CN106701902A

CN106701902A - FOXR2 gene and application of expression product to diagnosis and treatment of liver cancer

Info

Publication number: CN106701902A
Application number: CN201510791601.3A
Authority: CN
Inventors: 李砚东; 高勇; 王晓
Original assignee: Shanghai East Hospital
Current assignee: Shanghai East Hospital
Priority date: 2015-11-17
Filing date: 2015-11-17
Publication date: 2017-05-24
Anticipated expiration: 2035-11-17
Also published as: CN106701902B

Abstract

The invention discloses an FOXR2 gene and application of an expression product to diagnosis and treatment of the liver cancer. The FOXR2 gene is used to prepare a product for diagnosis and treatment of the liver cancer. The FOXR2 gene and the expression product of the FOXR2 gene can be used as a specific marker gene in the diagnosis of the liver cancer. The FOXR2 gene and the expression product of the FOXR2 gene can also be used as a target gene used for preparing a medicine for treating the cancers, particularly the liver cancer. A novel way is provided for the treatment of the cancer.

Description

The application of FOXR2 genes and expression product in diagnosing cancer of liver with treatment

Technical field

The invention belongs to gene therapy and diagnostic techniques field, more particularly to a kind of FOXR2 genes and Application of its expression product in the diagnosis and treatment of liver cancer.

Background technology

Hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is the common malignant tumour of China One of, in the world, it is the most common the fifth-largest tumour in the world, is to cause tumour related Dead the third-largest reason.And in China, the death rate of liver cancer is higher be only second to lung cancer, cancer of the esophagus and Occupy the 3rd.Due to lacking sensitive and special method of early diagnosis, clinically find to be generally late period, General curative effect is poor, the death rate is high, and China dies from the people of liver cancer about 110,000 every year, accounts for whole world PLC mortality The 45% of number.Liver cancer initial symptoms are not obvious, late period be mainly shown as hepatodynia, it is weak, become thin, The symptoms such as jaundice, ascites.Due to onset concealment, quickly grow, Most patients have reached when making a definite diagnosis To Locally Advanced or there is DISTANT METASTASES IN, the Natural Survival phase is very short.The early diagnosis of liver cancer is for having Effect treatment and long term survival are most important.

It has been generally acknowledged that cancer embryo and glycoprotein antigen, enzyme and isodynamic enzyme, cell factor, gene are used as Primary liver cancer markers.At present, in China to the etiologic diagnosis of liver cancer detecting Serum AFP (first tire Albumen) based on.The usual normal values of AFP are below 20 μ g/L.All AFP>500 μ g/L continue 1 month, Or AFP>200 μ g/L continue 2 months and without hepatopathy frequently flooded area, can exclude gestation and gonad embryonal carcinoma Person, answers strong suspicion liver cancer, can be diagnosed by Medical Imaging and be made a definite diagnosis.In hepatocarcinoma early diagnosis In development history, AFP has started the beginning of liver cancer immunity diagnosis, makes it possible hepatocarcinoma early diagnosis, And started the upsurge of tumor markers research.But hepatocarcinoma early diagnosis are examined from carrying out examination with AFP It is disconnected to be developed so far, fail to make a breakthrough again, i.e., cure rate or 5 years survival rates can not be enable to carry Height, or even occur that AFP is normal in inspection and tumour is to the phenomenon of liver cancer early stage.Although the sensitivity of AFP Property and specificity it is unsatisfactory, but because the specificity there is presently no other tumor markerses can Compared favourably with AFP, therefore, AFP is still most widely used hepatic carcinoma mark in current global range Will thing.By the end of current, the early diagnosis of liver cancer and the molecule parting of personalized treatment is instructed to diagnose still It is great challenge that we face, and finds sensitivity and specificity new tumor markers higher, It is the key for improving hepatocarcinoma early diagnosis level.

The therapeutic effect that recent two decades carry out liver cancer is obtained compared with much progress, and the raising of curative effect mainly has benefited from examining Cut off the water supply the development of flat raising, particularly Imaging Technology so that some cases can in early detection, Effected a radical cure.But for most of mid and late liver cancers, how to improve curative effect and come for every country Say all be problem.Therefore in addition to the conventional therapies such as operation, radiotherapy, intervention, the treatment means of liver cancer Comparatively compare many, including the office such as frost free heat exchanger, focusing ultrasound thermal therapy in RF ablation, knurl Portion's treatment means, formed at present " based on surgical intervention, supplemented by local treatment, multidisciplinary synthesis Therapeutic mode ".Wherein, the chemotherapeutics of liver cancer continues to use the medicine of other tumours substantially, for Property it is poor, general curative effect is not good, and in the urgent need to finding new action target spot, research and development liver cancer is special Different novel drugs, to improve the specificity and validity of chemotherapy.

The content of the invention

The invention discloses the application of a kind of people FOXR2 genes and its expression product, for preparing liver cancer Diagnosis and treatment product.

The first aspect of the present invention provides the purposes of a kind of FOXR2 genes or FOXR2 albumen, for making The reagent or kit of standby detection liver cancer；

In another preference, described kit includes：Quantitative inspection is carried out to FOXR2 albumen or mRNA The reagent of survey and corresponding label or specification.

In another preference, described reagent includes FOXR2 specific primers, specific antibody, probe And/or chip.

In another preference, above-mentioned reagent includes detection chip, including nucleic acid chip and protein Chip.

In another preference, described nucleic acid chip includes the liver cancer phase of substrate and point sample on substrate The specific oligonucleotide probe of correlation gene, the specific oligonucleotide of described liver cancer related gene is visited Pin includes the probe specifically bound with FOXR2 genes or mRNA.

In another preference, described protein-chip includes the liver cancer of substrate and point sample on substrate The specific antibody of GAP-associated protein GAP, the specific antibody of described hepatoma associated protein includes anti-FOXR2 The specific antibody of albumen.

In another preference, described FOXR2 albumen includes fusion protein and non pregnant women.

A kind of the second aspect of the present invention, there is provided diagnostic kit for detecting liver cancer, it is described Kit contains a container, the detection reagent containing detection FOXR2 albumen or mRNA in the container； And label or specification, the label or specification indicate the kit for detecting liver cancer.

In another preference, herein below is indicated in described label or specification：

When the relative mrna expression amounts and the FOXR2 of cancer beside organism with reference to albumen of the FOXR2 of detection object The ratio between relative mrna expression amount with reference to albumen >=1.5, the then probability for pointing out the detection object to suffer from liver cancer Higher than general population.

