CN114574578A - Lung squamous carcinoma adjuvant immunotherapy target gene C22orf15 detection kit and application thereof - Google Patents
Lung squamous carcinoma adjuvant immunotherapy target gene C22orf15 detection kit and application thereof Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention belongs to the technical field of biology, and relates to a target gene C22orf15 detection kit for adjuvant immunotherapy of squamous cell lung carcinoma and application thereof. The present study demonstrated a significant decrease in C22orf15 expression in lung squamous carcinoma. The expression level of C22orf15 is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMATESCORE, is positively correlated with infiltration of various immune cells and is positively correlated with the expression of immune checkpoint genes, so that the C22orf15 can be used as a new target for immunotherapy of squamous cell lung carcinoma.
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 C22orf15 in preparation of a preparation for squamous cell lung carcinoma detection and/or adjuvant immunotherapy, and a kit for detecting a target gene C22orf15 for adjuvant immunotherapy of squamous cell lung carcinoma and application thereof.
Background
Lung cancer is a malignant tumor with the current global morbidity and mortality in the front, wherein the curative effect of Lung squamous cell carcinoma (LUSC) is still unsatisfactory after comprehensive treatment such as surgery, radiotherapy, chemotherapy and the like. Immunotherapy is a new promising treatment mode for lung squamous cell carcinoma, but has the problems of low response rate, side effect 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 new target C22orf15 for lung squamous carcinoma immunotherapy, and provides application of a reagent for detecting the expression level of C22orf15 in preparation of a preparation for lung squamous carcinoma detection and/or adjuvant immunotherapy, and a kit for detecting a target gene C22orf15 for adjuvant immunotherapy of lung squamous carcinoma and application thereof.
The first aspect of the invention provides an application of a reagent for detecting the expression level of C22orf15 in preparing a preparation for lung squamous carcinoma detection and/or adjuvant immunotherapy.
Further, the detecting the expression level of C22orf15 includes detecting the mRNA expression level of C22orf15 and/or detecting the protein expression level of C22orf 15.
Further, the method for detecting the expression level of C22orf15 comprises the following steps: detecting the expression quantity of C22orf15 mRNA in squamous cell lung carcinoma and paracarcinoma tissues by an RT-qPCR method; detecting the expression level of C22orf15 mRNA in squamous cell lung carcinoma and paracarcinoma tissues by a molecular probe technology; the expression level of C22orf15 protein in squamous cell lung carcinoma and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of C22orf15 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C22orf15 gene, a PCR primer, or an antibody targeting C22orf 15.
According to the present invention, preferably, the reagent for detecting the expression level of C22orf15 is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-CCACAACTGGAGGAAGCGTA-3’(SEQ ID NO：1)
5’-CAGTGTGCAATCCATCCCCT-3’(SEQ ID NO：2)
The second aspect of the present invention provides a target gene C22orf15 detection kit for adjuvant immunotherapy of lung squamous carcinoma, which comprises: reagents for detecting the expression level of C22orf 15.
Further, the reagent for detecting the expression level of C22orf15 is an oligonucleotide probe, a PCR primer targeting a DNA sequence or an mRNA sequence of the C22orf15 gene, or an antibody targeting C22orf 15.
Specifically, the reagent for detecting the expression level of C22orf15 is a reagent 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.
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 squamous cell lung carcinoma detection and/or adjuvant immunotherapy.
The C22orf15 is a protein coding gene, and the research of the invention proves that the expression of C22orf15 in lung squamous carcinoma is obviously reduced. The expression level of C22orf15 is positively correlated with immune microenvironment indexes StromalScore, ImmuneScore and ESTIMATESCORE, is positively correlated with multiple immune cell infiltrations, and is positively correlated with the expression of immune checkpoint genes, so that the C22orf15 can be used as a new target for immunotherapy of squamous cell lung carcinoma. And based on this, a C22orf15 detection kit was designed.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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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.
Fig. 1 shows the change in expression of the target gene C22orf15 in squamous cell lung carcinoma. (A) Unpaired sample results showed significant down-regulation of C22orf15 expression in lung squamous carcinoma tissues; (B) paired sample results showed significant down-regulation of C22orf15 expression in lung squamous carcinoma tissues.
Figure 2 shows the correlation of C22orf15 expression with immune cell infiltration in lung squamous carcinoma. C22orf15 expression was significantly positively correlated with multiple immune cell infiltrates.
Figure 3 shows the correlation of C22orf15 expression with immune checkpoint genes in squamous cell lung carcinoma. C22orf15 expression was significantly positively correlated with the immune checkpoint gene.
Fig. 4 shows the change in expression of C22orf15 in squamous cell lung carcinoma.
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 significant down-regulation of the expression of the target gene C22orf15 in squamous cell lung carcinoma.
Analysis of C22orf15 expression in squamous cell lung carcinoma:
1. data downloading and sorting:
the UCSC XENA website (https:// xenoxybronower. net/datapages /) is used for downloading TCGA lung squamous carcinoma expression profile data and clinical data, and the C22orf15 expression profile data is subjected to log2 transformation and then subjected to group comparison.
2. Differential analysis of C22orf15 expression in squamous cell lung carcinoma
The difference in C22orf15 mRNA levels between lung squamous carcinoma tissue and normal tissue was analyzed using the R software package and the ggplot2 package was used for visualization as shown in fig. 1.
Fig. 1 shows the change in expression of the target gene C22orf15 in squamous cell lung carcinoma. (A) C22orf15 expression was significantly down-regulated in non-paired samples of squamous cell lung carcinoma; (B) c22orf15 expression was significantly down-regulated in squamous cell lung carcinoma paired samples. P < 0.001.
