CN114196756A - Lung squamous carcinoma adjuvant immunotherapy target gene LINC00890 detection kit and application - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to a lung squamous carcinoma adjuvant immunotherapy target gene LINC00890 detection kit and application thereof. The research of the invention proves that the LINC00890 expression in the squamous cell lung carcinoma is obviously reduced. The LINC00890 expression level is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMMATEScore, is positively correlated with infiltration of various immune cells and is positively correlated with the expression of immune checkpoint genes, so that the LINC00890 can be used as a new target for lung squamous carcinoma immunotherapy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting an expression level of LINC00890 in preparation of a preparation for squamous cell lung carcinoma detection and/or adjuvant immunotherapy, and a kit for detecting a target gene LINC00890 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 lung squamous carcinoma treatment mode with prospect, has the problems of low treatment response rate, side effect and the like, and has important significance for clinical tumor immunotherapy by developing a novel auxiliary immunotherapy target.
Disclosure of Invention
The invention aims to provide a new lung squamous carcinoma immunotherapy target LINC00890, and provides application of a reagent for detecting the expression level of LINC00890 in preparation of a preparation for lung squamous carcinoma detection and/or adjuvant immunotherapy, and a lung squamous carcinoma adjuvant immunotherapy target gene LINC00890 detection kit and application.
The invention provides application of an agent for detecting the expression level of LINC00890 in preparing a preparation for detecting squamous cell lung carcinoma and/or assisting immunotherapy.
Further, said detecting the expression level of LINC00890 comprises detecting the expression level of mRNA of LINC00890 and/or detecting the expression level of protein of LINC 00890.
Further, the method for detecting the expression level of LINC00890 comprises the following steps: detecting the expression quantity of LINC00890 mRNA in squamous cell lung carcinoma and tissues beside the lung carcinoma by an RT-qPCR method; detecting the expression quantity of LINC00890 mRNA in squamous cell lung carcinoma and tissues beside the lung carcinoma by a molecular probe technology; detecting the expression level of the LINC00890 protein in squamous cell lung cancer and paracarcinoma tissues by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of the LINC00890 is an oligonucleotide probe and a PCR primer of a DNA sequence or an mRNA sequence of a targeted LINC00890 gene, or an antibody of the targeted LINC 00890.
According to the present invention, preferably, the reagent for detecting the expression level of LINC00890 is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-TGCCCATTTTCCCACTCTGG-3’(SEQ ID NO：1)
5’-TGTTTGAGCCGAAGGAGCAT-3’(SEQ ID NO：2)
The second aspect of the invention provides a lung squamous carcinoma auxiliary immunotherapy target gene LINC00890 detection kit, which comprises: an agent for detecting the expression level of LINC 00890.
Further, the reagent for detecting the expression level of the LINC00890 is an oligonucleotide probe and a PCR primer of a DNA sequence or an mRNA sequence of a targeted LINC00890 gene, or an antibody of the targeted LINC 00890.
Specifically, the reagent for detecting the expression level of LINC00890 is a reagent with an amino acid 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 LINC00890 is a protein coding gene, and the research of the invention proves that the LINC00890 expression in lung squamous cell carcinoma is obviously reduced. The LINC00890 expression level is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMMATEScore, is positively correlated with infiltration of various immune cells and is positively correlated with the expression of immune checkpoint genes, so that the LINC00890 can be used as a new target for lung squamous carcinoma immunotherapy. And a LINC00890 detection kit is designed based on the LINC00890 detection kit.
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.
Fig. 1 shows the expression change of target gene LINC00890 in squamous cell lung carcinoma. LINC00890 expression was significantly down-regulated in squamous lung carcinoma tissues.
Figure 2 shows the correlation of LINC00890 expression in squamous lung cancer with the tumor immune microenvironment. (A) LINC00890 expression was significantly positively correlated with estimatiscore; (B) LINC00890 expression was significantly positively correlated with ImmuneScore; (C) LINC00890 expression was significantly positively correlated with StromalScore.
Figure 3 shows the correlation of LINC00890 expression with immune cell infiltration in squamous lung carcinoma. LINC00890 expression was significantly positively correlated with multiple immune cell infiltrates.
Figure 4 shows the correlation of LINC00890 expression with immune checkpoint genes in squamous lung carcinoma. LINC00890 expression was significantly positively correlated with immune checkpoint genes.
Fig. 5 shows the change in expression of LINC00890 in squamous 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 that the expression of the target gene LINC00890 is significantly down-regulated in squamous cell lung carcinoma.
LINC00890 expression analysis in squamous cell lung carcinoma
1. Data downloading and sorting:
TCGA squamous cell lung carcinoma expression profile data and clinical data are downloaded by using a UCSC XENA website (https:// xenoxybrowser. net/datapages /), and LINC00890 expression profile data are subjected to log2 transformation and then subjected to group comparison.
2. Expression difference analysis of LINC00890 in squamous cell lung carcinoma
The difference in LINC00890 mRNA levels between squamous lung 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 expression change of target gene LINC00890 in squamous cell lung carcinoma. LINC00890 expression is significantly down-regulated in squamous cell lung carcinoma. P < 0.0001.