It is described to include beta-actin with reference to albumen in another preference.

In another preference, described detection reagent includes：Specific primer, specific antibody, Probe and/or chip.

In another preference, described kit is used to detect human liver tissue sample or blood sample.

In another preference, described hepatic tissue sample includes liver cancer tissue, cancer beside organism or policy Hepatic tissue.

A kind of the third aspect of the present invention, there is provided the use of FOXR2 albumen, FOXR2 genes or its inhibitor On the way, the medicine of growth of cancer cells or propagation is suppressed for preparing, or for preparing the medicine for the treatment of liver cancer.

In another preference, described inhibitor includes：The antibody of FOXR2, the antisense of FOXR2 nucleic acid The activity inhibitor of RNA, siRNA, shRNA and FOXR2.

In another preference, described inhibitor includes the si-1 or si-2 of FOXR2 genes, preferably Ground, such as SEQ ID NO.:9-10 and/or SEQ ID NO.:Shown in 11-12.

The fourth aspect of the present invention, there is provided the suppression liver cancer cell growth or propagation of a kind of external non-therapeutic Method, including step：In the presence of FOXR2 albumen or its inhibitor, HCC is cultivated, so as to press down Liver cancer cell growth processed or propagation.

In another preference, described method includes pressing down to addition FOXR2 in the cultivating system of HCC Preparation, so as to suppress liver cancer cell growth or propagation.

The fifth aspect of the present invention, there is provided a kind of method of the candidate compound of screening treatment liver cancer, bag Include step：

In (a) test group, test compound is added in the cultivating system of cell, and observe the survey The expression quantity and/or activity of FOXR2 in the cell of examination group；In control group, in the culture of same cell Without test compound in system, and observe FOXR2 in the cell of control group expression quantity and/ Or activity；

Wherein, if the expression quantity of the FOXR2 of cell and/or activity are less than control group in test group, just Show that the test compound is the time of the treatment liver cancer for having inhibitory action to the expression of FOXR2 and/or activity Select compound.

In another preference, described HCC includes various conventional SMMC-7721s, for example HCC-LM3, WRL-68, YY-8103, Huh7, Hep3B, or L02.

In another preference, methods described also includes step：

B () further tests it to liver cancer cell growth for the candidate compound obtained in step (a) Or the inhibitory action of propagation.

In another preference, the step (b) includes step：In test group, the culture of cancer cell Test compound is added in system, and observes the quantity and/or growing state of cancer cell；In control group, Without test compound in the cultivating system of cancer cell, and observe quantity and/or the growth of cancer cell Situation；Wherein, if the quantity or the speed of growth of cancer cell are less than control group in test group, indicate that The test compound is the candidate compound of the treatment liver cancer for having inhibitory action to the growth of cancer cell or propagation Thing.

The sixth aspect of the present invention, additionally provides a kind of method for suppressing or treating liver cancer, including step： The purposes of the FOXR2 inhibitor of safe and effective amount is applied to the object (mammal) for needing to treat.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (strictly according to the facts Apply example) in specifically describe each technical characteristic between can be combined with each other, so as to constitute new or preferred Technical scheme.As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Figure 1A and B be embodiment 1 in real-time quantitative PCR detect FOXR2 genes in 41 hepatocarcinoma patient cancers Expression schematic diagram in tissue and cancer beside organism, wherein, " N " refers to cancer beside organism, and " C " refers to Liver cancer tissue.FOXR2 gene expression amounts are higher than during result shows 28 cancerous tissues of (68.3%) patient Corresponding cancer beside organism.

Fig. 1 C show that SABC means detect FOXR2 genes group by human liver cancer tissue sample and cancer The differential expression knitted, wherein, " N " refers to cancer beside organism, and " C " refers to liver cancer tissue, the cancer group of patient Middle FOXR2 gene expression amounts are knitted higher than corresponding cancer beside organism.

Fig. 2A shows the cDNA sequence of FOXR2 genes.

Fig. 2 B show the amino acid sequence of FOXR2 coded by said gene albumen.

Fig. 3 A show mistakes of the FOXR2 in hepatoma cell strain Hep3B, Huh7, YY-8103 and L02 Expression, illustrates FOXR2 successful expressions in carrier for expression of eukaryon.

Fig. 3 B show the FOXR2 albumen of overexpression enhance hepatoma cell strain Hep3B, Huh7, The growing ability of YY-8103 and L02.

Fig. 3 C show that the FOXR2 albumen of overexpression promotes the propagation of L02.

Fig. 4 A show that artificial synthesized siRNA effectively disturbs the expression of FOXR2 genes.

Fig. 4 B show the expression inhibiting HCC WRL68 of the method silence FOXR2 disturbed by RNA With the growth of HCC-LM3.

Fig. 5 shows the expression inhibiting of the method silence FOXR2 disturbed by RNA HCC The clonality of WRL68 and HCC-LM3 in soft agar, further relates to FOXR2 and promotes HCC Pernicious sign.

Fig. 6 A show that the FOXR2 genes of overexpression promote the thin YY-8103 of liver cancer to be formed in nude mice by subcutaneous The ability of tumour.FOXR2 overexpression group is all bigger than control group and heavy in volume and tumor weight, Two groups have statistical significance simultaneously.

Fig. 6 B show that silence FOXR2 gene expression inhibition HCCs WRL68 is formed in nude mice by subcutaneous The ability of tumour.In volume and tumor weight FOXR2 silences expression group all it is smaller than control group and Gently, while two groups have statistical significance.

Specific embodiment

The present inventor's in-depth study by extensive, first it was unexpectedly observed that FOXR2 is in cancerous tissue Low expression, and height is expressed in cancer beside organism and normal structure, therefore FOXR2 can be detected as liver cancer Mark be used for detect or complementary detection liver cancer.Additionally, the inhibitor of FOXR2 can suppress cancer cell The growth of (especially HCC).The present invention is completed on this basis.

FOXR2 albumen and polynucleotides

Jaw frame (Forkheadbox, Fox) protein family is that a class DNA lands have winged-helix knot The transcription factor of structure, there is 19 subtribes at present, and they not only activate or suppress purpose by combining DNA The transcriptional activity of gene, some can also directly combine with heterochromatin and participate in its reconstruct, cooperate with other to transcribe The factor participates in transcriptional regulatory.Fox albumen is in embryonic development, cell cycle regulating, carbohydrate and lipid generation Thank, play a role during the various biological such as biological aging and immunological regulation, sent out in tumour Also played an important role during exhibition.FOXR2 is important a member of FOX protein families.So far, it is relevant The report article of FOXR2 gene functions is few.The present invention elaborates expression of the FOXR2 in liver cancer first The propagation of situation and the research meanses proof FOXR2 promotion HCCs for passing through cell biology, is one Potential oncogene.

