CN114480641A - Colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit and application - Google Patents

Colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit and application Download PDF

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CN114480641A
CN114480641A CN202111646765.9A CN202111646765A CN114480641A CN 114480641 A CN114480641 A CN 114480641A CN 202111646765 A CN202111646765 A CN 202111646765A CN 114480641 A CN114480641 A CN 114480641A
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niban3
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赵小芳
李梦琳
李春明
张虎
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Jiangsu Vocational College of Medicine
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Abstract

The invention belongs to the technical field of biology, and relates to a colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit and application thereof. The present study demonstrated a significant decrease in NIBAN3 expression in colorectal cancer. The expression level of the NIBAN3 is positively correlated with immune microenvironment indexes of StromalScore, ImmuneScore and ESTIMMATEScore, is positively correlated with a plurality of immune cell infiltrates and is positively correlated with the expression of an immune inspection gene, and an NIBAN3 detection kit is designed.

Description

Colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit and application
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 NIBAN3 in preparation of a preparation for colorectal cancer detection and/or adjuvant immunotherapy, and a kit and application of a target gene NIBAN3 for colorectal cancer adjuvant immunotherapy.
Background
Colorectal cancer (CRC) refers to a cancerous change occurring in the colon or rectum, and may occur anywhere in the colon or rectum, but is most commonly found in the rectum and sigmoid colon. Colorectal cancer is the third most common cancer, and the cancer with the second highest mortality rate, and it is estimated that there are over 180 million new CRC cases and 88 million CRC-related death cases worldwide in 2018. In China, CRC is one of the most common digestive tract malignant tumors, the morbidity and mortality of CRC are third and fifth among all cancers, and the morbidity still continues to increase, especially in urban males at the fastest rate. Immunotherapy is a promising new colorectal cancer treatment mode, but the problems of low treatment response rate, side effects and the like still exist at present, 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 NIBAN3 for colorectal cancer immunotherapy, and provides application of a reagent for detecting the expression level of NIBAN3 in preparation of a preparation for colorectal cancer detection and/or adjuvant immunotherapy, and a kit for detecting a target gene NIBAN3 for adjuvant immunotherapy of colorectal cancer and application thereof.
The invention provides application of a reagent for detecting the expression level of NIBAN3 in preparing a preparation for colorectal cancer detection and/or adjuvant immunotherapy.
Further, the detecting the expression level of the NIBAN3 comprises detecting the expression level of mRNA of the NIBAN3 and/or detecting the expression level of protein of the NIBAN 3.
Further, the method for detecting the expression level of the NIBAN3 comprises the following steps: detecting the expression quantity of the NIBAN3 mRNA in the colorectal cancer and the para-carcinoma tissues by an RT-qPCR method; detecting the expression quantity of the NIBAN3 mRNA in the colorectal cancer and the tissues beside the cancer by a molecular probe technology; the expression level of the NIBAN3 protein in colorectal cancer and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of the NIBAN3 is an oligonucleotide probe, a PCR primer or an antibody of the DNA sequence or the mRNA sequence of the NIBAN3 gene, wherein the oligonucleotide probe or the PCR primer is targeted to the NIBAN 3.
According to the present invention, preferably, the reagent for detecting the expression level of NIBAN3 is SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-TGCTTGCTGCAGAGGATTGA-3’(SEQ ID NO：1)
5’-GCTCAGCCTCAGTTTCCTCA-3’(SEQ ID NO：2)
The second aspect of the invention provides a colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit, which comprises: reagents for detecting the expression level of NIBAN 3.
Further, the reagent for detecting the expression level of the NIBAN3 is an oligonucleotide probe, a PCR primer or an antibody targeting the DNA sequence or the mRNA sequence of the NIBAN3 gene, or the antibody targeting the NIBAN 3.
Specifically, the reagent for detecting the expression level of the NIBAN3 is a reagent with the nucleotide 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 as described above in the manufacture of a formulation for colorectal cancer detection and/or adjuvant immunotherapy.
The research of the invention proves that the expression of the NIBAN3 in Colon cancer (Colon cancer, COAD) and Rectal cancer (real) is obviously reduced. The expression level of the NIBAN3 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 the NIBAN3 can be used as a new target for colorectal cancer immunotherapy. And based on the result, an NIBAN3 detection kit is 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 NIBAN3 in colorectal cancer. (A) Significant downregulation of NIBAN3 expression in colorectal cancer tissues; (B) significant downregulation of NIBAN3 expression in colon cancer tissues; (C) NIBAN3 expression was significantly down-regulated in rectal cancer tissues.
Figure 2 shows the correlation of NIBAN3 expression with immune cell infiltration in colorectal cancer. (A) The EPIC method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (B) the Timer method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (C) the MCPcounter method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (D) the xCELL method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells.
Figure 3 shows the correlation of NIBAN3 expression with immune checkpoint genes in colorectal cancer. NIBAN3 expression was significantly positively correlated with the immune checkpoint gene.
Figure 4 shows the change in expression of NIBAN3 in colorectal 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 significant down-regulation of the expression of the target gene NIBAN3 in colorectal cancer.
Analysis of NIBAN3 expression in colorectal cancer:
1. data downloading and sorting:
the UCSC XENA website (https:// xenoxybronower. net/datapages /) is used for downloading TCGA colorectal cancer expression profile data and clinical data, and the NIBAN3 expression profile data are transformed by log2 and then are compared among groups.
2. Differential analysis of expression of NIBAN3 in colorectal cancer
The R software package was used to analyze the differences in the NIBAN3 mRNA levels between colorectal and normal tissues and the ggplot2 package was used for visualization as shown in fig. 1.
FIG. 1 shows the change in expression of the target gene NIBAN3 in colorectal cancer. (left) significant downregulation of NIBAN3 expression in colorectal cancer tissues; (mid) significant downregulation of NIBAN3 expression in colon cancer tissues; (right) NIBAN3 expression was significantly down-regulated in rectal cancer tissues. P <0.001, p < 0.0001.
Example 2
This example serves to demonstrate that NIBAN3 expression in colorectal cancer is significantly positively correlated with tumor immune microenvironment markers.
Correlation of NIBAN3 expression with tumor immune microenvironment in rectal cancer:
each colorectal cancer patient tissue, stromal score, ImmuneScore and estate scores, was calculated using the R software package estate, expressing Pearson's correlation coefficient with immune infiltration score using the corr. As shown in table 1.
Table 1 shows the correlation of NIBAN3 expression with tumor immune microenvironment in colorectal cancer. NIBAN3 expression was significantly positively correlated with each of the estamatiscore, the StromalScore and the ImmuneScore.
TABLE 1
Figure BDA0003444060260000051
Example 3
This example was used to verify that NIBAN3 expression was positively correlated with immune cell infiltration in colorectal cancer.
Correlation analysis of NIBAN3 expression with immune cell infiltration in colorectal cancer:
the EPIC, Timer, MCPcounter and xCELL methods of the R software package IOBR were used to calculate the immune cell infiltration score for each colorectal cancer patient separately, and the corr.test function of the R software package psych was used to calculate the pearson correlation coefficient of NIBAN3 with the immune cell infiltration score, ggplot2 package was used for visualization. As shown in fig. 2.
Figure 2 shows the correlation of NIBAN3 expression with immune cell infiltration in colorectal cancer. (A) The EPIC method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (B) the Timer method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (C) the MCPcounter method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells; (D) the xCELL method detects that the expression of the NIBAN3 is obviously and positively correlated with the infiltration of immune cells.
Example 4
This example was used to verify that NIBAN3 expression was positively correlated with immune checkpoint gene expression in colorectal cancer.
Correlation analysis of the expression of NIBAN3 and immune-related genes in colorectal cancer:
the correlation coefficients of the expression and the pilson of the marker genes of the five immune pathways are respectively calculated by using the corr.test function of the R software package psych, and ggplot2 is used for visualization. As shown in fig. 3.
FIG. 3 shows the correlation of NIBAN3 expression with immune-related genes such as immune checkpoints in colorectal cancer. NIBAN3 expression was significantly positively correlated with the immune checkpoint gene.
Example 5
This example illustrates the preparation of a kit for detecting the expression of NIBAN 3.
1. Primer design
PCR primers were designed based on the CDS region sequence of transcript of human NIBAN3 Gene (Gene ID:199786), 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 of NIBAN3 real-time fluorescent quantitative PCR specific primer 30.0. mu.L, 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
Figure BDA0003444060260000071
3. RT-PCR identification of expression changes of NIBAN3 in colorectal cancer
(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 12000rpm at 4 deg.C 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 contains 2 XPCR Mix 10. mu.L, upstream and downstream primers 0.4. mu.L each, cDNA 1. mu.L, ddH2O8.2. mu.L. Inverse directionThe 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. Expression difference between 9 colorectal cancer samples (CRC) and 9 paracancer 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. 4.
Figure 4 shows the change in expression of NIBAN3 in colorectal cancer. As can be seen, NIBAN3 expression was significantly reduced in colorectal 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
<110> Jiangsu medical profession college
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Claims (10)

