CN107674915A

CN107674915A - A kind of application of circular rna in colorectal cancer biomarker

Info

Publication number: CN107674915A
Application number: CN201710785421.3A
Authority: CN
Inventors: 不公告发明人
Original assignee: Beijing Kai Sheng Biotechnology Co Ltd
Current assignee: Beijing Kai Sheng Biotechnology Co Ltd
Priority date: 2017-09-04
Filing date: 2017-09-04
Publication date: 2018-02-09
Anticipated expiration: 2037-09-04
Also published as: CN107674915B

Abstract

The invention provides a kind of circular rna (Circular RNA the circRNA) application of (GSE1 circRNA) in colorectal cancer biomarker, this circular rna (GSE1 circRNA) long 219bp, between on No. 16 chromosome of the mankind 85633914 to 85634132, GSE1 is the covering gene of the circRNA.The present invention can be used for differentiate may suffer from colorectal cancer object or colorectal cancer pathological, then or determine previously the object through discriminating operation prognosis method.The present invention is prepared for examination circular rna (GSE1 circRNA) primer.Methods described includes the amount of the circular rna (GSE1 circRNA) in cancer and cancer beside organism's sample of the detection from the object, and the wherein GSE1 circRNA of relatively low amount are related to the possibility increase of the object colorectal cancer prognosis mala.With the application prospect for preparing clinical colorectal cancer biomarker.

Description

A kind of application of circular rna in colorectal cancer biomarker

Technical field

The invention belongs to the purposes of natural ribonucleotide, it is related to cyclic ribonucleotides (GSE1-circRNA) use On the way, more particularly to the purposes in the new biomarker of GSE1-circRNA colorectal cancer patients, for differentiate colorectal cancer and The object of more poor prognosis.

Background technology

Colorectal cancer is also known as colorectal cancer, is a kind of common malignant tumour.Colon cancer early stage and non-evident sympton, until Middle and advanced stage of falling ill is found.The early diagnostic rate for improving colorectal cancer is most important for treatment in time and improvement prognosis.Such as pass through What the GSE1-circRNA occurred in colorectal cancer was proved, it is known that have abnormal GSE1- in Colorectal Carcinoma CircRNA is expressed.It can be raised and lowered in colorectal cancer in view of GSE1-circRNA expression quantity, thus provide disease and enter The Signs of pathology of exhibition.

CircRNA is widely present in various biological cells, has Stability Analysis of Structures, abundance height and tissue specific expression etc. Feature.During being genomic DNA transcriptional expression, cyclisation circularizes RNA, a kind of non-coding RNA.Research shows, some CircRNA plays the effect of gene expression regulation as competitive endogenous RNA (ceRNA).CircRNA utilizes its microRNA (miRNA) response element combination miRNA, to block the inhibitory action that miRNA is expressed its target gene, so as to regulate and control other phases Close mRNA expression.The discovery prompting circRNA of circRNA important function in gene expression regulation in drug development and Had a good application prospect in diagnosis of disease.

CircRNA turned into the hot fields of tumor research as a kind of control RNA (Regulatory RNA) in recent years. Its expression of circRNAs possesses tissue specificity.Both some sends out as competitive endogenous RNA (ceRNA) in circRNA Wave the effect of gene expression regulation.Thomas B.Hansen et al. pass through the analysis and research to people AGO albumen and miR-7, it was demonstrated that Effects of the circRNA as efficient microRNA sponges.Expressions of the circRNAs in tumour and normal structure is poor Not very significantly, or even hundreds if not thousands of times can be reached, this significant difference causes it to have on as candidate markers Have a clear superiority.

The content of the invention

It is an object of the invention to provide circular rna (Circular RNA circRNA) GSE1-circRNA in colorectal cancer Application in biomarker, the long 219bp of English name GSE1-circRNA, GSE1-circRNA, positioned at No. 16 dye of the mankind Between colour solid 85633914 to 85634132, GSE1-circRNA base sequence structure, GSE1 is the covering base of the circRNA Cause.

The invention provides differentiate may suffer from colorectal cancer object or colorectal cancer by stages, then or to previously warp The method and its primer sequence structure, PCR primer recognition sequence that the prognosis of object of the discriminating with colorectal cancer is determined.

Methods described includes the GSE1-circRNA amounts in sample of the detection from the object, wherein relatively low amount Possibility bad with possibility increase of the object with colorectal cancer or colorectal cancer prognosis GSE1-circRNA increases Add correlation.

Beneficial effects of the present invention have：1) GSE1-circRNA purposes is proposed；2) research of the invention shows exception Amount the possibility increase of suffering from colorectal cancer of GSE1-circRNA and the object or colorectal cancer prognosis may be prompted not It is good.3) circRNA has the features such as Stability Analysis of Structures, abundance height and tissue specific expression.And circRNA is in Patients with Colorectal Cancer Cancerous tissue and cancer beside organism in expression quantity have differences, therefore GSE1-circRNA have turn into colorectal cancer biomarker Application prospect.

Brief description of the drawings

Comparison diagram of the PCR recognition sequences with PCR primer sequencing result that Fig. 1 is GSE1-circRNA.

Fig. 2 is GSE1-circRNA easy structure and primer location figure.

Fig. 3 is the result figure using GSE1-circRNA examination primer pair tissue of patient sample expression quantity examinations.

Embodiment

With reference to specific experiment step, the present invention is described further.

1. experiment material：

Clinical sample：The cancerous tissue and cancer beside organism's sample of 29 Patients with Colorectal Cancer are provided by 301 Hospital.