The purpose of the present invention is to disclose the application of a kind of people FOXR2 genes and its expression product, for making Standby diagnosing cancer of liver and the product for the treatment of.FOXR2 genes of the invention and its expression product, can be used as diagnosis The specificity marker gene of liver cancer, makes diagnosing cancer of liver more accurate, quick；FOXR2 genes of the invention And its expression product, it is alternatively arranged as preparing the molecular medicine for the treatment of liver cancer, there is provided new liver cancer treatment is on the way Footpath.Therefore, this area can be used for the GAP-associated protein GAP of diagnosing cancer of liver in the urgent need to developing, in order to effectively Suppress the growth of HCC, this area can be used to suppress the medicine of liver cancer cell growth in the urgent need to exploitation Thing, to improve the specificity and validity of chemotherapy.

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " FOXR2 albumen " can be mutual Change and use, refer to referred to as FOXR2).It should be understood that the term also including FOXR2 active fragment and Derivative.

In the present invention, " gene of the present invention ", " polynucleotides of the present invention " refer to coding FOXR2 albumen Or the nucleotide sequence of its active fragment and derivative, including justice and antisensenucleic acids.FOXR2 genes are determined Positioned at cell chromosome 6q15, cDNA total lengths are 786bp, 261 albumen of amino acid of encoding full leng.

In the present invention, term " FOXR2 albumen ", " FOXR2 polypeptides " or " liver cancer marker FOXR2 " It is used interchangeably, all refers to albumen or polypeptide with people's albumen FOXR2 amino acid sequences.

FOXR2, also known as SRSF12 (serine/arginine-rich splicing factor 12, it is rich Containing serine and arginic shear factor 12), because being originally found as the inhibiting factor and molecule of SR Measure and gained the name for 35kD.SR albumen is that a class is rich in serine and arginic with RRM RNA combinations The shear factor of domain, falls to include with regulation and control eukaryotic pre-mRNA transcripts alternative splicing Function that is sub and splicing extron.In vivo study finds that FOXR2 other SR albumen of antagonism and can swash Alternative splicing (Alison E, the et al.2001. of the least significant ends of pre-mRNA 5 ' of live adenovirus E1A THE JOURNAL OF BIOLOGICAL CHEMISTRY)。

The cDNA sequence of FOXR2 genes such as SEQ ID NO.:Shown in 1；Genbank accession number 135295, The amino acid sequence NP_542781.3 of the cDNA sequence CCDS47459.1 of FOXR2 and its coding.

The amino acid sequence of FOXR2 coded by said gene albumen such as SEQ ID NO.:Shown in 2.

As used herein, " separation " refers to that material is separated (if day from its primal environment Right material, primal environment is natural surroundings).Such as many nucleosides under the native state in active somatic cell Sour not isolated and purified with polypeptide but same polynucleotides or polypeptide are same such as from native state to be deposited Other materials in separate, then isolate and purify.

As used herein, " the FOXR2 albumen or polypeptide of separation " refers to that FOXR2 albumen is substantially free of Natural relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use The purified technology of protein purifying FOXR2 albumen of standard.Substantially pure polypeptide is in non-reduced polyacrylamide Single master tape can be produced on amine gel.In the present invention, FOXR2 albumen includes fusion protein and non-melts Hop protein.

Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide. Polypeptide of the invention may also include or not include the methionine residues of starting.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, base Because of group DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be compiled Code chain or noncoding strand.

The polynucleotides for encoding the mature polypeptide of FOXR2 include：The coded sequence of encoding mature polypeptide； The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence of mature polypeptide is (and optional Additional coding sequence) and non-coding sequence.Term " polynucleotides of coded polypeptide " can be included The polynucleotides for encoding this polypeptide, or the multinuclear for also including additional code and/or non-coding sequence Thuja acid.

The invention further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid with the present invention The polypeptide of sequence or the fragment of polypeptide, analogs and derivatives.The variant of this polynucleotides can be day The variant that the allelic variant or non-natural for so occurring occur.These nucleotide variants include that substitution becomes Allosome, Deletion variants and insert variation.As known in the art, allelic variant is a multinuclear The alternative forms of thuja acid, it is probably substitution, missing or the insertion of one or more nucleotides, but will not From the function of substantially changing its coded polypeptide.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization, including the just nucleic acid piece with antisense Section.As used herein, the length of " nucleic acid fragment " at least contains 15 nucleotides, preferably at least 30 Individual nucleotides, more preferably at least 50 nucleotides, more than preferably at least 100 nucleotides.Nucleic acid Fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or separate many of coding FOXR2 albumen Nucleotides.

People FOXR2 nucleotides full length sequence of the invention or its fragment can generally use PCR TRAPs, weight Group method or artificial synthesized method are obtained.For PCR TRAPs, can be according to published relevant nucleotides Sequence, especially open reading frame sequence design primer, and with commercially available cDNA storehouses or by this area CDNA storehouses prepared by conventional method known to technical staff obtain relevant sequence as template, amplification. When sequence is more long, it is often necessary to carry out twice or repeatedly PCR amplifications, then will amplify for each time again Fragment is stitched together by proper order.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This Carrier is typically cloned into, then is transferred to cell, then the host by conventional method from after propagation is thin Isolated relevant sequence in born of the same parents.

Additionally, can also synthesize relevant sequence with artificial synthesized method, when especially fragment length is shorter. Generally, by first synthesizing multiple small fragments, being then attached again can obtain sequence fragment very long.

It is optimized for obtaining gene of the invention using the method for round pcr DNA amplification/RNA.For The primer of PCR can be properly selected according to the sequence information of invention disclosed herein, and available conventional Method synthesizes.Can be with conventional method as separated by gel electrophoresis and purifying the DNA/RNA fragments for expanding.

The present invention also relates to the carrier comprising polynucleotides of the invention, and with carrier of the invention or The host cell that FOXR2 albumen coded sequences are produced through genetic engineering, and produce this to send out through recombinant technique The method of the bright polypeptide.

