CN107012213A - The biomarker of colorectal cancer - Google Patents

The biomarker of colorectal cancer Download PDF

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CN107012213A
CN107012213A CN201710185973.0A CN201710185973A CN107012213A CN 107012213 A CN107012213 A CN 107012213A CN 201710185973 A CN201710185973 A CN 201710185973A CN 107012213 A CN107012213 A CN 107012213A
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ifitm1
hervs
cell
colorectal cancer
hescs
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付雨栋
王�华
龚鹏
周忠诚
戚峰
刘林
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Nankai University
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Abstract

The biomarker of colorectal cancer.It is related to application of the interferon-induced transmembrane protein 1 in endogenous retrovirus in suppressing colorectal cancer.IFITM1 function is studied with CRISPR/Cas9 technique construction IFITM1 KO hESCs.Research finds that the human embryo stem cell (hESCs) of IFITM1 missings is bred in cell, cells pluripotency, telomere length, is more or less the same in terms of telomerase activation with IFITM1 WT.Human endogenous retrovirus expressing viral is raised in IFITM1 KO hESCs, and it is expressed to express than Colorectal Carcinoma in cancer beside organism and raised.Detected by ChIP qPCR, it is found that H3K9me3 is enriched with HERVs sites in IFITM1 KO hESCs and decline.These as shown by data are in hESCs and colorectal cancer, and IFITM1 expressions express negatively correlated with HERVs, and IFITM1 suppresses HERVs expression by regulating and controlling epigenetic.

