CN115992247A

CN115992247A - Detection method of DCLK1 gene and application thereof in colorectal cancer detection

Info

Publication number: CN115992247A
Application number: CN202211592866.7A
Authority: CN
Inventors: 袁脉; 施杰
Original assignee: Wuxi Medicos Biotechnology Co ltd
Current assignee: Wuxi Medicos Biotechnology Co ltd
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-04-21

Abstract

The invention discloses a detection method of DCLK1 gene and application thereof in colorectal cancer detection, relating to the biotechnology field; the detection method adopts RT-qPCR detection. The invention also discloses a biomarker for early diagnosis of colorectal cancer, which is DCLK1. The invention adopts an immunohistochemical method to detect the expression condition of DCLK1 in adult colorectal cancer and paracancerous normal tissues, analyzes the correlation between the expression condition of DCLK1 and clinical pathological characteristics of colorectal cancer patients, clarifies the important role of DCLKI in the evolution process of colorectal cancer, and can be used as a biomarker for early diagnosis of colorectal cancer.

Description

Detection method of DCLK1 gene and application thereof in colorectal cancer detection

Technical Field

The invention relates to the technical field of biology, in particular to a detection method of DCLK1 gene and application thereof in colorectal cancer detection.

Background

Colorectal cancer (colorectal cancer, CRC) is a common malignancy of the digestive system, with its incidence rising year by year worldwide. Surgical treatment is a radical treatment method for colorectal cancer, and patients with middle and advanced colorectal cancer rely on chemotherapy and radiotherapy to increase survival rate for 5 years. Therefore, the early diagnosis and early treatment which are timely and accurate have important clinical significance for improving the survival rate of colorectal cancer patients. Numerous methods have been explored by scholars at home and abroad for early diagnosis of colorectal Cancer such as the combined detection of colorectal Cancer patient serum malignancy-specific growth factors (Tumor Specific Growth Fanctor, TSGF), cancer-associated antigens (Cancer anti 242, CA 242) and carcinoembryonic antigen (CEA), the expression of Signal transduction and transcriptional activator 3 (Signal transducers and activators of transduction, STAT 3) in colorectal Cancer and adenomatous polyp tissues, and the like.

Doublecortin-like kinase-1, (Doublecortin-like kinase1, DCLK 1) is a member of the protein kinase superfamily and the duoublecortin family, whose coding genes are located on human chromosome 13q135. Recent studies have found that DCLK1 is expressed in the cytoplasm and stroma of most malignant tissues such as breast cancer, pancreatic cancer and prostate cancer epithelial cells and is associated with tumor metastasis and poor prognosis. At present, although a great deal of DCLK1 is reported to be related to breast cancer, pancreatic cancer, prostate cancer and colon cancer, there are few reports of DCIK1 in adult colorectal cancer tissues.

Disclosure of Invention

The invention aims to provide a detection method of DCLK1 gene and application thereof in colorectal cancer detection, so as to solve the problems in the prior art, and the invention discovers that DCIK1 has abnormally high expression in colorectal cancer, and prompts that the DCIK1 plays a role in colorectal cancer tumorigenesis and development, so that the DCIK1 can be used as a biomarker for early diagnosis.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a biomarker for early diagnosis of colorectal cancer, which is DCLK1.

The invention also provides application of the reagent for detecting the DCLK1 expression quantity in preparing colorectal cancer early diagnosis products.

Further, the product is a kit or a reagent.

The invention also provides an early colorectal cancer diagnosis product, which comprises a reagent for detecting the DCLK1 expression quantity.

Further, the product is a kit or a reagent.

Further, the reagent comprises a primer pair for RT-qPCR detection, wherein the upstream primer of the primer pair is: 5'-AGGCAGGTTACCATCACTGG-3', the downstream primer is: 5'-CATTGTTCTAGCTGCTCCCC-3'.

The invention also provides a detection method of the DCLK1 gene, the detection method adopts RT-qPCR detection, and the upstream primer of the primer pair used for the RT-qPCR detection is: 5'-AGGCAGGTTACCATCACTGG-3', the downstream primer is: 5'-CATTGTTCTAGCTGCTCCCC-3'. .

Further, the internal control of the RT-qPCR assay employs GAPDH.

Further, the upstream primer of the primer pair for detecting GAPDH is: 5' -TGCACCACCAACTGCTTAGC-3, the downstream primer is: 5'-GGCATGGACTGTGGTCATGAG-3'.

The invention also provides application of the method in colorectal cancer detection.

The invention discloses the following technical effects:

the invention adopts an immunohistochemical method to detect the expression condition of DCLK1 in adult colorectal cancer and paracancerous normal tissues, analyzes the correlation between the expression condition of DCLK1 and clinical pathological characteristics of colorectal cancer patients, clarifies the important role of DCLKI in the evolution process of colorectal cancer, and can be used as a biomarker for early diagnosis of colorectal cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the expression of DCLK1 in normal paracancerous tissue;

FIG. 2 shows the expression of DCLK1 in colorectal cancer tissue.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Example 1

1 materials and methods

1.1 general data

70 parts of case tissue of a patient diagnosed with colorectal cancer by pathology are collected. Of the patients, 42 men, 28 women, and ages 26-80 years.