By conventional recombinant DNA technology, can be used to express using polynucleotide sequence of the invention or Produce the FOXR2 albumen of restructuring.In general there are following steps：

(1) the polynucleotides (or variant) of encoding human FOXR2 albumen of the invention, or with containing should The recombinant expression carrier conversion of polynucleotides or suitable host cell of transduceing；

(2) host cell that is cultivated in suitable culture medium；

(3) separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used to build the DNA sequences encodings of FOXR2 containing people and conjunction The expression vector of suitable transcription/translation control signal.These methods include recombinant DNA technology in vi, DNA Synthetic technology, In vivo recombination technology etc..Described DNA sequence dna can be effectively connected to suitable in expression vector When in promoter, to instruct mRNA to synthesize.Ribosomes of the expression vector also including translation initiation is combined Site and transcription terminator.

Additionally, expression vector preferably includes one or more selected markers, to provide for selecting Select the phenotypic character of the host cell of conversion, such as the dihyrofolate reductase of eukaryotic culture, new mould Plain resistance and green fluorescent protein (GFP), or it is anti-for the tetracycline or ampicillin of Escherichia coli Property.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, Ke Yiyong In appropriate host cell is converted, marking protein is allowed it to.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast is thin Born of the same parents；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, strepto- The bacterial cell of Pseudomonas；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9； Zooblast of CHO, COS or 293 cells etc..

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.When When host is prokaryotes such as Escherichia coli, the competent cell that can absorb DNA can be after exponential phase of growth Harvest, use CaCl₂Method treatment, step used is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also be carried out with the method for electroporation.When host is eucaryote, can From following DNA transfection methods：Calcium phosphate precipitation, conventional mechanical methods such as microinjection, electricity Perforation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention. According to host cell used, culture medium used may be selected from various conventional mediums in culture.It is being suitable to Cultivated under conditions of host cell growth.After host cell growth is to appropriate cell density, use Suitable method (such as temperature transition or chemical induction) induces the promoter of selection, and cell is further cultured for into one section Time.

Recombinant polypeptide in the above methods can be expressed or be secreted into the cell or on cell membrane It is extracellular.If desired, its physics, chemistry and other characteristics can be utilized to pass through various separation methods Separate the albumen with purification of Recombinant.These methods are well-known to those skilled in the art.These methods Example is included but is not limited to：Conventional renaturation process, processed with protein precipitant (salting-out method), from The heart, the broken bacterium of infiltration, super treatment, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion The knot of displacement chromatography, high performance liquid chroma- tography (HPLC) and other various LC technologies and these methods Close.

Antibody

There is specific polyclonal antibody and monoclonal antibody present invention additionally comprises to people's FOXR2 albumen, Especially monoclonal antibody.Here, " specificity " refers to that antibody can be incorporated into people's FOXR2 gene outcomes Or fragment.It is preferred that referring to that those can be combined but nonrecognition and combination with people FOXR2 gene outcomes or fragment In the antibody of other non related antigen molecules.Antibody of the invention can be by those skilled in that art The various technologies known are prepared.

The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetence Antibody fragment, such as Fab ' or (Fab)₂Fragment；Heavy chain of antibody；Antibody light chain；It is genetically engineered Single Chain Fv Molecule A；Or chimeric antibody.

The antibody of anti-human FOXR2 albumen can be used in immunohistochemistry technology, in detection biopsy specimen People's FOXR2 albumen.

Inhibitor and pharmaceutical composition

Using albumen of the present invention, by various conventional screening assays, can filter out and occur with FOXR2 albumen The material of interaction, especially inhibitor etc..

The inhibitor (including antibody, antisensenucleic acids and other inhibitor) of FOXR2 albumen of the present invention, when When (administration) is administered in treatment, expression and/or the activity of FOXR2 albumen can be suppressed, and then press down The growth of HCC processed or propagation.Generally, can by these inhibitor be formulated in it is nontoxic, inert and In pharmaceutically acceptable aqueous carrier medium, wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, Although pH value can be varied from the property for being formulated material and illness to be treated.Prepare Pharmaceutical composition can be administered by conventional route, including (but being not limited to)：Stomach enteral, In knurl, intramuscular, intraperitoneal, intravenous, subcutaneous, intracutaneous or local administration.

Can be used for inhibitor of the invention includes：The antibody of FOXR2, the antisense RNA of FOXR2 nucleic acid, The activity inhibitor of siRNA, shRNA and FOXR2.Wherein, typical FOXR2 inhibitor is siRNA And shRNA.

Can be used for siRNA of the invention includes various siRNA with FOXR2 as target spot, art technology Personnel can carry out conventional screening according to the target spot, wherein, a kind of preferred siRNA includes such as SEQ ID NO:9 (positive-sense strands) and SEQ ID NO.:Sequence (si-1) shown in 10 (antisense strands)；Or preferred siRNA Including such as SEQ ID NO:11 (positive-sense strands) and SEQ ID NO.:Sequence (si-2) shown in 12 (antisense strands).

Typically, using FOXR2 genes as the target spot for preparing cancer treatment drug technical scheme include with Lower scheme：

1. chemical synthesis double stranded ribonucleic acid molecule, its sequence-specific is directed to FOXR2 gene orders, profit It is delivered in HCC disturb the expression of FOXR2 genes with liposome, observes soft-agar cloning shape Into the change of the characteristics of cell biology such as ability, cell propagation.Can be set using the conventional method in this area Meter and synthesis specificity are directed to the nucleotide sequence (such as siRNA) of FOXR2.

2. utilize various carriers, including DNA vector, slow virus carrier to disturb the expression of FOXR2 genes, Reach in vivo interference FOXR2 genes effect, detect they to nude mice by subcutaneous control curative effect into knurl body Really, so as to realize suppressing the purpose of Hepatocarcinoma Proliferation.

3. polypeptide, the monoclonal antibody for being capable of specificity suppression FOXR2 gene delivery activity are obtained, is reached Suppress the purpose of FOXR2 activity, so that reality suppresses the purpose of propagation in HCC body.

Present invention also offers a kind of pharmaceutical composition, it contains the FOXR2 eggs of the present invention of safe and effective amount White or its inhibitor and pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but not limiting In)：Salt solution, buffer solution, glucose, water, glycerine, ethanol, and combinations thereof.Pharmaceutical preparation should be with Administering mode matches.Pharmaceutical composition of the invention can be made into injection form, for example, use physiology salt Water or the aqueous solution containing glucose and other assistant agents are prepared by conventional method.Such as tablet and glue The pharmaceutical composition of capsule etc, can be prepared by conventional method.Pharmaceutical composition for example injection, solution, Tablet and capsule are preferably aseptically manufactured.The dosage of active component is therapeutically effective amount, for example often Its mg/kg body weight of about 1 microgram -10.

