CN106148495A

CN106148495A - The application in colorectal cancer biomarker of a kind of circular rna

Info

Publication number: CN106148495A
Application number: CN201510160437.6A
Authority: CN
Inventors: 张悦; 栗世铀; 贾宝庆; 刘洪�; 刘洪一; 宋福海; 薛勇敢
Original assignee: Beijing Institute of Genomics of CAS
Current assignee: Beijing Institute of Genomics of CAS
Priority date: 2015-04-08
Filing date: 2015-04-08
Publication date: 2016-11-23

Abstract

The invention provides a kind of circular rna (Circular RNA circRNA) (CPSF6-circRNA) application in colorectal cancer biomarker, this circular rna (CPSF6-circRNA) long 11434bp, being positioned on the 12nd article of chromosome of the mankind, CPSF6 is the covering gene of this circRNA.The present invention can be used for differentiating to suffer from the object of colorectal cancer or the pathological of colorectal cancer, then or the method that determines the previously operation prognosis of object through differentiating.The present invention is prepared for the primer of examination circular rna (CPSF6-circRNA).Described method includes detecting the amount from the circular rna (CPSF6-circRNA) in the cancer and cancer beside organism's sample of described object, and wherein the CPSF6-circRNA of higher amount increases relevant to the probability of described object colorectal cancer prognosis mala.There is the application prospect of the clinical colorectal cancer biomarker of preparation.

Description

The application in colorectal cancer biomarker of a kind of circular rna

Technical field

The invention belongs to the purposes of natural ribonucleotide, relate to the purposes of cyclic ribonucleotides (CPSF6-circRNA), Particularly relate to the purposes in the new biomarker of CPSF6-circRNA colorectal cancer patients, be used for differentiating colorectal cancer and less The object of good prognosis.

Background technology

Colorectal cancer, also known as colorectal cancer, is a kind of common malignant tumor.Colon cancer in early days and non-evident sympton, until morbidity Middle and advanced stage finds.Improve the early diagnostic rate of colorectal cancer for treating in time and to improve prognosis most important.As by straight at knot The CPSF6-circRNA occurred in intestinal cancer is proved, it is known that have abnormal CPSF6-circRNA table in Colorectal Carcinoma Reach.Can be raised and lowered in colorectal cancer in view of CPSF6-circRNA expression, be thus provided that the pathology of progression of disease Learn indication.

CircRNA is widely present in various biological cell, has the features such as Stability Analysis of Structures, abundance height and tissue specific expression. During being genomic DNA transcriptional expression, be cyclized into annular RNA, a class non-coding RNA.Research shows, some CircRNA plays the effect of gene expression regulation as competitive endogenous RNA (ceRNA).CircRNA utilizes it MicroRNA (miRNA) response element combines miRNA, to block the inhibitory action that its target gene is expressed by miRNA, from And regulate and control the expression of other relevant mRNA.The discovery prompting circRNA of circRNA important function in gene expression regulation Drug development and diagnosis of disease have a good application prospect.

CircRNA became the hot fields of tumor research in recent years as a kind of control RNA (Regulatory RNA). Its expression of circRNAs possesses tissue specificity.In circRNA, both some is sent out as competitive endogenous RNA (ceRNA) Wave the effect of gene expression regulation.Thomas B.Hansen et al. is by people's AGO albumen and the analysis and research of miR-7, card Understand the circRNA effect as efficient microRNA sponge.CircRNAs expression in tumor and normal structure Difference is very notable, even can reach hundreds if not thousands of times, and this significant difference makes it become tool in candidate markers Have a clear superiority in.

Summary of the invention

It is an object of the invention to provide circular rna (Circular RNA circRNA) CPSF6-circRNA raw in colorectal cancer Application in thing mark, the long 11434bp of English name CPSF6-circRNA, CPSF6-circRNA, it is positioned at the 12nd of the mankind the On bar chromosome 69644908 to 69656342, the base sequence structure of CPSF6-circRNA, CPSF6 is this circRNA Covering gene.

The invention provides and differentiate to suffer from the object of colorectal cancer or colorectal cancer by stages, then or to previously suffering from through differentiating There are method and primer sequence structure, PCR primer recognition sequence that the prognosis of the object of colorectal cancer is determined.

Described method includes detecting to be measured from the CPSF6-circRNA in the sample of described object, wherein higher amount CPSF6-circRNA and described object suffer from the probability that probability increases or colorectal cancer prognosis is the best of colorectal cancer to be increased Relevant.

Beneficial effects of the present invention has: 1) propose the purposes of CPSF6-circRNA；2) research of the present invention shows abnormal The probability that the CPSF6-circRNA of amount and described object suffer from colorectal cancer increases or colorectal cancer prognosis may be pointed out the best. 3) circRNA has the features such as Stability Analysis of Structures, abundance height and tissue specific expression.And circRNA is at Patients with Colorectal Cancer In cancerous tissue and cancer beside organism, expression there are differences, and therefore CPSF6-circRNA has becomes colorectal cancer biomarker Application prospect.

Accompanying drawing explanation

Fig. 1 is the PCR recognition sequence comparison diagram with PCR primer sequencing result of CPSF6-circRNA.

Fig. 2 is to use the CPSF6-circRNA examination primer result figure to tissue of patient sample expression examination.

Fig. 3 is easy structure and the primer location figure of CPSF6-circRNA.

Detailed description of the invention

Below in conjunction with specific experiment step, the present invention is described further.

1) experiment material:

Clinical sample: cancerous tissue and cancer beside organism's sample of 16 example Patients with Colorectal Cancers are provided by 301 Hospital.