Description

The biomarker of colorectal cancer
Technical field：
The invention belongs to field of biomedicine technology, and in particular to the HERVs based on detection IFITM1 and its regulation and control is related The expression of gene is come the method that diagnoses colorectal cancer.
Background technology：
1. in recent years, multiple studies have shown that IFITM1 high expression in Several Kinds of Malignancy, such as IFITM1 cervix cancer, High expression in the cancer of the esophagus, oophoroma, the cancer of the brain, and the up-regulated expression and be considered as colorectal cancer molecule mark in colorectal cancer Will thing.More and more people pay close attention to its effect in the generation development of malignant tumour and tumour early stage are attacked.In colorectal cancer, It is only woven with IFITM1 expression, the activation energy of Wnt/ β-catenin signal paths in intestinal canal tumour forming process in tumor group Induce IFITM1 expression；In chronic myelogenous leukemia, it is low in high-risk group of expression, and low danger group expression is high, table Up to level, high patient has more preferable prognosis, therefore, it is believed that IFITM1 is probably patients with chronic myeloid leukemia prognosis Molecular marker.
2. human endogenous retrovirus (Human endogenous retroviruses, HERVs) can swivel base In gene, the genome that may be inserted into people, 8% ratio is accounted in human genome.Its quilt in embryo development procedure Activation, IFITM1 can protect embryo and reproduction cell from the infection of endogenous retrovirus virus and exogenous virus.ERVs Include long terminal repeat (LTRs), its expression can be suppressed by H3K9me3 histone modifications, and can pass through Increase genomic DNA methylation level to suppress HERVs expression.HERVs, which is overexpressed, can cause genomic instability, unconventionality expression HERVs is even associated with the generation of cancer.
3. proposing IFITM1 in forefathers' research as colorectal cancer molecular marker, found through overtesting in colorectal cancer Middle IFITM1 expression expresses negatively correlated with HERVs, is come in the present invention using IFITM1 and HERVs expression as double standards It is used as the mark of colorectal cancer.
The content of the invention：
It is an object of the invention to provide a kind of purposes of the IFITM1 and its HERVs of regulation and control in colorectal cancer mark. Specifically, the application the present invention relates to IFITM1 albumen and its HERVs of regulation and control in diagnosis of colorectal carcinoma kit.The present invention Implementation the diagnosis of more accurate colorectal cancer can be provided.
Technical scheme
A kind of albumen and its gene of regulation and control are that human endogenous retrovirus exists in preparation for detecting in subject The purposes in kit in the in-vitro method of colorectal cancer, the purposes includes：Detect the gene of the IFITM1 and its regulation and control i.e. Expression of the human endogenous retrovirus (HERVs) in the measure cell of subject is Colon and rectum biopsy test sample product；Will be described Measured value compared with reference value, IFITM1 is significantly lower than reference value apparently higher than reference value and HERVs, shows subject Colorectal cancer may be suffered from.
The reference value level is IFITM1 and HERVs related genes in normal cell i.e. Colon and rectum biopsy test sample product Level.The normal cell is the cell with determining cell same type in same subject.The normal cell is subject In with determining the cell of cell same type, the subject do not have cancer.The sample is known or suspected thin comprising tumour Born of the same parents.
1. the present invention be at least partially based on to the IFITM1 of high expression in people's Colon and rectum and in colorectal cancer tumour it is general All over the identification of the HERVs genes of low expression.The method of the present invention is useful for the detection of colorectal cancer.
2. diagnose colorectal cancer by the use of IFITM1 and HERVs as biomarker.
3. the presence for the colorectal cancer that methods described herein can be used in diagnosis subject.In some specific implementations In scheme, described method includes, and sample is obtained from subject, assesses IFITM1 and HERVs in the sample and exists and/or water It is flat, described presence and/or level are compared with the reference of one or more, for example, represent normal IFITM1 and HERVs water Level in flat control reference, such as normal cell from unaffected subject or from same individual.
4. sample：The present invention method some specific embodiments in, described sample be include known to or by The tumour cell of suspection, e.g. biopsy samples.In some specific embodiments, described sample is freezing, fixed And/or through infiltration processing, e.g. formalin fixes the sample of the sample or frost of FFPE (FFPE) in liquid nitrogen.
