CN109735622A - LncRNA relevant to colorectal cancer and its application - Google Patents

Abstract

The invention discloses a kind of lncRNA relevant to colorectal cancer and its applications.Inventor passes through the cancerous tissue and cancer beside organism's progress transcript profile lncRNA-mRNA sequencing analysis to Colon and rectum patient, find the apparent 1ncRNAs of differential expression and its co-expression gene, discovery and colorectal cancer closely related LOC105374879 and its co-expression gene.The application provides new molecular marker for the early diagnosis of colorectal cancer, lays the foundation for clinical application.

Description

LncRNA relevant to colorectal cancer and its application

Technical field

The invention belongs to Cancer Molecular diagnostic fields, and in particular to lncRNA relevant to colorectal cancer and its preparation disease Application in sick diagnostic reagent.

Background technique

Colorectal cancer (colorectal, CRC) is one of most common malignant tumour, and domestic disease incidence is in recent years continuous Increase, has become common one of the nine big tumours in China.In recent decades, the improvement of various detection methods is so that colorectal cancer Diagnosis have great progress.The current colorectal cancer screening in China is mainly or using screening.Use high risk factor Questionnaire survey combination immunization fecal occult blood inspection (FOB) is primary dcreening operation scheme, and questionnaire is positive or the crowd of the FOB positive is considered as Primary dcreening operation Positive Populations will receive enteroscopy.The screening scheme is considered as the knot that can be promoted under the current national conditions in China Carcinoma of the rectum screening scheme, but there is also the spaces further increased.The morbidity and mortality of U.S.'s colorectal cancer are all declining, This has benefited from the improvement of screening scheme.Domestic scientific research personnel are attempting to establish using gene tester always a kind of simple noninvasive , the screening means of colorectal cancer specificity, it has been reported that gene have GAPLINC, KRAS, SHROOM4 (ZL2016100689102), SLC26A9 (ZL2015110045037) etc..

Long-chain non-coding RNA (long non-coding RNAs, IncRNAs) is that length is 200-100000 alkali base-pair Non-coding RNA molecule, by carrying out specific research and analysis, discovery to some IncRNAs with specific expression levels 1ncRNAs plays a significant role in terms of the adjusting of gene.The occurrence and development of LncRNAs and tumour have important relationship, may Important symbol object and target spot as tumor diagnosis and therapy.In terms of diagnosis, due to the 1ncRNAs in tumor tissues express it is past Toward exception, and there is tissue and species specificity, makes it possible to become tumor markers, especially early stage some tumours, Since its is small in size, traditional Imaging Method is not easy to find.By the 1ncRNAs in detection blood, can easily understand Patient suffers from a possibility that certain tumour.

Inventor carries out transcript profile lncRNA-mRNA sequencing point by cancerous tissue to Colon and rectum patient and cancer beside organism Analysis finds the apparent 1ncRNAs of differential expression and its co-expression gene, and carries out quantitative fluorescent PCR verifying, searches out straight with knot The relevant 1ncRNA of intestinal cancer --- LOC105374879 and its co-expression gene.The application provides for the early diagnosis of colorectal cancer New molecular marker, lays the foundation for clinical application.

Summary of the invention

The purpose of the present invention is to provide a kind of long-chain non-coding RNA LOC105374879 relevant to colorectal cancer, sequences Column have 90% or more sequence homology with SEQ ID NO.1.

Term " homologous " be primarily referred to as it is homologous in sequence, that is, be used to illustrate two or more protein or DNA Sequence ancestors having the same.Homologous sequence generally has similar function.The homology of protein and DNA are often through them The similitude of sequence determines, similitude refer to be used to describe during sequence alignment it is between detection sequence and target sequence identical The height of DNA base or amino acid residue sequence proportion.In general, when similarity degree is higher than 50%, often speculate inspection Sequencing column and target sequence may be homologous sequence；When degree of similarity is lower than 20%, just it is difficult to determine if to have same Source property.

Preferably, LOC105374879 sequence and SEQ ID NO.1 have 95% or more sequence homology；It is furthermore preferred that Long-chain non-coding RNA sequence is SEQ ID NO.1.

