CN105463123A - Molecular marker for rectal adenocarcinoma diagnosis and treatment - Google Patents

Molecular marker for rectal adenocarcinoma diagnosis and treatment Download PDF

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CN105463123A
CN105463123A CN201610068910.2A CN201610068910A CN105463123A CN 105463123 A CN105463123 A CN 105463123A CN 201610068910 A CN201610068910 A CN 201610068910A CN 105463123 A CN105463123 A CN 105463123A
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shroom4
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rectal
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宋宏涛
王晓云
杨承刚
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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Abstract

The invention discloses a molecular marker for rectal adenocarcinoma diagnosis and treatment. The molecular mark is a SHROOM4 gene which is in a low-expression mode in tissue of a patient with rectal adenocarcinoma, and rectal adenocarcinoma apoptosis can be promoted by improving expression of SHROOM4. According to the molecular marker for the rectal adenocarcinoma diagnosis and treatment, sensibility and specificity of the rectal adenocarcinoma diagnosis are greatly improved, and meanwhile a new target site is provided for a gene therapy of the rectal adenocarcinoma.

Description

A kind of molecular marked compound for rectal adenocarcinoma diagnosis and treatment
Technical field
The invention belongs to biomedicine field, relate to a kind of molecular marked compound for rectal adenocarcinoma diagnosis and treatment, described molecular marked compound is SHROOM4.
Background technology
Rectal adenocarcinoma is whole world alimentary system malignant tumour occurred frequently, and its sickness rate rises year by year.In the U.S., its sickness rate and case fatality rate occupy the 3rd of malignant tumour.In China, it accounts for the 4th in men and women's mortality of malignant tumors, has serious harm to the health and lives of the mankind.The generation of rectal adenocarcinoma, development are that a polygene changes, the complex process of multistage carcinogenesis, comprise the sudden change etc. of the activation of gene, the inactivation of cancer suppressor gene or disappearance, mismatch repair gene.Adenomas is developed to malignant change from benign lesion through precancerous lesion, until the evolution process of the complexity of invasion and m etastasis.
Rectal adenocarcinoma early clinic symptom is hidden, and not easily finds, arrives middle and advanced stage during most patient assessment, lose best opportunity of operation.Have report to show, after early stage rectal adenocarcinoma operation in patients, 5 years survival rates are about 90%, and patients with terminal is only 15%.Therefore, early find, early treatment is the key reducing rectal adenocarcinoma patient high mortality, improve prognosis.Rectal adenocarcinoma early diagnosis depends on intestines mirror, imaging examination and serologic marker thing carcinomebryonic antigen (CEA) etc. clinically, and intestines mirror is the most reliable method of rectal adenocarcinoma diagnosis, but has invasive, somewhat expensive, and patient compliance is poor; CT and ultrasonic examination not easily find less pathology, are subject to a definite limitation to the early diagnosis of rectal adenocarcinoma; CEA is the blood serum designated object of wide clinical application, and its Sensitivity and Specificity is lower, is mainly used in the Treatment monitoring of rectal adenocarcinoma patient.
At present, the histodiagnosis of specifying rectal adenocarcinoma mainly relies on HE stained, does not also have a kind of marker that can be used as independent body's diagnosis, searches out a kind of novel specific marker for rectal adenocarcinoma diagnosis, prognosis and treatment and just becomes very necessary.In recent years, along with molecular biological development, some new biomarkers occur, not only provide a great help to the pathological diagnosis of rectal adenocarcinoma, and for its Index for diagnosis, treatment plan selection provide more fully foundation, played unique effect to a certain extent.The oncogene that further searching is relevant to rectal adenocarcinoma and cancer suppressor gene are as evaluating the auxiliary characteristics of its biological behaviour and having great importance for research rectal adenocarcinoma as the target spot of targeted therapy.
Summary of the invention
In order to make up the deficiencies in the prior art, the object of the present invention is to provide a kind of molecular marked compound-SHROOM4 gene that can be used for rectal adenocarcinoma diagnosis and treatment.Compare the Diagnosis and Treat method of traditional rectal adenocarcinoma, use gene marker to carry out Diagnosis and Treat rectal adenocarcinoma and there is promptness, specificity and non-invasive.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides SHROOM4 gene and the application of expression product in the product preparing Diagnosis of Rectal gland cancer thereof.