5. the method for detection：A. any of method in this area can be used to detect and/or quantify herein described Biomarker level.IFITM1 and HERVs mRNA (transcript) can be for example assessed using methods known in the art Level, such as Northern traces, RNA in situ hybridizations (RNA-ISH), rna expression determine, such as microarray analysis, RT- (for example utilizing random primer or widow T primers) is sequenced in PCR, RNA, deep sequencing (deep sequencing), clone, Northern traces, and amplification transcript, for example, utilize quantitative real-time polymerase chain reaction (qRT-PCR).B. have at some In body embodiment, the level of the protein of IFITM1 and HERVs codings can be detected.Commented using methods known in the art Estimate presence and/or the level of protein, such as using quantitative immunoassay method, such as EUSA (ELISAs), Immunoprecipitation, immunofluorescence, immunohistochemistry, enzyme immunoassay (EIA) (EIA), radiommunoassay (RIA) and western blot Analysis.C. in some specific embodiments, described method includes the reagent and sample for making optionally to be combined with biomarker Product are contacted, with biomarker described in evaluate sample, such as IFITM1 and HERVs transcripts/mRNA or protein (such as few core Thuja acid probe, antibody or its antigen-binding portion thereof) level.In some specific embodiments, described reagent is carried and can examined The mark of survey.The straight of (i.e. physical connection) is mutually coupled on labeled reagent, including by the reagent and detectable material Mark is connect, and indirect labelling is carried out by the reactivity of the reagent and detectable material.The example of detectable material Be it is known in the art, including chemiluminescent, fluorescence, it is radioactive or colour developing mark.Such as detectable material bag Include various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material and radioactive material.The example of suitable enzyme includes peppery Root peroxidase, alkaline phosphatase, beta galactosidase, or acetylcholinesterase；The example bag of suitable prosthetic group complexes Include streptavidin/biotin, and avidin/biotin；The example of suitable fluorescent material includes umbelliferone, glimmering Light element, fluorescein isothiocynate, rhodamine), dichlorotriazine ammonia fluorescein (dichlorotriazinylamine Fluorescein), dansyl Cl, quantum dot (quantum dots) or phycoerythrin；The example of luminescent material includes Rumi Promise；The example of bioluminescent material includes luciferase, fluorescein and aequorin；
The example of suitable radioactive substance includes125I,131I,35S or3H.In general, using antibody detection protein Matter.Antibody can be polyclonal antibody, more preferably monoclonal antibody.Complete antibody or its antigen-binding fragment can be applied (for example Fab or F (ab')2)。
Brief description of the drawings：
Fig. 1 is immunofluorescence figure and block diagram, proves IFITM1 in colorectal cancer by immunofluorescence and quantitative PCR experiment Really high expression in tumor locus and hESCs (two cell lines of WA26 and RUES2).
Fig. 2 is to include immunofluorescence figure (D, E), block diagram (G, I), gel electrophoresis figure (H), cellular morphology figure (C), exposure Picture (B, F, J).The IFITM1 that Fig. 2 checkings are built knocks out cell line, and examines cellular change after IFITM1 knockouts.Wherein A shows Show that CRISPR/Cas9 knocks out gene schematic diagram.B, D identification IFITM1 knock out cell line.C shows cellular morphology after IFITM1 knockouts It is unchanged.F shows that stem cell versatility is without significant change after IFITM1 knockouts.I, J show IFITM1 knock out after cell telomere without Significant change.G, H show Cell Telomerase Activity thing significant change after IFITM1 knockouts.
Fig. 3 is block diagram, intracellular HERVs expression after detection IFITM1 is knocked out.Wherein A shows HERVs structure, black Color arrow shows the substantially site of our designed quantitative PCR (the same quantitative PCR of ChIP-qPCR primers) primers.B is shown in In hESCs, HERVs expression rises (P15 generations) after IFITM1 is knocked out.C is by ChIP-qPCR description of tests in hESCs (RUES2) in, combinations of the H3K9me3 in HERVs sites declines (P15 generations) after IFITM1 is knocked out.
Fig. 4 is immunofluorescence figure and scatter diagram, intracellular DNA methylation situation of change after detection IFITM1 is knocked out.Pass through Immunofluorescence experiment, and count after cell fluorescence intensity it is known that 5mC/5hmC declines in IFIFM1-KO hESCs (i.e. DNA methylation declines), then can illustrate that IFITM1 methylates to regulate and control HERVs expression by regulating DNA.
Fig. 5 is block diagram, HERVs expression in detection patient samples.Illustrate the tumor locus HERVs in colorectal cancer Expression is lower than normal portions.
Fig. 6 is DNA methylation situation in immunofluorescence figure and scatter diagram, detection tumor sample.Illustrate in colorectal cancer, Tumor locus 5mC (DNA methylation degree is high) rise is known that by 5mC/5hmC ratio, DNA methylation can suppress HERVs expression, so IFITM1 can suppress HERVs expression by regulating DNA methylation.
Specific embodiment：
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention And without limiting the scope of the present invention.Unless otherwise described, implementation of the invention will use molecular biology and immunologic normal Rule technology, these are known to those skilled in the art.