The purpose of the present invention is to provide a kind of reagent for detecting colorectal cancer, reagent detects LOC105374879 in sample Expression.

Further, reagent is using sequencing technologies, nucleic acid hybridization technique or nucleic acid amplification technologies detection LOC105374879 Expression.

Sequencing technologies are mainly that second generation sequencing technologies (mainly include 454 pyrosequencing of Roche, Illumina Solexa sequencing and ABI SOLID sequencing) and third generation sequencing technologies (mainly include single molecular fluorescence sequencing and nano-pore survey Sequence).

Further, the nucleic acid amplification technologies are selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence The amplification (NASBA) of column.Wherein, PCR needs directly expand RNA reverse transcription at DNA (RT-PCR), TMA and NASBA before amplification Increase RNA.

Further, it is detected using sequencing technologies, probe hybridization technique, biochip technology or fluorescent quantitative PCR technique The expression of LOC105374879.

Further, reagent includes a pair of primer for being used for quantitative fluorescent PCR nucleic acid amplification, sequence be SEQ ID NO.2 and SEQ ID NO.3。

In general, PCR uses the annealing of denaturation, primer pair and opposite strand and multiple circulations of primer extend, with index side The copy number of formula increase target nucleic acid sequence；Reverse transcriptase (RT) is then used for the DNA (cDNA) complementary from mRNA preparation by RT-PCR, Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in substantially constant temperature, ionic strength and pH Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein multiple RNA copy of target sequence is autocatalytically given birth to At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA process Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides Acid is covalently attached in thermal denaturation, hybridization and the multiple circulations of the repetition of connection by DNA ligase, to generate detectable double-strand Connect oligonucleotide product；SDA uses multiple circulations of following steps: primer sequence pair and the opposite strand of target sequence move back Fire carries out primer extend under there are dNTP α S to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting The polymerase-mediated primer extend that the mouth end 3' carries out is to replace existing chain and generate for next round primer annealing, nicking and displacement Chain, so as to cause product geometry expand.

In the present invention " probe " refer to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.It removes Non- to indicate otherwise, term " probe " is often referred to match and another polynucleotides (often referred to as " target multicore glycosides by complementary base Acid ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence it is complementary Property target polynucleotide combine.Probe can make direct or indirect label, and range includes primer.Crossing system, including, but not It is limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.

The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence 80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA, It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Term " hybridization " in the present invention is used to refer to the pairing of complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid Association intensity) influenced by factor such as below: the stringency of complementarity, related condition between nucleic acid is formed Hybrid Tm and nucleic acid in G:C ratio.The individual molecule of pairing in its structure containing complementary nucleic acid is known as " self Hybridization ".

Further, the sample of above-mentioned detection colorectal cancer reagent detection is cancerous tissue or blood.

The purpose of the present invention is to provide the reagents of detection LOC105374879 gene expression dose in preparation colorectal cancer Application in diagnostic tool.

The purpose of the present invention is to provide application of the mentioned reagent in preparation diagnosis of colorectal carcinoma tool.

Further, colorectal cancer is colon cancer or the carcinoma of the rectum.

The purpose of the present invention is to provide a kind of diagnosis of colorectal carcinoma reagent, reagent hybridizes skill by sequencing technologies, nucleic acid Art, nucleic acid amplification technologies or the method for immunoassays detect gene expression dose relevant to LOC105374879, relevant base Because one or several in following gene: HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1, HEATR1, TMEM201, ADK、RANBP1、GUCA2A、CLDN23、CD79A、RAB27A、OTUD1、TMEM54、ABHD3、RIMKLA、USP2、CD244。

Relevant gene includes positive correlation gene or negative correlation gene.