Detection method and kit

The invention further relates to the diagnosis examination of quantitative and detection and localization people FOXR2 protein levels or mRNA level in-site Proved recipe method.These experiments are known in the art.The people's FOXR2 protein levels detected in experiment, Can be used for diagnosing liver cancer.

A kind of method in detection sample with the presence or absence of FOXR2 albumen is using the specificity of FOXR2 albumen Antibody is detected that it includes：Sample is contacted with FOXR2 protein specific antibodies；See whether shape Into antibody complex, form antibody complex and mean that in sample there is FOXR2 albumen.

FOXR2 albumen or its polynucleotides can be used for the diagnosis and treatment of FOXR2 protein related diseases.This Part or all of the polynucleotides of invention can be fixed on microarray or DNA chip as probe, used The Differential expression analysis of gene and gene diagnosis in tissue is analyzed.The antibody of anti-FOXR2 can be fixed on On protein-chip, for the FOXR2 albumen in quantitative determination sample.

Present invention also offers a kind of kit for detecting liver cancer, it contains drawing for specific amplification FOXR2 Thing pair and/or FOXR2 specific antibodies, preferred primer such as SEQ ID NO.:5 and SEQ ID NO.: Shown in 6.

Screening technique

Method present invention also offers drug screening is carried out based on FOXR2.A kind of method is first to screen Influence (promotion) FOXR2 expression or the compound of activity, then further test the compound for filtering out Its rejection to HCC.A kind of screening technique can be based on the expression of the mRNA of FOXR2.

Wherein, representational cancer cell includes (but being not limited to)：HCC.

Universal method：

(1) acquisition of clinical tissue sample

Liver cancer and cancer beside organism take from the liver cancer patient of operative treatment, are signed with patient before sample is obtained Informed Consent Form.The liver of surgery excision once in vitro, rapid excised tumor primary tumor and surrounding 5cm with Outer cancer beside organism, it is quick-frozen and move to -80 DEG C of Refrigerator stores in input liquid nitrogen, it is stored in liquid nitrogen during transport. Cancer makes last diagnostic with cancer beside organism by pathologist.Sample is according to Edmondson grade scales point It is I-III grades.

(2) tissue and cell RNA are extracted

Using TRIzol Reagent (Invitrogen) reagent extracting RNA, concrete operations are as follows：

1) vessel such as mortar, stone roller pestle and homogenizer are cleaned, and use ddH again respectively₂O and DEPC H₂O is rinsed, so Dried in 180 DEG C of baking ovens afterwards about 4 hours, to remove RNase；

2) add appropriate liquid nitrogen to be allowed to precooling in mortar, tissue is taken out rapidly from liquid nitrogen, cut about 50-100mg sizes, the grind into powder in mortar；

3) in ground tissue powder completely being moved into the EP pipes without RNase as far as possible with curet, EP Pipe is added in advance appropriate volume (1ml) TRIzol reagents, fully homogenate；

4) room temperature is placed 5 minutes, in proportion to addition chloroform (200 μ l/1ml TRIzol) in centrifuge tube, Rapid acutely vibration 15 seconds, are stored at room temperature 2-3 minutes, 4 DEG C, are centrifuged 15 minutes under the conditions of 12000 × g；

5) upper strata aqueous phase is transferred to as much as possible in the new EP pipes without RNase, is added isometric different Propyl alcohol, overturns and mixes 5 times, is stored at room temperature 10 minutes, 4 DEG C, is centrifuged 10 minutes under the conditions of 12000 × g, Now visible RNA precipitate；

6) supernatant is outwelled, adds 75% ethanol (1ml/1ml TRIzol), mixed, wash RNA, 4 DEG C of centrifugation, is centrifuged 5 minutes under the conditions of 7500 × g；

7) supernatant is abandoned, residual ethanol is eliminated as far as possible, precipitation spontaneously dries 5-10min, and (attention has been sure not White drying)；Add 30-50 μ l DEPC H₂O, pressure-vaccum several times, dissolves RNA precipitate；

8) ELIASA determines RNA concentration and purity OD 260/280 (1.8-2.0)；Gel electrophoresis observation has Without degraded, -80 DEG C of preservations.

Cell line RNA is extracted, the cell in growth period of taking the logarithm, and nutrient solution is drawn, according to the area of culture dish Add TRIzol reagents (the 1ml TRIzol/10cm of respective amount²) cell lysis, piping and druming several times, will The cell being cleaved is collected into the EP pipes without RNase, and remaining is according to above-mentioned steps 4) -8) complete chloroform - isopropanol method isolates and purifies RNA.

(3) reverse transcription of RNA

With M-MLV Reverse Transcriptase (Promega) reverse transcription, operate as follows：

1) following components is added in the EP pipes of nuclease free：

It is placed in PCR instrument, 70 DEG C, 5 minutes, immediately after in cooled on ice 5min.

2) following components is added in above-mentioned system：

After gently mixing, it is placed in PCR instrument, 37 DEG C, 60min.

The cDNA that reverse is obtained is placed in 4 DEG C of preservations.

(4) real-time quantitative PCR

Real-time quantitative PCR reaction is usedPremix Ex Taq^TM(Perfect Real Time) is tried The reaction system of agent box (TaKaRa Biotechnology Co., Ltd. Dalian, China), utilizes Thermal Cycler Dice^TMReal Time System (TP800 real-time fluorescence quantitative PCR instrument, TaKaRa) Operated.The amplified production length of quantitative PCR (can the most properly be extended to 80bp-150bp 300bp)。

Reaction system is as follows：

Reaction condition：

Solubility curve analytical procedure：

95℃ 15sec

60℃ 30sec

95℃ 15sec

Dissociation time is 4sec.

The default value that autofluorescent background signal and threshold value are set using instrument, each PCR reactions can be automatic after terminating Generation, Ct values represent that the fluorescence signal in each reaction tube reaches given threshold (the 10 of Baseline fluorescence intensity The period experienced when again)；Genes of interest FOXR2 each template does 3 multiple pipes, the Ct values for obtaining Average；The Ct average values of FOXR2 genes subtract the reference gene (β-actin) of corresponding template Ct average values, obtain Δ Ct.The Δ Ct of liver cancer group subtracts the Δ Ct of corresponding adjacent tissues, obtains Δ Δ Ct Value, the multiple proportion of the FOXR2 genes in liver cancer group and cancer side group uses 2^-ΔΔCtRepresent.