Example 2
This example demonstrates that LINC00890 expression in squamous lung carcinoma is significantly positively correlated with tumor immune microenvironment markers.
Correlation analysis of LINC00890 expression and tumor immune microenvironment in squamous cell lung carcinoma:
the R software package, estamate, was used to calculate the StromalScore, immneccore and estamate scores of 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 LINC00890 expression with the immune infiltration score. As shown in fig. 2.
Figure 2 shows the correlation of LINC00890 expression in squamous lung cancer with the tumor immune microenvironment. (A) LINC00890 expression was significantly positively correlated with estimatiscore; (B) LINC00890 expression was significantly positively correlated with ImmuneScore; (C) LINC00890 expression was significantly positively correlated with StromalScore.
Example 3
This example was used to verify that LINC00890 expression was positively correlated with immune cell infiltration in squamous cell lung carcinoma.
Correlation analysis of LINC00890 expression and immune cell infiltration in lung squamous carcinoma:
the MCPcounter, Timer, EPIC and xCELL methods of the R software package IOBR are used for calculating the immune cell infiltration score of each patient with squamous cell carcinoma of lung respectively, the corr.test function of the R software package psych is used for calculating the Pearson correlation coefficient of LINC00890 and the immune cell infiltration score, and the ggplot2 package is used for visualization. As shown in fig. 3.
Figure 3 shows the correlation of LINC00890 expression with immune cell infiltration in squamous lung carcinoma. (A) An EPIC method is used for detecting that LINC00890 expression is obviously and positively correlated with immune cell infiltration; (B) the Timer method detects that LINC00890 expression and immune cell infiltration are obviously and positively correlated; (C) the MCPcounter method detects that LINC00890 expression is obviously and positively correlated with immune cell infiltration; (D) the xCELL method detects that LINC00890 expression is obviously and positively correlated with immune cell infiltration.
Example 4
This example was used to verify that LINC00890 expression was positively correlated with immune checkpoint gene expression in squamous cell lung carcinoma.
And (3) calculating the Pearson correlation coefficient of LINC00890 expression and an immune checkpoint gene respectively by using a corr.test function of the R software package psych, wherein ggplot2 is used for visualization. As shown in fig. 4.
Figure 4 shows the correlation of LINC00890 expression with immune checkpoint genes in squamous lung carcinoma. LINC00890 expression was significantly positively correlated with immune checkpoint genes.
Example 5
This example illustrates the preparation of a kit for detecting the expression level of LINC 00890.
1. Primer design
PCR primers were designed based on the CDS region sequence of the transcript of the human LINC00890 Gene (Gene ID:401613), and the primer sequences 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 LINC00890 real-time fluorescent quantitative PCR specific primer 30.0 mu L, and 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. RT-PCR identification of expression change of LINC00890 in squamous cell lung carcinoma
(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-ΔΔCTCalculating the relative expression of NSMCE 2: 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 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. 5.
Fig. 5 shows the change in expression of LINC00890 in squamous lung carcinoma. It can be seen that LINC00890 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
<110> New countryside medical college
<120> lung squamous carcinoma adjuvant immunotherapy target gene LINC00890 detection kit and application
<130> BJI2103232JSYY
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tgcccatttt cccactctgg 20
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<213> Artificial Sequence (Artificial Sequence)
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tgtttgagcc gaaggagcat 20
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ggcacccagc acaatgaaga 20
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actcctgctt gctgatccac 20
Claims (10)
1. Application of an agent for detecting the expression level of LINC00890 in preparing a preparation for lung squamous carcinoma detection and/or adjuvant immunotherapy.
2. The use of claim 1, wherein said detecting the expression level of LINC00890 comprises detecting the mRNA expression level of LINC00890 and/or detecting the protein expression level of LINC 00890.
3. The use of claim 1, wherein the method of detecting the expression level of LINC00890 comprises: detecting the expression quantity of LINC00890 mRNA in squamous cell lung carcinoma and tissues beside the lung carcinoma by an RT-qPCR method; detecting the expression quantity of LINC00890 mRNA in squamous cell lung carcinoma and tissues beside the lung carcinoma by a molecular probe technology; detecting the expression level of the LINC00890 protein in squamous cell lung cancer and paracarcinoma tissues by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the agent for detecting the expression level of LINC00890 is an oligonucleotide probe, a PCR primer or an antibody targeting LINC00890 gene DNA sequence or mRNA sequence.
5. The use of claim 4, wherein the agent for detecting the expression level of LINC00890 is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
6. A lung squamous carcinoma auxiliary immunotherapy target gene LINC00890 detection kit, which comprises: an agent for detecting the expression level of LINC 00890.
7. The kit of claim 6, wherein the reagent for detecting the expression level of LINC00890 is an oligonucleotide probe, a PCR primer or an antibody targeting LINC00890 gene DNA sequence or mRNA sequence.
8. The kit of claim 7, wherein the reagent for detecting the expression level of LINC00890 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.
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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