1. Use of a reagent for detecting the expression level of NIBAN3 in the preparation of a formulation for colorectal cancer detection and/or adjuvant immunotherapy.
2. The use of claim 1, wherein said detecting the expression level of NIBAN3 comprises detecting the mRNA expression level of NIBAN3 and/or detecting the protein expression level of NIBAN 3.
3. The use of claim 1, wherein the method for detecting the expression level of NIBAN3 comprises: detecting the expression quantity of the NIBAN3 mRNA in the colorectal cancer and the para-carcinoma tissues by an RT-qPCR method; detecting the expression quantity of the NIBAN3 mRNA in the colorectal cancer and the tissues beside the cancer by a molecular probe technology; the expression level of the NIBAN3 protein in colorectal 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 NIBAN3 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the NIBAN3 gene, a PCR primer, or an antibody targeting NIBAN 3.
5. The use of claim 4, wherein the reagent for detecting the expression level of NIBAN3 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 colorectal cancer adjuvant immunotherapy target gene NIBAN3, which comprises: reagents for detecting the expression level of NIBAN 3.
7. The kit of claim 6, wherein the reagent for detecting the expression level of the NIBAN3 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the NIBAN3 gene, a PCR primer, or an antibody targeting NIBAN 3.
8. The kit of claim 7, wherein the reagent for detecting the expression level of NIBAN3 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 for the manufacture of a formulation for colorectal cancer detection and/or adjuvant immunotherapy.
CN202111646765.9A 2021-12-29 2021-12-29 Colorectal cancer adjuvant immunotherapy target gene NIBAN3 detection kit and application Withdrawn CN114480641A (en)

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Application publication date: 20220513