(5) construction of eukaryotic expression vector

1) template：The cDNA library of people's liver immortalized cells L02.

2) selection of carrier for expression of eukaryon：pcDNA^TM3.1/myc-His (-) A, 5522nucleotides.

3) according to FOXR2mRNA (NM_198451.3) sequence, with reference to expression vector pcDNA^TM The restriction enzyme site design primer of 3.1/myc-His (-) A, primer sequence is Forward: 5-ccaccATGGACTTAAAACTAAAAGACTG(SEQ ID NO.:3)；Reverse: 5-gttcGGTACCAAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO.:4).Wherein in reverse primer The terminator codon of FOXR2 is removed so that c-myc and 6xHis labels on the C-terminal band of FOXR2. Using high fidelity DNA polymerase PrimeSTAR^TMHS DNA Polymerase (TaKaRa), with L02 CDNA is template amplification gene FOXR2 total length opening code-reading frames, and 50 μ l overall reaction system compositions are as follows：

Using two-step PCR (95 DEG C, 10sec；60 DEG C, 90sec), expand 35 circulations.PCR (gel purification kit is reclaimed in primer size about 1.0kb, 1% agarose gel electrophoresis identification size, rubber tapping： MACHEREY-NAGEL the PCR primer of clip size) is met.

4) EcoRV, KpnI (TaKaRa Biotechnology Inc.Dalian, China) double digestion is returned Receive PCR primer and vector plasmid pcDNA^TM3.1/myc-His (-) A, endonuclease reaction system is as follows：

37 DEG C of endonuclease reactions 1 hour；Digestion products are reclaimed in rubber tapping.

5) connect：The PCR primer that digestion is reclaimed is with carrier according to mole ratio (4:1) ratio mixing, DNA Connection enzyme system link, also includes (the TaKaRa Code of 2.5 4 × Solution of μ l I in system：D102A), ddH₂O polishings are to 10 μ l, 16 DEG C of connection 2h or even overnight；

6) convert：Take 10 μ l connection products and 100 μ l competence bacterium (TOP10 or DH5 α) are mixed Close, 30min, 42 DEG C of heat shock 90sec are placed on ice, be immediately placed on 5min on ice, add 800 μ l LB nutrient solutions without antibiotic, 37 DEG C, 200rpm shaken cultivation 30min make thalline recover and expand Increase a generation, 3000rpm centrifugation 2min, the most of supernatant of removal stays 50-100 μ l bacterium solutions, gently Piping and druming precipitation is mixed, and is then uniformly applied on the LB flat boards of amicillin resistance (Amp+), 37 DEG C Culture 12-16 hours.

7) clone identification：Picking by after ammonia benzyl resistance screening grow bacterium colony plus ampicillin liquid Amplification Culture in culture medium, extracting plasmid carries out digestion identification：Take that 1-2 μ g are small to take out plasmid EcoRV, KpnI double digestions, agarose gel electrophoresis identification endonuclease bamhi size, carrier pcDNA^TM3.1/myc-His(-) A clip sizes about 5.5kb, FOXR2 reading frames clip size about 1000bp, the clone for meeting size send survey Sequence confirms the correctness of Insert Fragment sequence.

(6) measure of cell growth curve

1) different types of HCC cells are pressed into 3-5 × 10 according to its growth characteristics³/ 100 μ l/ holes calculate Cell total amount, after abundant vitellophag, is diluted to required concentration, is inoculated in 96 orifice plates.Every group daily Three wells, by 5-7 days inoculating cells；

2) observation of cell state and number after cell is substantially adherent.With CCK-8 developers (Cell Counting Kit-8, DOJINDO, Japan) chromogenic reaction is carried out, every 100 μ l nutrient solutions add 10 μ L CCK-8,37 DEG C, 5%CO₂Incubator is placed and is incubated 1h, and ELIASA determines the absorbance at 450nm, Record, determines the actual initial density of cell, used as growth relative zero.

3) half amount changes liquid daily or every other day, specific depending on requirement of experiment；

4) basis of microscopic observation cellular morphology, Fixed Time Interval measurement, records cell growth condition；

5) it is general to survey 5 to 7 days.After end to be determined, collect data and processed, figure is drawn with Excel Table.

(7) cell clonal formation experiment

1) transfect：Using Lipofectamine^TM2000 (Invitrogen) transfectional cells, overexpression Or in silenced cell FOXR2 genes expression；

2) cell after transfecting, in 6 orifice plates or 35mm culture dishes with disappearing after normal nutrient solution culture 24h Change and count, 100mm culture dishes (different cell line numbers are different) are seeded to by certain amount, continue to train 24h is supported, the G418 (600-1000 μ g/ml) of debita spissitudo is then added according to cell category, to sieve The positive cell clone of choosing transfection；

3) cultivate 2-3 weeks, changed fresh medium every 3-5 days therebetween and add G418 screenings, until having Macroscopic cell clonal formation；

4) the training liquid in culture dish is sucked, 1 × PBS is washed twice, coomassie brilliant blue R_250 dyeing 2h is used After water is gently rinsed, then with coomassie brilliant blue staining destainer decolouring 30-60min；

5) Clone formation coloration result is taken pictures, and each is cultivated according to identical standard (cell clone size) Cell clone on ware is counted.

(8) Western blotting (Western Blot)

1) prepared by protein sample：After the cell of culture sucks culture supernatant, washed twice with the 1XPBS of precooling, 2 × SDS lysates (100mM Tris-Cl, pH=6.8,4%SDS, 20% glycerine) is added, is fully split Xie Hou, boiling water bath heating 10min, 12000 × g centrifugation 10min, supernatant is transferred in new pipe, BCA Protein Assay Kit are quantified to the albumen for obtaining, -80 DEG C of preservations；

2) protein electrophoresis is separated：The appropriate sample-loading buffer containing 200mM DTT is added in protein sample (loading buffer), boiling water bath heating 10min, is slightly centrifuged, SDS-PAGE protein gels electricity Swimming separates sample；

3) transferring film：Running gel, nitrocellulose membrane, thickness (thin) filter paper backing plate are dipped in (24 in transferring film buffer solution MM Tris, 192mM glycine, 20% methyl alcohol) balance 15-20min.By the thickness filter paper backing plate of positive pole -1 The order of the thin filter paper backing plate-negative pole of-nitrocellulose membrane--2 layers of running gel is put well, wet to turn instrument (XCell SureLock^TM, invitrogen) and 30 volts of transferring film 30-40min；

4) close：5% skimmed milk power/0.1%PBST is used as confining liquid, horizontal shaker, room temperature closing 30min-2h；

5) primary antibody：Primary antibody dilutes (reference antibody specification recommended density) with confining liquid, is incubated at room temperature 2h Or 4 DEG C of overnight incubations, 0.1%PBST washes three times, each 5min；

6) secondary antibody：Fluorescence secondary antibody dilutes (1 with confining liquid:1000) 30min, 0.1%PBST, are incubated at room temperature Wash three times, each 5min；

7) film is swept：ODYSSEY infrared imaging systems scan nitrocellulose filter, preserve image.

