CN107674915B

CN107674915B - Application of circular RNA in colorectal cancer biomarker

Info

Publication number: CN107674915B
Application number: CN201710785421.3A
Authority: CN
Inventors: 不公告发明人
Original assignee: Beijing Tricision Biotherapeutics Inc
Current assignee: Beijing Tricision Biotherapeutics Inc
Priority date: 2017-09-04
Filing date: 2017-09-04
Publication date: 2021-11-23
Anticipated expiration: 2037-09-04
Also published as: CN107674915A

Abstract

The invention provides application of Circular RNA (Circular RNA \ circRNA) (GSE 1-circRNA) in a colorectal cancer biomarker. The Circular RNA (GSE 1-circRNA) is 219bp long and is located between 85633914 and 85634132 on the 16 th chromosome of human, and GSE1 is a cover gene of the circRNA. The Circular RNA (GSE 1-circRNA) can be used for identifying an object possibly suffering from colorectal cancer or a pathological type of the colorectal cancer, and then or determining a method for surgical prognosis of the previously identified object. A primer for screening the Circular RNA (GSE 1-circRNA) is prepared. The method comprises the step of detecting the amount of the Circular RNA (GSE 1-circRNA) in a cancer and para-carcinoma tissue sample from the object, wherein the lower amount of GSE 1-circRNA is related to the increase of the possibility of poor prognosis of the colorectal cancer of the object. The application has the application prospect of preparing the clinical colorectal cancer biomarker.

Description

Application of circular RNA in colorectal cancer biomarker

Technical Field

The invention belongs to the use of natural ribonucleotides, relates to the use of cyclic ribonucleotides (GSE1-circRNA), and particularly relates to the use of a novel biomarker of GSE1-circRNA colorectal cancer patients for identifying colorectal cancer and objects with poorer prognosis.

Background

Colorectal cancer is also known as colorectal cancer, and is a common malignant tumor. Colon cancer has no obvious symptoms in early stage until the middle and late stage of the disease. Improving the early diagnosis rate of colorectal cancer is crucial to timely treatment and prognosis improvement. Abnormal GSE1-circRNA expression in colorectal cancer tissues is known, as demonstrated by GSE1-circRNA present in colorectal cancer. Given that GSE1-circRNA expression levels can be increased or decreased in colorectal cancer, pathological indications of disease progression are provided.

The circRNA is widely present in various biological cells and has the characteristics of stable structure, high abundance, tissue-specific expression and the like. Is a non-coding RNA of a circular RNA in the transcription and expression process of genome DNA. Studies have shown that some circrnas act as competitive endogenous rnas (cernas) to exert regulation of gene expression. The circRNA utilizes its microrna (miRNA) response element to bind to miRNA, to block the inhibitory effect of miRNA on its target gene expression, thereby regulating the expression level of other related mrnas. The discovery of the important role of circRNA in gene expression regulation suggests that circRNA has good application prospects in drug development and disease diagnosis and treatment.

As a regulatory RNA (regulatory RNA), circRNA has recently become a hot spot in tumor research. The expression of the circRNAs has tissue specificity. Some of the circRNAs function as competitive endogenous RNAs (cepRNAs) to regulate gene expression. Hansen et al, show the effect of circRNA as a high-efficiency microRNA sponge through analytical studies on human AGO protein and miR-7. The expression level of the circRNAs in tumor and normal tissues is very different, even can reach hundreds or even thousands of times, and the obvious difference makes the circRNAs have obvious advantages in becoming candidate markers.

Disclosure of Invention

The invention aims to provide application of Circular RNA (Circular RNA \ Circular RNA) GSE 1-Circular RNA in colorectal cancer biomarkers, wherein the Circular RNA is the application of the Circular RNA (Circular RNA \ Circular RNA) GSE 1-Circular RNA, the Circular RNA is the English name GSE 1-Circular RNA, the length of the GSE 1-Circular RNA is 219bp, the Circular RNA is positioned between the No. 16 chromosome 85633914 and the No. 85634132 of a human, the GSE 1-Circular RNA has a base sequence structure, and the GSE1 is a covering gene of the Circular RNA.

The invention provides methods of identifying a subject likely to have colorectal cancer or the stage of colorectal cancer, and determining the prognosis of a subject previously identified as having colorectal cancer, and primer sequence structures and PCR product recognition sequences thereof.

The method comprises detecting an amount of GSE1-circRNA in a sample from the subject, wherein a lower amount of GSE1-circRNA correlates with an increased likelihood of the subject having colorectal cancer or an increased likelihood of a poor prognosis of colorectal cancer.

The invention has the following beneficial effects: 1) the application of GSE1-circRNA is provided; 2) the study of the present invention shows that abnormal amounts of GSE1-circRNA are associated with an increased likelihood that the subject will suffer from colorectal cancer or may indicate a poor prognosis of colorectal cancer. 3) The circRNA has the characteristics of stable structure, high abundance, tissue-specific expression and the like. And the expression quantity of the circRNA in the cancer tissue and the para-cancer tissue of the colorectal cancer patient is different, so the GSE1-circRNA has the application prospect of becoming a colorectal cancer biomarker.

Drawings

FIG. 1 is a comparison of the PCR recognition sequence of GSE1-circRNA and the results of sequencing the PCR products.

FIG. 2 is a diagram showing the simple structure and primer positions of GSE 1-circRNA.