Example 2
This example demonstrates that C22orf15 expression in squamous cell lung carcinoma is significantly and positively correlated with tumor immune microenvironment markers.
Correlation analysis of C22orf15 expression in lung squamous carcinoma with tumor immune microenvironment:
the R software package, estamate, was used to calculate the stromal score, ImmuneScore, and estamate scores for each patient with squamous cell lung carcinoma, and the corr.test function of the R software package, psych, was used to calculate the pearson correlation coefficient of C22orf15 expression with the immune infiltration score. As shown in table 1.
Table 1 shows the correlation of C22orf15 expression in lung squamous carcinoma with the tumor immune microenvironment. C22orf15 expression was significantly positively correlated with each of estimatiscore, ImmuneScore, and stromal score.
TABLE 1
Example 3
This example was used to verify that C22orf15 expression was positively correlated with immune cell infiltration in squamous cell lung carcinoma.
Correlation analysis of C22orf15 expression in lung squamous carcinoma with immune cell infiltration:
the Pearson correlation coefficient of C22orf15 and immune cell infiltration score is calculated by using the corr.test function of the R software package GSVA immune infiltration algorithm ssGSEA and psych, and the ggplot2 package is used for visualization. As shown in fig. 2.
Figure 2 shows the correlation of C22orf15 expression with immune cell infiltration in lung squamous carcinoma. Squamous cell lung carcinoma C22orf15 expression was significantly positively correlated with multiple immune cell infiltrates.
Example 4
This example was used to verify that C22orf15 expression is positively correlated with immune checkpoint gene expression in squamous cell lung carcinoma.
Correlation analysis of C22orf15 expression with immune checkpoint genes in squamous cell lung carcinoma:
the correlation coefficient between C22orf15 expression and the Pearson correlation coefficient of the immune checkpoint gene were calculated respectively by using the corr.test function of the R software package psych, and ggplot2 was used for visualization. As shown in fig. 3.
Figure 3 shows the correlation of C22orf15 expression with immune checkpoint genes in squamous cell lung carcinoma. C22orf15 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 C22orf 15.
1. Primer design
PCR primers were designed based on the CDS region sequence of the transcript of the human C22orf15 Gene (Gene ID:150248), and the sequences of the primers are shown in Table 2.
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 C22orf15 real-time fluorescent quantitative PCR specific primer 30.0 mu L, and the primer sequence is shown in Table 2;
(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 2.
TABLE 2 fluorescent quantitative RT-PCR primer sequences
3. Identification of expression changes of C22orf15 in squamous cell lung carcinoma 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 (65 ℃ for 10-15 min). 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: 2min at 94 ℃, 15s at 94 ℃, 40s at 60 ℃ and 40 cycles.
(4)2-ΔΔCTCalculating the relative expression of NSMCE 2: ACTB as an internal reference Gene, and the 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 convert Δ CTAnd control group Delta CTSubtracting to obtain Δ Δ CT(taking paracarcinoma sample Δ CTHas an average value of Δ CTControl), the relative expression of NSMCE2 in each group was calculated using the Power function in the Excell table. The difference in expression, P, between 9 squamous cell lung carcinoma samples (LUSC) and 9 paracarcinoma samples (Normal) was analyzed by T-test, plotted using the software GraphPad Prism 6.0<A difference of 0.05 is statistically significant. The results are shown in FIG. 4.
Fig. 4 shows the change in expression of C22orf15 in squamous cell lung carcinoma. It can be seen that C22orf15 expression is significantly reduced in squamous cell lung carcinoma.
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 C22orf15 in the preparation of a formulation for lung squamous carcinoma detection and/or adjuvant immunotherapy.
2. The use of claim 1, wherein the detecting the expression level of C22orf15 comprises detecting the mRNA expression level of C22orf15 and/or detecting the protein expression level of C22orf 15.
3. The use of claim 1, wherein the method for detecting the expression level of C22orf15 comprises: detecting the expression quantity of C22orf15 mRNA in squamous cell lung carcinoma and paracarcinoma tissues by an RT-qPCR method; detecting the expression level of C22orf15 mRNA in squamous cell lung carcinoma and paracarcinoma tissues by a molecular probe technology; the expression level of C22orf15 protein in squamous cell lung carcinoma 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 C22orf15 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C22orf15 gene, a PCR primer, or an antibody targeting C22orf 15.
5. The use of claim 4, wherein the reagent for detecting the expression level of C22orf15 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 target gene C22orf15 for adjuvant immunotherapy of lung squamous carcinoma, which comprises: reagents for detecting the expression level of C22orf 15.
7. The kit of claim 6, wherein the reagent for detecting the expression level of C22orf15 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C22orf15 gene, a PCR primer, or an antibody targeting C22orf 15.
8. The kit according to claim 7, wherein the reagent for detecting the expression level of C22orf15 is a nucleic acid having the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table 2.
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 lung squamous carcinoma detection and/or adjuvant immunotherapy.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008154333A2 (en) * | 2007-06-08 | 2008-12-18 | Asuragen, Inc. | Mir-34 regulated genes and pathways as targets for therapeutic intervention |
| WO2011052748A1 (en) * | 2009-10-30 | 2011-05-05 | 学校法人慶應義塾 | Method for determining sensitivity to an anticancer agent |
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008154333A2 (en) * | 2007-06-08 | 2008-12-18 | Asuragen, Inc. | Mir-34 regulated genes and pathways as targets for therapeutic intervention |
| CN101801419A (en) * | 2007-06-08 | 2010-08-11 | 米尔纳疗法公司 | Gene and path as the miR-34 regulation and control for the treatment of the target of intervening |
| WO2011052748A1 (en) * | 2009-10-30 | 2011-05-05 | 学校法人慶應義塾 | Method for determining sensitivity to an anticancer agent |
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
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