Further, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection SHROOM4 gene and expression product thereof with the product of Diagnosis of Rectal gland cancer.
Further, the product of described RT-PCR Diagnosis of Rectal gland cancer at least comprises the primer of a pair specific amplified SHROOM4 gene; The product of described real-time quantitative PCR Diagnosis of Rectal gland cancer at least comprises the primer of a pair specific amplified SHROOM4 gene; The product of described immunodetection Diagnosis of Rectal gland cancer comprises: the antibody be combined with SHROOM4 protein-specific; The product of described in situ hybridization Diagnosis of Rectal gland cancer comprises: with the probe of the nucleic acid array hybridizing of SHROOM4 gene; The product of described chip Diagnosis of Rectal gland cancer comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with SHROOM4 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of SHROOM4 gene.
Further, described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to rectal adenocarcinoma multiple genes) comprising SHROOM4 gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to rectal adenocarcinoma multiple protein) comprising SHROOM4 albumen.By being detected by multiple mark with rectal adenocarcinoma simultaneously, the accuracy rate of rectal adenocarcinoma diagnosis greatly can be improved.
The invention provides a kind of product of Diagnosis of Rectal gland cancer, described product can carry out Diagnosis of Rectal gland cancer by the expression level detecting SHROOM4 gene in rectal tissue.
Further, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit, protein immunization detection kit.
Further, described gene detecting kit can be used for detecting the expression level of the multiple genes (such as, relevant to rectal adenocarcinoma multiple genes) comprising SHROOM4 gene.Described protein immunization detection kit can be used for detecting the expression level of the multiple protein (such as relevant to rectal adenocarcinoma multiple protein) comprising SHROOM4 albumen.Multiple marks of rectal adenocarcinoma are detected simultaneously, greatly can improve the accuracy rate of rectal adenocarcinoma diagnosis.
The invention provides SHROOM4 gene and the application of expression product in the pharmaceutical composition of preparation treatment rectal adenocarcinoma thereof.
Further, described pharmaceutical composition comprises increases SHROOM4 genetic expression, strengthen the reagent of SHROOM4 expressive function and/or enhancing SHROOM4 expression product activity.
Further, described reagent comprises: reagent, the activator of SHROOM4 albumen, the reagent containing SHROOM4 protein of the nucleic acid containing energy encode functional SHROOM4 albumen.
Wherein, the reagent of the described nucleic acid containing energy encode functional SHROOM4 albumen can be single-chain nucleic acid (as mRNA) or the double-strandednucleic acid (as DNA) of the SHROOM4 albumen translating into activity form under favourable condition, described nucleic acid can be connected on expression vector or recombinate in host cell, as long as active SHROOM4 albumen can be encoded into, the carrying mode of any one SHROOM4 gene.The reagent that described SHROOM4 protein activator refers to stimulates SHROOM4 protein-active, increase SHROOM4 protein-active, promote SHROOM4 protein-active, strengthen SHROOM4 protein activation, make the sensitization of SHROOM4 protein-active or rise SHROOM4 protein-active, the agonist (as activated antibody) etc. of transcriptional activation agent as specific in demethylation reagent, SHROOM4 promotor and/or enhanser, SHROOM4 albumen.
Further, aforementioned pharmaceutical compositions also comprises pharmaceutically acceptable carrier, and described carrier can be one also can be multiple, and described carrier includes but not limited to that thinner is as lactose, sodium-chlor, glucose, urea, starch, water etc.; Tackiness agent is as starch, pregelatinized Starch, dextrin, Star Dri 5, sucrose, gum arabic, gelatin, methylcellulose gum, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyoxyethylene glycol, PVP, Lalgine and alginates, xanthan gum, hydroxypropylcellulose and Vltra tears etc.; Tensio-active agent is as polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulphate, glyceryl monostearate, cetyl alcohol etc.; Humectant is as glycerine, starch etc.; Absorption carrier is as starch, lactose, bentonite, silica gel, kaolin and soap clay etc.; Lubricant is as Zinic stearas, glyceryl monostearate, polyoxyethylene glycol, talcum powder, calcium stearate and magnesium, polyoxyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, single bay sucrose acid ester, sodium laurylsulfate, magnesium laurylsulfate, Stepanol MG etc.; Weighting agent is as N.F,USP MANNITOL (granular or powdery), Xylitol, sorbyl alcohol, maltose, erythrose, Microcrystalline Cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate etc.; Disintegrating agent is as cross-linked ethylene pyrrolidone, sodium starch glycolate, low-substituted hydroxypropyl ylmethyl, croscarmellose sodium, soybean polysaccharide etc.