1.2 inclusion criteria and exclusion criteria

Inclusion criteria: (1) Fibrocolonoscopy and taking a biopsy pathology confirmed colorectal cancer patient; (2) no treatment such as radiotherapy, chemotherapy and the like is carried out before operation; (3) complete clinical data.

Exclusion criteria: (1) age <18 years; (2) pregnant and parturient women and women in lactation period; (3) Pathological examination proves that the tumor is interstitial tumor, malignant melanoma, squamous carcinoma, carcinoid and the like; (4) incomplete clinical data.

1.3 immunohistochemical staining

Paraffin tissue of colorectal tumor and corresponding paracancerous tissue was taken and 5 μm paraffin sections were prepared. After deparaffinization and hydration of the sections, DCLK1 expression was assessed by immunohistochemical staining of streptavidin-peroxidase (SP) according to the immunohistochemical kit instructions procedure. Incubation DCLK1 antibody dilution was 1:200.DAB developed and light microscopy randomly photographed 5 fields.

1.4Western blot experiments

The RIPA buffer extracts a protein sample from a tissue or cell and quantitates the extracted protein sample with the BCA protein quantitation kit. An equal amount of protein was separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated protein was transferred to PVDF membrane. Blocking the membrane with 5% Bovine Serum Albumin (BSA) at room temperature for 2h, adding primary antibodies (DCLK 1, TGF-. Beta.1, p-Smad2, p-Smad7, smad2 and Smad7; diluted 1:1000 in each case) and incubating for 1.5h; after TBST washing, the secondary antibody (1:2000) was incubated for 1h at room temperature. The target protein bands were exposed by ECL chemiluminescence. Beta-actin was used as an internal control and ImageJ software measured the relative gray scale of the protein of interest.

1.5RT-qPCR experiments

TRIzol reagent extracts total RNA from tissue and cell specimens. The concentration of the RNA samples was measured using a Nanodrop1000 spectrophotometer (Thermoscientific, USA). cDNA was synthesized by reverse transcription using PrimeScript kit. The cDNA and primers were subjected to RT-qPCR reactions in 7900Real-time CRSysteme (applied biosystems, USA) by means of a SYBRGreen PCRMasterMix kit. Primer sequence: DCLK1F:5'-AGGCAGGTTACCATCACTGG-3', R:5'-CATTGTTCTAGCTGCTCCCC-3'. GAPDH was used as an internal control with the primer sequences as follows: GAPDHF:5' -TGCACCACCAACTGCTTAGC-3, R:5'-GGCATGGACTGTGGTCATGAG-3'. The relative expression level of DCLK1 mRNA was calculated using the 2- ΔΔct method.

1.6 statistical methods

Statistical analysis was performed using GraphPad Prism6 software. The data are expressed by mean ± standard deviation (x ± s), the comparison between the two groups adopts t test, and the comparison between the multiple groups adopts single factor analysis of variance; p <0.05 is statistically significant for the differences.

2 results

2.1 immunohistochemical staining results are shown in FIGS. 1-2, which show that DCLK1 is localized mainly in the colon tumor cytoplasm, in the form of a brown-yellow granule (FIG. 2).

2.2 colorectal cancer and normal colon tissue DCLK1 expression results are shown in Table 1. The results showed that DCLK1 had a high expression rate of 38.57% in colorectal cancer tissue and 2.85% in normal paracancerous tissue, and that there was a statistical difference between the two groups (P < 0.0001).

TABLE 1

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A biomarker for early diagnosis of colorectal cancer, characterized in that the biomarker is DCLK1.

2. An application of a reagent for detecting DCLK1 expression quantity in preparing colorectal cancer early diagnosis products.

3. The use according to claim 2, wherein the product is a kit or a reagent.

4. A colorectal cancer early-diagnosis product comprising an agent for detecting the expression level of DCLK1.

5. The early colorectal cancer diagnosis product according to claim 4, wherein the product is a kit or a reagent.

6. The early diagnostic colorectal cancer product of claim 4, wherein the reagent comprises a primer pair for RT-qPCR detection, the upstream primer of the primer pair being: 5'-AGGCAGGTTACCATCACTGG-3', the downstream primer is: 5'-CATTGTTCTAGCTGCTCCCC-3'.

7. The detection method of the DCLK1 gene is characterized by adopting RT-qPCR detection, wherein the upstream primer of a primer pair used for the RT-qPCR detection is as follows: 5'-AGGCAGGTTACCATCACTGG-3', the downstream primer is: 5'-CATTGTTCTAGCTGCTCCCC-3'.

8. The method of claim 7, wherein the internal control of the RT-qPCR assay employs GAPDH.

9. The method of claim 7, wherein the upstream primer of the primer pair for detecting GAPDH is: 5' -TGCACCACCAACTGCTTAGC-3, the downstream primer is: 5'-GGCATGGACTGTGGTCATGAG-3'.

10. Use of a method according to any one of claims 7-9 for colorectal cancer detection.