Further, positive correlation gene be HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1, HEATR1, TMEM201, ADK、RANBP1。

Further, negative correlation gene be CD244, USP2, RIMKLA, ABHD3, TMEM54, OTUD1, RAB27A, CD79A, CLDN23、GUCA2A。

The purpose of the present invention is to provide detect application of the above-mentioned diagnostic reagent in preparation diagnosis of colorectal carcinoma tool.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used for determining the structure of chromosome.RNA ISH is for measuring with position tissue slice or entirely MRNA and other transcripts (for example, ncRNA) in organization embedding.Usually sample cell and tissue are handled in situ solid Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base The probe of label is positioned and is quantified.ISH can also be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

It would be recognized by those skilled in the art that the invention is not limited to any specific variants to LOC105374879 Gene expression is quantified.In some embodiments, have same or similar with LOC105374879 sequence at least 85% CDNA sequence, such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or at least 99% the same or similar cDNA sequence.

Detailed description of the invention

Fig. 1 is the co-expression gene network of LOC105374879；Triangle be with LOC105374879 negative correlation gene, Ellipse is and LOC105374879 positive correlation gene；

Fig. 2 is the qRT-PCR verification result figure of difference expression gene LOC105374879.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, or according to the normal condition proposed by manufacturer.

The acquisition of 1 sample of embodiment is extracted and data analysis

It collects and carries out 4 rectal adenocarcinoma patient with operation cancers and cancer beside organism in this hospital, be put into and used 0.1%DEPC water logging It steeps overnight and in the 1.5ml sterile EP tube sterilized, and is quickly put into -80 DEG C of liquid nitrogen container and saves.It is constructed after extracting sample rna Then the library lncRNA and mRNA is surveyed mRNA with llumina hiseq x-ten second generation high throughput sequencing technologies Sequence.By the processes such as removing connector, going low quality, depollute complete the processing of data, sequencing data is obtained.

MRNA, which is carried out, using software cuffquan, cuffdiff expresses quantitative and Differential expression analysis.Cuffquant is It is specifically used to carry out expression quantity assessment and standardized tool in Cufflinks external member, cuffdiff is in Cufflinks external member For calculating a tool of differential expression, cuffdiff utilizes the more each gene/transcript of quantitative result of cuffquant Differential expression between two groupings.Significant difference lncRNA and mRNA screening conditions: P<0.05, | log2FC |>1.Significant LOC105374879 is selected to carry out subsequent authentication experiment as target gene in difference lncRNA.

The co-expression gene of embodiment 2LOC105374879 is analyzed

As a kind of non-coding RNA, function is mainly reflected in the regulation to target gene lncRNA, mainly include to away from Trans acting regulatory (trans-regulate) from protein coding gene farther out, meanwhile, the gene with identical expression pattern, Functionally there is strong correlation.Therefore pass through the target base to lncRNA and mRNA coexpression, trans analysis and research lncRNA Cause.

By the Pearson correlation coefficients (Pearson for calculating differential expression lncRNA and mrna expression amount Correlation coefficient, PCC), analyze lncRNA and mRNA coexpression relationship, threshold value using 0.98, p < 0.001 screening with LOC105374879 coexpression mRNA (being specifically shown in Fig. 1), HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1、HEATR1、TMEM201、ADK、RANBP1、GUCA2A、CLDN23、CD79A、RAB27A、OTUD1、TMEM54、 ABHD3, RIMKLA, USP2, CD244, wherein positively related gene are as follows: HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1, HEATR1,TMEM201,ADK,RANBP1；Negatively correlated gene are as follows: CD244, USP2, RIMKLA, ABHD3, TMEM54, OTUD1、RAB27A、CD79A、CLDN23、GUCA2A。

The quantitative fluorescent PCR of 3 differential expression lncRNA of embodiment is verified

1, sample collection and extraction

Sample collection chooses this hospital and is diagnosed as the peripheral blood 28 of colon cancer or rectal cancer patient (wherein colon cancer is 13, the carcinoma of the rectum is 15), healthy population peripheral blood 24 for physical examination are compareed, QIAGEN blood rna extracts kit is utilized Extract the RNA in serum, specific steps reference book.

2, reverse transcription:

1) the total serum IgE template of 10pg-1 μ g and 2 10 × buffers of μ l, 2 μ l dATP (10mM), 0.5 μ l polyA is more Poly- enzyme, 0.5 μ l ribalgilase (RNase) inhibitor and deoxyribonuclease water (RNase free water) mixing, volume It is finally 20 μ l, 37 DEG C of incubation 1h.