Described pharmaceutical composition can use different additives to be prepared, such as stablizer, sterilant, buffer reagent, isotonic agent, sequestrant, pH control agent and tensio-active agent.
Stablizer comprises Human serum proteins, L-amino acid, sugar and derivatived cellulose.L-amino acid can also comprise any one in glycine, halfcystine and L-glutamic acid.Carbohydrate comprises monose, such as glucose, seminose, semi-lactosi, fructose etc.; Sugar alcohol, such as N.F,USP MANNITOL, Inositol nf12 99, Xylitol etc.; Disaccharides, such as sucrose, maltose, lactose etc.; Saccharan, such as dextran, hydroxypropylated starch, sulfuration chrondroitin, hyaluronic acid etc. and their derivative.Derivatived cellulose comprises methylcellulose gum, ethyl cellulose, Natvosol, hydroxypropylcellulose, HPMC and sodium cellulose glycolate.
Tensio-active agent comprises ion or nonionogenic tenside, such as polyoxyethylene alkyl ester, sorbitanic monoacyl ester, glycerin fatty acid ester.
Additive buffer reagent can comprise boric acid, phosphoric acid, acetic acid, citric acid, L-glutamic acid and corresponding salt (their basic metal or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent comprises Repone K, sodium-chlor, sugar and glycerine.Sequestrant comprises sodium ethylene diamine tetracetate and citric acid.
Present invention also offers a kind of pharmaceutical composition for the treatment of rectal adenocarcinoma, described pharmaceutical composition comprises increase SHROOM4 genetic expression, strengthen the reagent of SHROOM4 expressive function and/or enhancing SHROOM4 expression product activity.
Medicine of the present invention may be used for disappearance or the deficiency of supplementary endogenic SHROOM4 albumen, by improving the expression of SHROOM4 albumen, thus treats because SHROOM4 albumen reduces the rectal adenocarcinoma caused.
The carrier carrying gene of the present invention is various carrier known in the art, as commercially available carrier, comprises plasmid, clay, phage, virus etc.
Further, in the present invention, the nucleic acid of SHROOM4 albumen or coding SHROOM4 albumen can be given by liposome, and the effect of described liposome is by drug targeting in specific tissue, and increases the transformation period of medicine.Liposome comprises emulsifying agent, pore forming material, liquid fatty substance, Solid lipid, insoluble monolayer, phospholipid dispersions, tensio-active agent etc.Can also comprise in described liposome and can be combined or other treatment or immunogenic composition by the acceptor molecule in the cell of target.
Pharmaceutical composition of the present invention can be formulated into any administration fashion, such as, utilize in the intradermal injection of syringe or other device, subcutaneous injection, intravenous injection, peritoneal injection, intrapleural injection, Intravesical administration, coronary artery or intra-tumoral injection, oral administration, rectal administration.
The mode of drugs delivery tissue of the present invention or cell can be divided into the mode in external or body.Vitro formats comprises by the medicine containing SHROOM4 gene or containing in the drugs delivery cell of SHROOM4 protein, then by Transplanted cells or feed back in body.In body, mode comprises directly by the medicine containing SHROOM4 gene or containing in the infusion of medicine in-vivo tissue of SHROOM4 protein.
Medicine of the present invention also can with the drug combination of other treatment rectal adenocarcinoma, other treatment compound can with main activeconstituents (such as, the nucleic acid of SHROOM4 albumen or encoding said proteins) administration simultaneously, even administration simultaneously in same composition.Other therapeutic compound can also be given separately with independent composition or the dosage form different from main activeconstituents.The Fractional of main component (nucleic acid as SHROOM4 albumen or encoding said proteins) can with the administration simultaneously of other therapeutic compound, and other dosage can be individually dosed.
