tissues beside the cancer by an RT-qPCR method; detecting the expression quantity of SHISAL2AmRNA in breast cancer and tissues beside the breast cancer by a molecular probe technology; the expression level of SHISAL2A protein in breast cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the reagent for detecting the expression level of SHISAL2A is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of SHISAL2A gene, a PCR primer, or an antibody targeting SHISAL 2A.
5. The use of claim 4, wherein the reagent for detecting the expression level of SHISAL2A is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
6. A kit for detecting a breast cancer adjuvant immunotherapy target gene SHISAL2A, which comprises: reagents for detecting the expression level of SHISAL 2A.
7. The kit of claim 6, wherein the reagent for detecting the expression level of SHISAL2A is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of SHISAL2A gene, a PCR primer, or an antibody targeting SHISAL 2A.
8. The kit according to claim 7, wherein the reagent for detecting the expression level of SHISAL2A is a nucleic acid having the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
9. The kit of claim 6, wherein the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethanol, RNase-free water, random primers, 5 XM-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, a primer having the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and (4) ACTB real-time fluorescence quantitative PCR specific primers of the nucleotide sequence shown in the specification.
10. Use of a kit according to any one of claims 6 to 9 in the manufacture of a formulation for breast cancer detection and/or adjuvant immunotherapy.
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