Embodiment 1：
Detect expression (Fig. 1) of the IFITM1 in colorectal cancer and in hESCs
Materials and methods：
1. randomly select Patients with Colorectal Cancer, Patients with Colorectal Cancer frozen tissue section (including tumor locus and normal is taken Position), carry out immunofluorescence dyeing.
2. randomly selecting Patients with Colorectal Cancer, RNA is extracted, passes through its IFITM1 of quantitative PCR detection mRNA level in-site；And take HESCs cell lines (WA26 and RUES2) RNA, detects IFITM1 expressions.
3. result：IFITM1 tumor locus expression quantity in colorectal cancer is significantly higher than normal structure, and it is in hESCs Expressed in cell line high (control is used as using HEF cells).
Embodiment 2：
The hESCs cell lines (Fig. 2) knocked out using CRISPR/Cas9 technique constructions IFITM1
1. cell culture：Using (invitrogen) nutrient solutions of Essential 8 in 37 DEG C, 5%CO2Under the conditions of cultivate training HESCs cells are supported, it is necessary to change liquid daily.
2. build plasmid：Primer is designed according to the CDS region sequences of the NCBI IFITM1 albumen announced, passes through amplification, connection Etc. being inserted into px459 plasmids, compared through positive colony PCR identifications, sequencing, BLAST, as a result show px459-IFITM1 structures Build up work(.
3. cell transfecting and identification：HESCs cell lines are in the nutrient solutions of Essential 8, in 37 DEG C, 5%CO2Incubator Middle culture.Plasmid is gone in cell using consideration convey technology, cell is then cultivated.It is thin by drug screening and picking monoclonal Born of the same parents, then obtain IFITM1 by sequencing and western blot experiments and immunofluorescence experiment checking and knock out cell.
Embodiment 3：
Verify the influence (Fig. 2,3,4) to hESCs after IFITM1 knockouts
1. by western blot experiments cells pluripotency after IFITM1 is knocked out is detected not change；By quantitative PCR experiment and TRAP experiments detect cellular telomerase after IFITM1 is knocked out and not changed；Pass through quantitative PCR (T/S ratio) Experiment detects cell telomere length and not changed.
2. we are detected after IFITM1 knockouts by PCR experiment, cell extraction RNA is collected, detection knows intracellular HERVs is raised.Detect that the primer used is as follows：
3. tested (with primer in 2) by ChIP-qPCR, it has been found that after IFITM1 knockouts, H3K9me3 is in HERVs Site enrichment degree declines, because H3K9me3 has the effect for suppressing HERVs expression, therefore causes HERVs expression rises.
4. doing immunofluorescence (5mC and 5hmC) with generation cell in collecting 3, the fluorescence intensity of cell is counted, passes through 5mC/ 5hmC ratios are it is recognised that cell DNA methylation is reduced after IFITM1 is knocked out, and DNA methylation can suppress HERVs Expression.
5 conclusions：IFITM1 can suppress HERVs expression by way of adjusting epigenetic.
Embodiment 4：
Verify the expression (Fig. 5,6) of HERVs in Colon and rectum
1. taking Patients with Colorectal Cancer tumor tissues and normal structure, RNA is then extracted, is found by quantitative PCR detection, (IFITM1 expression is high) HERVs expression is lower than normal structure (IFITM1 expression is low) in tumor tissues.
2. after being cut into slices after tissue paraffin is embedded, immunofluorescence dyeing (5mC and 5hmC) counts the fluorescence intensity of cell, By 5mC/5hmC ratios it is recognised that (IFITM1 expression is high) DNA methylation degree is high in tumor tissues.
3. conclusion：With in hESCs, IFITM1 can suppress HERVs expression by way of adjusting epigenetic, And determine that tumor tissues IFITM1 expression is high in Colon and rectum, and HERVs expression reductions.

Claims (5)

1. a kind of biomarker of colorectal cancer, is related to the gene i.e. human endogenous retrovirus of albumen and its regulation and control in system The purposes in the kit in the in-vitro method that there is colorectal cancer in detection subject is ready for use on, the purposes includes：Detection should IFITM1 and its gene of regulation and control are measure cell i.e. Colon and rectum biopsy of the human endogenous retrovirus (HERVs) in subject Expression in test sample product；Described measured value is compared with reference value, IFITM1 is apparently higher than reference value and HERVs is obvious Less than reference value, show that subject may suffer from colorectal cancer.
2. purposes according to claim 1, wherein the reference value level is IFITM1 and HERVs related genes normal Cell is the level in Colon and rectum biopsy test sample product.
3. purposes according to claim 2, wherein the normal cell be in same subject with to determine cell mutually similar The cell of type.
4. purposes according to claim 2, wherein the normal cell is with determining cell same type in subject Cell, the subject does not have cancer.
5. purposes according to claim 1, wherein the sample is known or suspected including tumour cell.
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US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
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US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
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