Over the course for the treatment of, according to the physiologic response of the severity of symptom, the frequency of recurrence and treatment plan, the dosage of pharmaceutical composition of the present invention can be adjusted.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of conventional prokaryotic host cell comprises intestinal bacteria, Bacillus subtilus etc.Conventional eukaryotic host cell comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative for the probe of SHROOM4 gene in the present invention.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
The specific antibody of the albumen of SHROOM4 described in the present invention comprises monoclonal antibody, polyclonal antibody.The specific antibody of described SHROOM4 albumen comprises complete antibody molecule, any fragment of antibody or modification, such as, and chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with SHROOM4 albumen.Be well known to a person skilled in the art for detecting the preparation of the antibody of protein level, and the present invention can use any method to prepare described antibody, as described in fragment or recombinant DNA technology can be utilized to synthesize by chemical method de novo synthesis.
In the context of the present invention, " SHROOM4 gene " comprises the polynucleotide of any function equivalent of people SHROOM4 gene and people SHROOM4 gene.SHROOM4 gene comprises and has more than 70% homology with SHROOM4 gene (NC_000023.11) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of SHROOM4 gene comprises any one DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) DNA sequence dna limited with (1) is under strict conditions hybridized and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described SHROOM4 gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, SHROOM4 gene expression product comprises the partial peptide of people SHROOM4 albumen and people SHROOM4 albumen.The partial peptide of described SHROOM4 albumen contains the functional domain relevant to rectal adenocarcinoma.
" SHROOM4 albumen " comprises any function equivalent of SHROOM4 albumen and SHROOM4 albumen.Described function equivalent comprises SHROOM4 albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of people SHROOM4 under high or low stringent condition.
Preferably, SHROOM4 albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described SHROOM4 albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, in a protein, one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is SHROOM4 albumen.Peptide or protein with SHROOM4 protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of SHROOM4 albumen.
SHROOM4 albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of SHROOM4 albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " treatment rectal adenocarcinoma " comprises generation and the recurrence of any symptom eliminating, alleviate, alleviate, reverse or prevent or postpone illness, namely comprises the therapeutic intervention to disease and Primary preventive intervention.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention SHROOM4 gene expression dose is relevant to rectal adenocarcinoma, by detecting the expression of SHROOM4 in experimenter's rectal tissue, can judge whether experimenter suffers from rectal adenocarcinoma, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-SHROOM4 gene of rectal adenocarcinoma, utilize molecular marked compound to realize the Diagnosis and Treat of disease, compare traditional means, have more promptness, specificity, non-invasive.
Accompanying drawing explanation
Fig. 1 display utilizes QPCR to detect the expression of SHROOM4 gene in rectal adenocarcinoma tissue;
Fig. 2 display utilizes QPCR to detect the expression of SHROOM4 gene in Rectal Adenocarcinoma Cells;
Fig. 3 display utilizes MTT to detect SHROOM4 genetic expression to the impact of Rectal Adenocarcinoma Cells multiplication capacity.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene marker relevant to rectal adenocarcinoma
1, sample collection
The routine rectal adenocarcinoma cancer beside organism of each collection 8 and rectal adenocarcinoma tissue samples.Above-mentioned sample is the excision sample of rectal adenocarcinoma patient, obtaining all by the agreement of the council of organizational ethics of above-mentioned all samples.
2, the preparation of RNA sample (utilizes E.Z.N.A. kit operates)
The tissue of above-mentioned acquisition to shred in rear input liquid nitrogen and is ground to Powdered, according to the specification sheets extraction and isolation RNA in test kit.Specific as follows:
1) separation of RNA:
A. RNA-is added in tissue homogenate or cell reagentII1ml;
B. room temperature places 3min, acutely shakes 15s after adding 0.2ml chloroform;
C. be placed in and prevent 10min on ice;
D.12000g, 4 DEG C of centrifugal 15min;
E. the aqueous phase shifting 80% enters in new 2mlEP pipe, adds the dehydrated alcohol of 1/2 amount, jolting;
F. the aforesaid liquid being less than 700 μ l is transferred to rNAMinicolumn, the centrifugal 60s of 10000g room temperature after jolting.
2) RNA purifying:
A. to rNAMinicolumn adds 500 μ lRWCWashBuffer, the centrifugal 30s of 10000g;
B. add 500 μ lRWBWashBuffer, the centrifugal 30s of 10000g, after repeating twice, take maximum centrifugal complete drying rNAMinicolumn;
C. add to pillar the DEPC water that 15 μ l are preheated to 70 DEG C, room temperature is centrifugal at full speed after placing 2min.