Reagent: CPSF6-circRNA examination primer is closed by Sangon Biotech's Beijing primer combining unit Become；Trizol (article No. 15596-018) is purchased from Invitrogen；Chloroform (article No. 20100927) is purchased from Beijing chemical industry, isopropyl Alcohol (article No. 1205031) is purchased from western Gansu Province chemical industry, and ethanol (article No. 101860) is sold purchased from northization；Random primers (goods Number C1181), M-MLV (article No. M1705),Ribonuclease Inhibitor (article No. 2511), Nuclease-Free Water (article No. 2511), dNTP mix (article No. P1195) are purchased from Promega；Premix Ex TaqTM II (article No. DRR081A) is purchased from Takara；Mineral oil (article No. D7295) is purchased from Sigma.

2) experimental technique:

A, colorectal cancer canceration tissue/cancer beside organism RNA extracts

Weigh 1 gram of organization material, after liquid nitrogen grinding, be transferred in the centrifuge tube of 1.5 milliliters of RNase-free, room temperature after mixing Stand about 5 minutes.Add the chloroform of 200 microlitres, mixing.11000 rpms are centrifuged 10 minutes.Take supernatant to add The chloroform of 200ul, 11000 rpms are centrifuged 10 minutes.Take supernatant and add the isopropanol of 500 microlitres ,-20 DEG C of precipitations 10 minutes.11000 rpms are centrifuged 15 minutes, precipitation are transferred in 1.5 milliliters of RNase-free centrifuge tubes, pre-cooling 75% washing with alcohol 2 times, dries, and RNase-free water dissolution (the most suitably dissolves, usually 20-35 microlitre). The OD value of Nanodrop measuring and calculating gained crude extract and concentration.

B, expression examination

1.cDNA synthesizes

1-2 microgram RNA is in 14 microlitre RNase-free water, then adds and add after 1 microlitre random primer softly mixes In 0.2mlPCR pipe.PCR instrument 70 DEG C hatches 5 minutes.Quickly it is placed on 2 minutes on ice.

Configuration reactant mixture:

Hatch 1 hour, hatch 15 minutes for 70 DEG C, 4 DEG C of insulations for 42 DEG C.

2.Real-time PCR

CDNA adds the ddH of 50ul₂O。

QPCR mixture is prepared on ice:

Pipe often adds 10 microlitre mineral oil.Centrifugal 1 minute of 1000 rpms of centrifuge.Sample is placed into QPCR instrument Middle operation, program is as follows:

3) experimental result:

See PCR recognition sequence and the comparison diagram of PCR primer sequencing result that Fig. 1 and Fig. 2, Fig. 1 are CPSF6-circRNA. Test result indicate that PCR recognition sequence and PCR primer sequencing result comparison success, the CPSF6-circRNA of CPSF6-circRNA The cancerous tissue and cancer beside organism of Patients with Colorectal Cancer are expressed.Fig. 2 is for using CPSF6-circRNA examination primer to patient's group The result figure of tissue samples expression examination.Test result indicate that CPSF6-circRNA is by the cancerous tissue and cancer of Patients with Colorectal Cancer Differential expression in tissue is relatively big, and cancerous tissue is lowered relative to cancer beside organism and also existed from 0.1905349 times to 0.9904351 times The patient changing wherein 6/11 lowers multiple below 0.5；Raise the expression quantitative change having 1.599753 times to 18.333660 times Changing, wherein the patient of 4/5 raises multiple within 5 times, and the patient of 1/5 raises more than 10 times.The biggest differential expression Give the CPSF6-circRNA application prospect as colorectal cancer biomarker.

Claims

1. circular rna (Circular RNA circRNA) CPSF6-circRNA is in preparation colorectal cancer biomarker Application, it is characterised in that the long 11434bp of described CPSF6-circRNA, is positioned on the 12nd article of chromosome of the mankind 69644908 To 69656342, the base sequence of described CPSF6-circRNA is as shown in SEQ ID NO:1.

Applying the most as claimed in claim 1, it includes detecting in the sample of the object that may suffer from colorectal cancer CPSF6-circRNA measures, and wherein the CPSF6-circRNA of higher amount and described object suffer from the probability increasing of colorectal cancer Add or probability that colorectal cancer prognosis is the best increases relevant.

Applying the most as claimed in claim 2, after treatment of colorectal cancer, the CPSF6-circRNA of reduced levels represents that treatment acts as With.

Applying the most as claimed in claim 2 or claim 3, the amount of described CPSF6-circRNA is cancerous tissue and cancer beside organism in sample Absolute magnitude.

Applying the most as claimed in claim 2 or claim 3, the amount of wherein said CPSF6-circRNA is from the blood serum sample from object Middle determine.

Applying the most as claimed in claim 2 or claim 3, the amount of wherein said CPSF6-circRNA is from the plasma sample from object Middle determine.

7. the primer detecting colorectal cancer biomarker, it is characterised in that described primer is used for identifying CPSF6-circRNA, The long 11434bp of described CPSF6-circRNA, is positioned on the 12nd article of chromosome of the mankind 69644908 to 69656342, The base sequence of described CPSF6-circRNA is as shown in SEQ ID NO:1.

8. detection primer as claimed in claim 7, it is for obtaining the CPSF6-circRNA expression of cancerous tissue and cancer beside organism, By the risk that the two contrast suffers from colorectal cancer in denoted object is increased or prognosis mala.

9. the test kit detecting colorectal cancer biomarker, it is characterised in that include for identifying CPSF6-circRNA's The combination of primer, probe, primer or probe combinations, the long 11434bp of described CPSF6-circRNA, be positioned at the 12nd of the mankind the On bar chromosome 69644908 to 69656342, the base sequence of described CPSF6-circRNA such as SEQ ID NO:1 institute Show.