2) 1 μ l 0.5 μ g/ μ l Oligo (dT) specific RT primer, 70 DEG C of incubation 5min are added in reaction tube.

3) it is incubated at least 2min on ice immediately, interrupts the secondary structure of RNA and primer.

4) by above-mentioned 20 μ l reaction mixture and 45 × buffers of μ l, 1 μ l dNTP (10mM), 0.5 μ l M-MLV is reversed Record enzyme, 0.5 μ l ribalgilase (RNase) inhibitor, 10 μ l polyA reaction mixtures and 4 μ l deoxyribonuclease water (RNase free water) mixing, 42 DEG C of incubation 1h.

3、QPCR

(1) design of primers

QPCR amplimer is designed according to LOC105374879 sequence (XR_001743918) in Genbank, is given birth to by Shanghai The synthesis of work biotechnology Services Co., Ltd, internal reference select GAPDH.Specific primer sequence is as follows:

LOC105374879 gene:

Forward primer is 5 '-GTGTTGACCTGGAGAATC-3 ' (SEQ ID NO.2)

Reverse primer is 5 '-CAATAATCTGGAGAAGTGAGT-3 ' (SEQ ID NO.3)

Amplification length 107bp.

(2) PCR reaction system is prepared according to table 1:

Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.

Table 1PCR reaction system

(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 10s, 55 DEG C of 50s) × 35 circulations.Using SYBR Green as Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis Determine purpose band, Δ Δ CT method carries out relative quantification.

1.5 statistical method

Experiment is all to be repeated 3 times according to each sample to complete, and result data is all the side with mean+SD Formula indicates, using SPSS13.0 statistical software come for statistical analysis, difference between the two is examined using t, it is believed that works as P There is statistical significance when < 0.05.

1.6 result

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2- Δ Ct × 100%.As a result as shown in Fig. 2, compared with Healthy People group, there is aobvious LOC105374879 gene expression amount in illness group It writes and increases, be 2.6 times of Healthy People group, difference has statistical significance (P < 0.05).With 24 healthy population cell means To detect baseline, in 28 colorectal cancer patients, 23 LOC105374879 gene expression amounts have a significant raising, and 3 LOC105374879 gene expression difference is not statistically significant, and 2 expression are lowered, and compares colon cancer and rectal cancer group LOC105374879 gene is not statistically significant in two groups of differential expression.Show LOC105374879 gene pairs colorectal cancer Diagnosis has certain guidance meaning, is expected to become the candidate markers of colorectal cancer molecular diagnosis in clinic or human health screening.

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

<110>No.3 Central Hospital of Tianjin City

<120>lncRNA relevant to colorectal cancer and its application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6170

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gtgtgcttac atttgcatca catatgtatg tgtctgtgtg cacatatgca cgtgtataca 60