(9) soft-agar cloning forms experiment

1) 1% and 2% low melting point Agarose (TaKaRa companies), autoclave sterilization are prepared respectively；

2) 2 × DMEM nutrient solutions (2.5 × DMEM, containing 20%FBS) is prepared；

3) 2%Agarose and 2 × DMEM nutrient solutions by 37 DEG C of insulations are mixed by same volume, with every hole 0.5ml is added in 24 orifice plates, is placed in 4 DEG C of refrigerators, it is to be solidified after use；

4) fully the cell of digestion culture, into individual cells, is counted, and is diluted to same concentrations (3000-5000 Individual/0.5ml)；

5) cell suspension is mixed with the 1%Agarose of 37 DEG C of insulations with same volume, is added to and has completed In 24 orifice plates of lower floor's glue, per hole 0.5ml, 4 DEG C of refrigerator 10min are placed in；

6) 0.2ml nutrient solutions are added on the soft agar of solidification, 37 DEG C, 5%CO are placed in₂In incubator, Continue to cultivate 2-3 weeks；

7) growing state of each hole of basis of microscopic observation the inside cell clone, counts, and is analyzed.

(10) tumor formation in nude mice

1) mouse used by is the 5-6 weeks male BLAB/c nu nude mice of size, by Shanghai Si Laike experimental animals Co., Ltd provides, and raises in southern model animal Culture Center；

2) cell after treatment is taken, subcutaneous (the different cell categories inoculation numbers of mouse is inoculated in equal number It is different), to avoid error caused by individual difference, allogenic cell different disposal from symmetrical being inoculated in together One mouse；

3) after visual tumors to appear, every 3 days monitoring tumor sizes, slide measure reads tumour major diameter and short Footpath, calculates gross tumor volume as follows：Volume=major diameter × minor axis²；

4) after continuing to monitor about 8-9 times, disposal data, statistics.

(11) antibody is obtained and immune detection

1) antigen protein is obtained

The cDNA sequence of people's FOXR2 genes is obtained from Genebank databases, is expanded by PCR and obtained Encoder block, in insertion prokaryotes or eukaryotic expression vector, expresses FOXR2 albumen, and by gene work The purification system purifying protein of journey expression product.

2) Antibody preparation

Antibody can be prepared using following several method：

A cell fusion methods：With the FOXR2 protein immune animals (including rabbit, goat etc.) of above-mentioned preparation, Spleen cell is obtained, then is merged with myeloma cell, and routinely monoclonal antibody technology of preparing prepares Dan Ke Grand antibody.

B utilizes phage display storehouse, the spleen IgG variable regions of the immune animal of clone to be simultaneously expressed as gene Engineered monoclonal antibody.

C prepares polyvalent antibody using the protein immune animal of purifying.

3) detect

Antibody (resisting more or monoclonal antibody) prepared by a, the pathological examination of liver cancer is carried out with histochemical method, Negative signal is liver cancer.

B takes patients serum, is detected with ELISA method, and negative reaction is the suspicious patient of liver cancer.

C using FOXR2 antibody as protein-chip one of probe, for kinds of tumors diagnosis.

Embodiment 1：Expression patterns of the FOXR2 in clinical sample

By lot of experiments, it is found that jaw frame albumen FOXR2 substantially rises in liver cancer tissue.41 In to clinical sample, detected by real-time quantitative PCR and find there is FOXR2 in 28 pairs of sample liver cancer tissues Apparently higher than corresponding cancer beside organism, rise rate reaches 68.3%, and statistical analysis show p for expression<0.01 (Figure 1A), illustrates the reliability of result.Meanwhile, have detected a number of liver by ImmunohistochemistryMethods Methods The expression pattern of FOXR2 in cancerous tissue, as shown in Figure 1 C, FOXR2 gene expressions in the cancerous tissue of patient Amount is higher than corresponding cancer beside organism.The primer sequence of the real-time quantitative PCR of detection FOXR2 expression used in experiment It is classified as：5-TCAGTGTGCAGGAGATCTAC-3(SEQ ID NO.:5) and： 5-AAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO.:6).As the primer of the β-actin of internal reference Sequence is：5-cctggcacccagcacaatg-3(SEQ ID NO.:7) and： 5-gggccggactcgtcatact-3(SEQ ID NO.:8)。

Embodiment 2：Overexpression FOXR2 genes promote the propagation of HCC

By NCBI internet retrievals, cDNA sequence CCDS 35308.1 (Fig. 2A) (the SEQ ID NO. of FOXR2: 1) and its coding amino acid sequence NP_940853.1 (Fig. 2 B) (SEQ ID NO.:2).In order to test Functions of the card FOXR2 in liver cancer generation, we construct its carrier for expression of eukaryon first, by FOXR2's CDNA clone enters pcDNA^TM3.1/myc-His (-) A, transfects the plasmid that builds into carrying out after HCC Western blot detect that discovery FOXR2 is successfully expressed (Fig. 3 A).Ensuing functional experiment growth Curve and Clone formation result show, the overexpression of FOXR2 promote HCC Hep3B, Huh7, The propagation of YY-8103 and L02, clonality (Fig. 3 B, C).For building FOXR2 eukaryotic expressions The primer sequence of carrier is Forward:5-ccaccATGGACTTAAAACTAAAAGACTG-3(SEQ ID NO.: 3)；Reverse:5-gttcGGTACCAAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO.:4).