Reagent：GSE1-circRNA examinations primer is synthesized by Sangon Biotech's Beijing primer Portion synthesizes；Trizol (article No. 15596-018) is purchased from Invitrogen；Chloroform (article No. 20100927) is purchased from Beijing chemical industry, different Propyl 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) are purchased from Sigma.

2. experimental method：

1) colorectal cancer canceration tissue/cancer beside organism RNA extractions

Weigh 1 gram of organization material, after liquid nitrogen grinding, be transferred in 1.5 milliliters of RNase-free centrifuge tube, mix rear chamber Temperature stands 5 minutes or so.200 microlitres of chloroform is added, is mixed.11000 rpms centrifuge 10 minutes.Supernatant is taken to add 200ul chloroform, 11000 rpms centrifuge 10 minutes.The isopropanol for taking supernatant to add 500 microlitres, -20 DEG C of precipitations 10 Minute.11000 rpms centrifuge 15 minutes, precipitation are transferred in 1.5 milliliters of RNase-free centrifuge tubes, the 75% of precooling Ethanol washs 2 times, dries, RNase-free water dissolving (according to circumstances appropriate dissolving, usually 20-35 microlitres).Nanodrop The OD values and concentration of measuring and calculating gained crude extract.

2) expression quantity examination

A.cDNA is synthesized

In 1-2 micrograms RNA to 14 microlitres of RNase-free water, then 1 microlitre of Random primer is added to be added after softly mixing In 0.2ml PCR pipes.70 DEG C of PCR instrument is incubated 5 minutes.Quickly it is placed on 2 minutes on ice.

Configure reactant mixture：

42 DEG C are incubated 1 hour, and 70 DEG C are incubated 15 minutes, 4 DEG C of insulations.

b.Real-time PCR

50ul ddH is added in cDNA₂O。

QPCR mixtures are prepared on ice：

10 microlitres of mineral oil are often added in pipe.1000 rpms of centrifuge centrifuges 1 minute.Sample is placed into ABI Run in 7500 quantitative real time PCR Instruments, program is as follows：

3) analysis of experimental data：According to qRT-PCR amplification situations, obtained GSE1-circRNA Ct values, according to internal reference Gene GAPDH amplification situation, relative organization itself GAPDH relative expression is calculated, further according to 2^-ddctMethod calculates tumour The expression difference of the relatively same patient's Carcinoma side normal tissues of GSE1-circRNA in tissue.

3. experimental result：

Referring to Fig. 1 and Fig. 3, Fig. 1 is GSE1-circRNA PCR recognition sequences and the comparison diagram of PCR primer sequencing result. Test result indicates that GSE1-circRNA PCR recognition sequences compare success with PCR primer sequencing result, GSE1-circRNA exists Expressed in the cancerous tissue of Patients with Colorectal Cancer and cancer beside organism.Fig. 3 is to use GSE1-circRNA examination primer pair tissue of patient The result figure of sample expression quantity examination.Using patient itself Carcinoma side normal tissue as control, GSE1-circRNA is calculated in tumour Relative expression levels in tissue.By test result indicates that GSE1-circRNA groups by the cancerous tissue of Patients with Colorectal Cancer and cancer Differential expression in knitting is larger, 24/29 patient's cancerous tissue relative to cancer beside organism lower there is from 0.0386688 times to 0.7918976 times of change, wherein 22/29 patient lowers multiple below 0.5；Up-regulation is arrived in the presence of 1.35170863 times 9.122064711 times of expression quantity change, its 4/29 patient raise multiple within 3 times, 1/29 patient up-regulation 3 times with On.So big differential expression has given application prospects of the GSE1-circRNA as colorectal cancer biomarker.

Claims

(Circular RNA the circRNA) applications of GSE1-circRNA in colorectal cancer biomarker 1. circular rna, The long 219bp of English name GSE1-circRNA, GSE1-circRNA, No. 16 chromosome 1172247533 positioned at the mankind arrive Between 172251541, GSE1-circRNA base sequence structure, GSE1 is the best covering gene of the circRNA.
2. differentiate the object that may suffer from colorectal cancer or the pathological of colorectal cancer, then or to previously through differentiating with knot Method and its primer sequence structure that the prognosis of the object of the carcinoma of the rectum is determined, PCR primer recognition sequence, methods described include Detect the GSE1-circRNA amounts in the sample from the object, the wherein GSE1-circRNA of relatively low amount and the object knot The bad possibility increase of carcinoma of the rectum prognosis is related.
3. the method for claim 2 is used for the treatment of colorectal cancer in tracing object, such as the GSE1- of reduced levels after the treatment CircRNA represents that treatment is worked.
4. the amount of the method for Claims 2 or 3, wherein GSE1-circRNA is the absolute of cancerous tissue and cancer beside organism in sample Amount.
5. claim 2-4 method, wherein the GSE1-circRNA is determined in the blood serum sample from object.
6. claim 2-4 method, wherein the GSE1-circRNA is determined in the plasma sample from object.
7. for detection primer and PCR primer recognition sequence in claim 2-5 method, it is additionally comprised for this method Operation instruction.
8. the detection primer of claim 6, it additionally comprises cancer beside organism's expression quantity, the cancer obtained using the detection primer The GSE1-circRNA of tissue expression quantity increases with suffering from the risk of colorectal cancer in the contrast denoted object of cancer beside organism's expression quantity Add or prognosis mala.
9. according to the application described in claim 1-8, it is characterized in that：The detection kit of preparation is with using by GSE1-circRNA In its primer, probe, primer combination or probe combinations of identification.