tgtgcctgtg tatgtgcaca taaacagtgt ctttgagggc ttggcaatct gtgttgatgt 120

tattgaagct ctgcacctga cttctggtca cttttctgtt actgaccagc taaataactt 180

tgagtaagtc attgaactta tttggacact agttccatca actgttagat atgaggatgg 240

gaatggattg tctcaaagat ccttttgagt tcaaaaagtc tatgatacca gtgagaatgt 300

tccaaagcct taatgacacc tcactgaatg cagcttaaaa tggctgcaag ctgaccagac 360

gattgcattc tccaaggtga gctgcccttc atacctcatg cccttgaata atgcagcgta 420

agtgtgttta ttgtgccagc tcttcttgta gttttattct aatccctcgt ctcaggcaac 480

atttgttttg gctgcagttc gatacttcct ccttcagagc tatgcttggt tttacctcta 540

attctgtggc cctcaggggc tggttgtact gataaacttt tacaagtaaa aggagctgtg 600

gagatgacct ccagctgggg ctgaaggcct atacagcagc accttcttgc tgaagctaag 660

tgatcctctt ggggaggagg gttgggggtc cagggcccct gaagtcagga ttccccagtg 720

agttggtcaa tgtggactta gatcttgttg aaatatccag aagtatttct aagtagaata 780

tttttgatca cagaaacaag gtaaatattt ttgtcttcct tcagtggagg actttggagc 840

tctcaggtgg ctgctctcca tcccaggcct ggctgtagga ggctacctgg gtggggtgca 900

ggcccatgcc tggggtcctg gctccagagg caccacatgc cactcagtga ggataagaat 960

ccattctgca gcttctttca gccccacatc aataaatgga gacagtggga agggagctgt 1020

gctactttga aggatttggg ggcagactaa gaagaaaaca catgtgaaag tgctttgtaa 1080

ggatgagtgc atggcggggc gacgaggggc gttggaggca catgcagctg ggtaggagtt 1140

gcactttggt cattggccag ttatgtggct ttgggaactg ttcagacttc tggatcttgg 1200

cttccttttt tgttaagtag gtgatgatag ggatctcatt gggctgctgt gaggatgagg 1260

tgtgtttaca gtgcttggca aagagtgggg ctccatgcac ataactccat ttcctctaca 1320

tctacattgc tctgcaaacg aagggtgcta tgacaataaa cccaccccaa ggccaaagga 1380

ggggcgtgct gggggtggag agggatggct gggaactaac tagccggcat cagcaaagga 1440

gggattgttt tggtctttgt aggaaattag gaggattgcc tttctcatct attacttcca 1500

atgagttctg atgttctggg gaatcttctc tgaatgaatc ctggtattgc ttcatttata 1560

ctataactct gtagtataac tatatcctgt gtaatccaag ccaaagatag agggtttggg 1620

gacgacagat atttcaacaa tagtcactga ggcaaggaat tgtggattct aggatgaggc 1680

agcaggtgaa gggctttggt gcccacacag aggagaagag ggcaactgga gcgaggaaat 1740

gtcttgctgt cctgccgttt agctctggaa gtgtctcttg ggacaatctt ggagaaacgt 1800

caactgaagt gctagtttag tagcagggta gctgagtcct agcagtgttt cgcatttctc 1860

cagtgaaagc cctagaggac ttggatgtcc ccatctctaa agacaccggt ggcggcagag 1920

gcccagcttc taaacactca gggtcacaca ggcattggtg cttggtaaac gttttcttat 1980

ataatttcaa agccatctgt gacatggctg tatcatcccc attctactag tgagaaaacc 2040

aatgccaggg gatggaaaaa caacttcctt atggtcacac tgccagtcag tgcctggtgg 2100

gacccaagct gggccccaat cccccatgtc ccccttggca cctgtcctgc tttagggaag 2160

atggtctgtg gtcaaagctt aatctttgct ttgataaagg attaattcaa attttggaat 2220

gaaaagaaga gattttgaag ccctaaaact gtactgtgtg acaaaactcc tgttcttggg 2280

atatggtggc agaagtaagg gaaagtgtca gaaaaagtcc cgcaaggtga gaaaaactga 2340

aaaagagacc cctttcctat agaggctcac gaaggggaaa ttgcaactgt gcgaccctgt 2400

ctgctagctt tctctgtcac ttcatccccc attcctgttg ctgtgtgcac ttttatccaa 2460

ccctcttaat gtccatggct gcgctaccag caaaatctaa ccccatctgc ttggctaagc 2520

ttcttgcgct cacctatacc tgaggagtac gacatcaggc ttcagggctt attgaagtca 2580

ttactcacat cattgcacta atcacaaaca gaagtgaaag ttgtaaattg tatataatta 2640

catttcatag agaaaaaagt agatttaaaa atggcaatta atattcttaa tagccaaaag 2700

ttattgctaa ttaataagaa aaacataagc actccaattg aaaaaaaatg agcaaaggtc 2760

ataaagacat aaaactcgcc tgtggaaaca tacgtagagg aaacacagaa ttagtgagtg 2820