Embodiment 3：The expression inhibiting cell growth of silence FOXR2

In order to further verify the cell growth promotion functions of FOXR2, take RNA disturb silence its expression Mode detects the change of cell growth status.For disturbing the siRNA of FOXR2 expression by Shanghai Ji code system medicine Co., Ltd synthesizes, and sequence is：Si-1 positive-sense strands 5-GCUCCCUAGAUGAGAUACAdTdT-3 (SEQ ID NO.: 9)；Si-1 antisense strands 5-UGUAUCUCAUCUAGGGAGCdTdT-3 (SEQ ID NO.:10).Si-2 justice Chain 5-GGUGUUAAGUAAAGUUAGAdTdT-3 (SEQ ID NO.:11)；Si-2 antisense strands 5-UCUAACUUUACUUAACACCdTdT-3(SEQ ID NO.:12).As the non-spies of NC of interference control Specific nucleotide sequence is：Positive-sense strand 5-UUCUCCGAACGUGUCACGUdTdT-3 (SEQ ID NO.:13)； Antisense strand 5-ACGUGACACGUUCGGAGAAdTdT-3 (SEQ ID NO.:14).Examined through real-time quantitative PCR Survey shows that artificial synthesized interference siRNA can effectively lower the expression (Fig. 4 A) of FOXR2, The downward that growth curve experiment also demonstrates FOXR2 can suppress the life of HCC WRL68 and HCC-LM3 (Fig. 4 B) long, strong support FOXR2 promotes the conclusion of growth of tumour cell.

Embodiment 4：FOXR2 promotes the pernicious sign of HCC

Soft-agar cloning Forming ability is a strong In vitroindex for reflecting degree of malignancy of tumor cell. In order to detect influences of the FOXR2 to HCC grade malignancy, have chosen WRL-68 and HCC-LM3 (is purchased from Cell institute of Chinese Academy of Sciences cell bank) as the research object of silence expression.By thin in soft agar to being grown in Born of the same parents clone's number analysis (Fig. 5), it is final to determine that silence expression FOXR2 effectively suppress WRL-68 and HCC-LM3 The grade malignancy of cell, shows that FOXR2 promotes the pernicious of HCC.

Embodiment 5：FOXR2 influences HCC in the one-tenth knurl ability of nude mice by subcutaneous

Experiment in vitro has clearly indicated that FOXR2 promotes the growth of HCC, next using tumor bearing nude mice Model studies influence of the expression of FOXR2 to HCC nude mice by subcutaneous one-tenth knurl ability.By 1 × 10⁶Individual mistake YY-8103 (the source Shanghai City Zhong Shan hospitals) cells and compared with control cells for expressing FOXR2 are symmetrically injected into nude mice Subcutaneous abdomen, observe knurl body growing state, and with slide measure record every 3 days knurl body length and Width.After 1 month, kill nude mice taking-up knurl body and weigh.Result shows that the overexpression of FOXR2 is effectively facilitated One-tenth knurl ability (Fig. 6 A) of the YY-8103 cells in nude mice by subcutaneous.Equally, by 1 × 10⁶Individual silence expresses FOXR2 WRL68 cells and compared with control cells to be symmetrically injected into mice belly subcutaneous, observation knurl body growing state is simultaneously recorded. Final result shows that the downward of FOXR2 effectively inhibits WRL68 cells nude mice by subcutaneous into the ability of knurl (Fig. 6 B).

Present invention experiment confirms expression of the FOXR2 genes in liver cancer tissue apparently higher than cancer beside organism, and The growth of HCC, therefore FOXR2 genes can be remarkably promoted by external source FOXR2 gene overexpressions And its expression product can examine liver cancer as the mark of diagnosing liver cancer and the drug target for liver cancer treatment It is disconnected more accurate, quick.In a word, FOXR2 genes of the present invention and application thereof for prevent and treat liver cancer provide it is new Therapy target and effective new drug.

The all documents referred in the present invention are all incorporated as reference in this application, just as each piece Document is individually recited as with reference to such.In addition, it is to be understood that reading above-mentioned instruction of the invention After content, those skilled in the art can make various changes or modifications to the present invention, these shapes of equal value Formula equally falls within the application appended claims limited range.

Claims

1. the purposes of a kind of FOXR2 genes or FOXR2 albumen or its detection reagent, it is characterised in that for making The reagent or kit of standby detection liver cancer.

2. purposes as claimed in claim 1, it is characterised in that described kit includes：To FOXR2 eggs White or mRNA carries out the reagent and corresponding label or specification of quantitative determination.

3. purposes as claimed in claim 1, it is characterised in that described reagent draws including FOXR2 specificity Thing, specific antibody, probe and/or chip.

4. purposes as claimed in claim 2, it is characterised in that indicated in described label or specification with Lower content：

The relative mrna expression amounts with reference to albumen of FOXR2 and cancer beside organism's (or normal structure) when detection object The ratio between relative mrna expression amounts with reference to albumen of FOXR2 >=1.5, then point out the detection object to suffer from liver cancer Probability is higher than general population.

5. the purposes of a kind of FOXR2 albumen, FOXR2 genes or its inhibitor, it is characterised in that be used for system The standby medicine for suppressing growth of cancer cells or propagation, or for preparing the medicine for the treatment of liver cancer.

6. purposes as claimed in claim 5, it is characterised in that described inhibitor includes：FOXR2's is anti- Body, the antisense RNA of FOXR2 nucleic acid, the activity inhibitor of siRNA, shRNA and FOXR2.

7. a kind of method for suppressing liver cancer cell growth or propagation of external non-therapeutic, it is characterised in that including Step：In the presence of FOXR2 albumen or its inhibitor, HCC is cultivated, so as to suppress liver cancer cell growth Or propagation.

8. a kind of method that the candidate compound of liver cancer is treated in screening, it is characterised in that methods described includes step Suddenly：

In (a) test group, test compound is added in the cultivating system of cell, and observe the test group Cell in FOXR2 expression quantity and/or activity；In control group, in the cultivating system of same cell Without test compound, and observe the expression quantity and/or activity of FOXR2 in the cell of control group；

Wherein, if the expression quantity of the FOXR2 of cell and/or activity are less than control group in test group, with regard to table The bright test compound is the candidates of the treatment liver cancer that has inhibitory action to the expression of FOXR2 and/or activity Compound.

9. method as claimed in claim 8, it is characterised in that methods described also includes step：

(b) for the candidate compound that obtains in step (a), further test it to liver cancer cell growth or The inhibitory action of propagation.

10. method as claimed in claim 9, it is characterised in that the step (b) includes step：Survey In examination group, test compound is added in the cultivating system of cancer cell, and observe quantity and/or the life of cancer cell Situation long；In control group, without test compound in the cultivating system of cancer cell, and it is thin to observe cancer The quantity and/or growing state of born of the same parents；Wherein, if the quantity or the speed of growth of cancer cell are less than in test group Control group, indicates that the test compound is the treatment liver cancer for having inhibitory action to the growth of cancer cell or propagation Candidate compound.