FIG. 3 is a graph showing the results of screening the expression level of a tissue sample of a patient using GSE1-circRNA screening primers.

Detailed Description

The invention is further described below in connection with specific experimental procedures.

1. Experimental materials:

clinical samples: cancer tissue and paracarcinoma tissue samples from 29 colorectal cancer patients were provided by the 301 hospital.

Reagent: GSE1-circRNA screening primer is synthesized by Beijing primer synthesis part of Biotechnology engineering (Shanghai) GmbH; trizol (cat # 15596-; chloroform (cat # 20100927) from Beijing chemical, isopropanol (cat # 1205031) from Xilongdan chemical, and ethanol (cat # 101860) from Beijing chemical; random primers (cat # C1181), M-MLV (cat # M1705),

ribonucleae Inhibitor (cat 2511), Nuclear-Free Water (cat 2511), dNTP mix (cat P1195) were purchased from Promega;

premix Ex TaqTM II (cat # DRR081A) was purchased from Takara; mineral oil (cat # D7295) was purchased from Sigma.

2. The experimental method comprises the following steps:

1) RNA extraction from cancerous tissue/tissue adjacent to cancerous tissue of colorectal cancer

Weighing 1 g of tissue material, grinding with liquid nitrogen, transferring to a centrifuge tube of 1.5 ml RNase-free, mixing uniformly, and standing at room temperature for about 5 minutes. Add 200. mu.l of chloroform and mix well. Centrifuge at 11000 rpm for 10 minutes. The supernatant was extracted with 200. mu.l of chloroform and centrifuged at 11000 rpm for 10 minutes. The supernatant was taken and 500. mu.l of isopropanol were added and precipitated at-20 ℃ for 10 minutes. Centrifuging at 11000 rpm for 15 min, transferring the precipitate to 1.5 ml RNase-free centrifuge tube, washing with 75% ethanol for 2 times, air drying, and dissolving with RNase-free water (appropriately dissolving according to the conditions, generally 20-35 μ l). And (4) calculating the OD value and the concentration of the obtained crude extract by using the Nanodrop.

2) Screening for expression amount

A.cDNA Synthesis

1-2. mu.g of RNA was added to 14. mu.l of RNase-free water, 1. mu.l of Random primer was added thereto, gently mixed, and then added to a 0.2ml PCR tube. The PCR instrument was incubated at 70 ℃ for 5 minutes. Quickly placed on ice for 2 minutes.

Preparing a reaction mixture:

incubate at 42 ℃ for 1 hour, 70 ℃ for 15 minutes, and incubate at 4 ℃.

b.Real-time PCR

50ul of ddH was added to the cDNA₂O。

Preparation of QPCR mixture on ice:

10 microliters of mineral oil was added to each tube. Centrifuge 1000 rpm for 1 minute. The samples were placed into an ABI 7500 fluorescent quantitative PCR instrument and run as follows:

3) and (3) analyzing experimental data: according to the amplification condition of qRT-PCR, the Ct value of the obtained GSE1-circRNA, according to the amplification condition of the internal reference gene GAPDH, the relative expression of the GAPDH of the relative tissue is calculated, and then according to 2^-ddctThe method calculates the difference of the expression level of GSE1-circRNA in the tumor tissue relative to the paracancerous normal tissue of the same patient.

3. The experimental results are as follows:

referring to FIGS. 1 and 3, FIG. 1 is a graph comparing the PCR recognition sequence of GSE1-circRNA with the results of sequencing the PCR products. The experimental result shows that the PCR recognition sequence of the GSE1-circRNA is successfully compared with the sequencing result of the PCR product, and the GSE1-circRNA is expressed in the cancer tissue and the para-cancer tissue of the colorectal cancer patient. FIG. 3 is a graph showing the results of screening the expression level of a tissue sample of a patient using GSE1-circRNA screening primers. Relative expression levels of GSE1-circRNA in tumor tissues were calculated, using patient's own paracancerous normal tissues as controls. The experimental result shows that the expression of GSE1-circRNA in cancer tissues and paracarcinoma tissues of colorectal cancer patients is greatly different, and the cancer tissues of 24/29 patients have the change from 0.0386688 times to 0.7918976 times compared with the cancer tissue downregulation, wherein the downregulation time of 22/29 patients is below 0.5; the upregulation varies from 1.35170863-fold to 9.122064711-fold, with the upregulation of 4/29 within 3-fold and 1/29 above 3-fold. The large expression difference gives the application prospect of GSE1-circRNA as a colorectal cancer biomarker.

Claims

1. The application of a reagent for detecting GSE1-circRNA in preparing a detection kit using GSE1-circRNA as a colorectal cancer biomarker in a tissue sample is characterized in that the GSE1-circRNA is circular RNA, in the base sequence structure of the GSE1-circRNA, GSE1 is the best covering gene of the circRNA, and the corresponding DNA sequence of the GSE1-circRNA is shown as follows:

2. use according to claim 1, for identifying a subject likely to suffer from colorectal cancer.

3. The use according to claim 2, comprising detecting the amount of GSE1-circRNA in a sample from said subject.

4. The use of any one of claims 1 to 3, wherein the reagents comprise detection primers and PCR product recognition sequences.

5. The use according to any one of claims 1 to 3 wherein the reagent comprises a primer or probe for identifying GSE 1-circRNA.

6. The use according to any one of claims 1 to 3, wherein the reagent comprises a primer combination or a probe combination for identifying GSE 1-circRNA.