tgtgtatgga aataaatgga aggataatgc ttgctaccca gtgtgggctt tgggggtaga 2880

tctacctgga ctgagttctg gattcactat ttactagctc catgaccttc agcaaagtaa 2940

cctctctaag cctttgtttt tatctgtaaa atagggatac ttattttccc tgccttgagg 3000

ggttattgtg aggataaaag aaaataattc tgtaaagggc ttagtaaagt gcctggtgca 3060

gagaaatttt tcagtgcaga ttagcaattg tcactgctag tagtatattt gcattgataa 3120

caattagtaa tttaacatct cactgaaatc aaagaaatgc aattaaaaca acaaatacat 3180

attatttgtt acttatcaga ttggaaaaca ggtgaaaaga ttgacagtaa ccactgttgg 3240

ttagatgtgg agagatgggt tctcttgtgc aattctggga tggtaggacc tgatgcccca 3300

tttctgcagg gcaatttggc aatatacatt aacgttgaac attctgatag tttttgaccc 3360

agtcattctt ctctcagaaa cttattcctg ggagataact gaacaaatgc acaataatga 3420

atatataaga atgtttatcc tggcattttt ttttatagaa acaaaaattc agaagcaact 3480

taaacatcct tcagtgagat tgactaataa gtcctgcttc atttttatga cagaatgcta 3540

ctactaatag cagtgttcta gattttcatc caccatcatg gataaagctc tccgtcatgt 3600

ggatgggtaa gaaaacatgg agtggaaaat gacatctata ctgtgcccat gtatgtgtat 3660

caatctttta gtttagtaaa agacacaagt ttgggaaaat gtgctttaaa ttgcttacat 3720

tgtcctcttc ttgggatcta atttcgttat ttgattttta tgagtgtctg ttatcttttt 3780

actcagaaaa aaaaggaaaa ggaaagtttt tttttttttg tcctttagca gcaaaacctt 3840

acacaaagat ctacacgttg atttcgctcc tatgaactga cttggagtta tgttccaaga 3900

acgcatcacc acagcttgct tccttttctt tcttccttgt ttgttactca gaagtgtttc 3960

ttcattcttt agtctatgat tcatgcaaga agacagaacc aatgttttgc aagaccttcc 4020

catgaagtag aaagaacagt agtgttcact ccataagagg cgtgcgctac cacttgcctt 4080

gcctcatggg caatgaggaa gttgcatggg tttcctacgg ctctgcaggg gtggagtgag 4140

ccagaaagaa ctggtcagcc ctagacattc aggtgctatc aaataccagc cctagggtca 4200

agaacactta aaaacaaaac aaacttgaaa cttcctttgc ttgtgttgct tagaatcact 4260

gcggtaggag gattcctgag gcattacctg gatagtctca tctggctgga catgcaggag 4320

aactgggaga ctgccgagac tgccacgtgg ggtgacttga gggatctttg acactctctg 4380

tcttaatctg ttttgtgttg ctataacaga atacacagac taagtaattt ataaagaaag 4440

gaaatttact tctcacagtt ttggaggctg agaagtccaa gactgagggg ctggcatctg 4500

acgagggcct tcttgctgct tcattccacg gcaaaatggc aaagagaaga tgagaagaaa 4560

ggctcaaagg gacggtggga cttgactgag gacccagcac ctcctggacc tgaatcaagg 4620

ccctctgtcc cgctgctttt gttgttgttt accctgcaac ttcacagccc ttcagaaggc 4680

cctggacatc atgtgcaagc tccgtggatg gcctgagatt catccttcca agttgttttc 4740

tgactgagag gcaacatttt aaattgtgat ccccccttac cccaccacac acactgaaac 4800

aaaagtttca agaagtcatt accacacaat gactcagata tgttctagtc tagtctgtct 4860

attccattcc attccagtct ctttccaaac gaaaataaaa ggaaggaaat gctgcatgcg 4920

acttggtaag ttaatttcag gacaccttgg gactgcttcc tgctgcatga ggcatcagca 4980

cacagcaggg gatattttgc attcctgtgt gcaccgtggg ttagacaggc tgcttggccc 5040

taagtggaga atggaactcc agtagaaagg ctgagatgca ctgcaagtaa ccactgcaga 5100

aaggagagga tgcccacagg caggctgaag tggacccgtg tggaaggaac acttcaagac 5160

cactggccaa gagacttcct tcatttttat cccttcctgt ttttcgcctg tgctgaggat 5220

tcaccaccag tgtggggagc ctctgactgt aaaaggaaag ctgactctgg ctccttcgtg 5280

ttttctgtta agttggggcc taagtttttc tgccagcctt tgcggaggaa atgcagcttc 5340

cctggcacac tgcccggtca agggacgttg aatccattgc aatcagcagt tgaagatcca 5400

cactacaggc catgttctta aaataagtgg ctccaggact tccaagaggc agaaggggct 5460

tcagctggcc atgggcagga tccaagtgga gcttcatccc tgcagagcaa tgcctggctc 5520

tcagcggagg cagtctggag cagggcttct acccttttca gctcatagat tactttggaa 5580

tctgatgaac actgggagcc ttctccttag aagaacatac atatgcacga gaatgagaca 5640

attacataac attccttggg tttcacagat gctgaagcca atgacttgct tgaagcctgg 5700

aagagggtgt tgacctggag aatcctaaag gccaaaattt agagaaaatg gtgccagtaa 5760

atgaattcac aaggagaaat gcttccttgc caactcactt ctccagatta ttgagtgcat 5820

ctatctgttt gcttttggct tcacgcaatt ggccctcttc agttccgtac tctttcctaa 5880

ataccttgca agttttcctc ttctctcatg ccatttataa tatgctgacg cctgattaga 5940

ggaaggaagg actcagctgc acttgcactg ggggaagggc cacttacagg aagcctacct 6000

ggggctgagc tgggtctcac tgcctcctag acctaagggc cccagctttt ccctcatctc 6060

ttgttgcagc caccacttgt tctagatctg catccatatc tgtaaaatgg gggtgataat 6120

accttcccat aaagtcactg tgaggattaa atgtccggca caatatttga 6170

<210> 2

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gtgttgacct ggagaatc 18

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

caataatctg gagaagtgag t 21

Claims (10)

1. a kind of reagent for detecting colorectal cancer, which is characterized in that reagent detects the expression of LOC105374879 in sample.

2. reagent according to claim 1, which is characterized in that reagent uses sequencing technologies, nucleic acid hybridization technique or nucleic acid The expression of amplification technique detection LOC105374879.

3. reagent according to claim 1, which is characterized in that reagent uses sequencing technologies, probe hybridization technique, gene core The expression of chip technology or fluorescent quantitative PCR technique detection LOC105374879, it is preferred that quantitative fluorescent PCR nucleic acid amplification Primer sequence be SEQ ID NO.2 and SEQ ID NO.3.

4. reagent according to claim 2, which is characterized in that the nucleic acid amplification technologies are selected from one of the following or several Kind: polymerase chain reaction, reverse transcriptase polymerase chain reaction, the amplification of transcriptive intermediate, ligase chain reaction, strand displacement expand Increasing and the amplification based on nucleic acid sequence.

5. reagent according to claim 1, which is characterized in that it is cancerous tissue or blood that reagent, which detects sample,.

6. application of the reagent described in claim 1-5 any one in preparation diagnosis of colorectal carcinoma tool.

7. application according to claim 6, which is characterized in that colorectal cancer is colon cancer or the carcinoma of the rectum.

8. a kind of diagnosis of colorectal carcinoma reagent, which is characterized in that reagent uses sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification Technology or the method for immunoassays detect gene expression dose relevant to LOC105374879, and relevant gene is following gene In it is one or several: HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1, HEATR1, TMEM201, ADK, RANBP1, GUCA2A、CLDN23、CD79A、RAB27A、OTUD1、TMEM54、ABHD3、RIMKLA、USP2、CD244。

9. diagnostic reagent according to claim 8, relevant gene includes positive correlation gene or negative correlation gene, is positively correlated Gene is HSPA5, TDP1, COA7, C1QBP, ASNS, CCNE1, HEATR1, TMEM201, ADK, RANBP1, and negative correlation gene is CD244、USP2、RIMKLA、ABHD3、TMEM54、OTUD1、RAB27A、CD79A、CLDN23、GUCA2A。

10. application of the diagnostic reagent described in claim 8 or 9 any one in preparation diagnosis of colorectal carcinoma tool.