3, high-throughput transcript profile order-checking
1) the RNA-seq section of reading location
First the low-quality section of reading is removed and obtain the clean section of reading, then utilize TopHatv1.3.1 will clean fragment to mate with reference to genome (hg19) with UCSCH.sapiens, the index built in advance of H.sapiensUCSChg19 version is downloaded from TopHat homepage, and as reference genome, when utilizing TopHat to mate with genome, each section of reading (defaulting to 20) is allowed to have multiple coupling site, maximum 2 mispairing.TopHat sets up possible shearing site storehouse according to exon region and GT-AG shear signal, will not navigate to the genomic section of reading navigate on genome according to these shearing site storehouses.We use the system default parameter of TopHat method.
2) transcript abundance assessment
What match reads segment file by Cufflinksv1.0.3 process, and RNA-seq segment number is carried out the relative abundance of standardized calculation transcript by Cufflinksv1.0.3.FPKM value refers in each 1,000,000 sequenced fragments the segment number matching the long exon region of specific gene 1kb.The fiducial interval of FPKM estimated value is calculated by Bayesian inference method.The GTF comment file of the reference that Cufflinks uses downloads (Homo_sapiens.GRCh37.63.gtf) from Ensembl database.
3) detection of difference expression gene
By the EnsemblGTF file of download be transferred to Cuffdiff by the source document that TopHat mates, Cuffdiff uses original matching files again to estimate the gene expression abundance of the transcript listed in GTF file, and checkout discrepancy is expressed.In Cuffidff exports, only have q value < 0.01, test display is successfully more just considered to differential expression.
4, result
RNA-seq result shows, and the expression amount of SHROOM4 gene in rectal adenocarcinoma tissue is significantly lower than the expression amount in cancer beside organism.
The differential expression of embodiment 2QPCR sequence verification SHROOM4 gene
1, large sample QPCR checking is carried out to SHROOM4 gene differential expression.Each 70 examples are organized according to the sample collection way selection rectal adenocarcinoma cancer beside organism in embodiment 1 and rectal adenocarcinoma.
2, QPCR concrete operation step is as follows:
(1) RNA extracts
RNA extraction step as described in Example 1.
(2) reverse transcription
A. in Microtube, configure mixed solution: OligodT (50 μMs) 1 μ l, dNTP mixed solution (10mM) 1 μ l, RNA template 5 μ g, adds ddH 2o to 10 μ l, mixes;
B. in PCR instrument, sex change, annealing reaction is carried out according to following reaction conditions: after 65 DEG C of 5min, place immediately on ice;
C. in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared:
Reaction solution 10 μ l after above-mentioned sex change, annealing, 5 × buffer4 μ l, RNase inhibitor (40U/ μ l) 0.5 μ l, rTase (200U/ μ l) 1 μ l, ddH 2o (RNase-free) 4.5 μ l, mixes;
D. in PCR instrument, reverse transcription reaction is carried out according to following reaction conditions:
(30 DEG C of 10min) × 3, (42 DEG C of 45min) × 4, (95 DEG C of 5min) × 5, after process, be placed on ice;
E. PCR reaction solution is prepared by following formula on ice:
premixExTaqII (TliRNaseHPlus) (2 ×) 12.5 μ l, forward primer (10 μMs) 1 μ l, reverse primer (10 μMs) 1 μ l, DNA profiling (<100ng) 2.0 μ l, ddH 2o8.5 μ l;
The cycling condition of F.PCR is as follows:
95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) × 40, choose β-actin as internal reference.The primer sequence of PCR is as follows:
The primer sequence of SHROOM4:
Forward primer: 5 '-ACAGTGTCTAAGATTGAAG-3 ' (SEQIDNO.3),
Reverse primer: 5 '-GGAGTGCCATTGATATTC-3 ' (SEQIDNO.4)
The primer sequence of β-actin:
Forward primer: 5 '-CCTGGGCATGGAGTCCTGTG-3 ' (SEQIDNO.5)
Reverse primer: 5 '-TCCTTCTGCATCCTGTCG-3 ' (SEQIDNO.6)
Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
4, result
Result as shown in Figure 1, compared with rectal adenocarcinoma cancer beside organism, lower in rectal adenocarcinoma tissue, and difference has statistical significance (P<0.05), consistent with RNA-sep result by SHROOM4 gene.
The process LAN of embodiment 3SHROOM4 gene
1, cell cultures
Human rectal adenocarcinoma cell strain HRC-99, with containing 10% calf serum and 1%P/S RPMI1640 substratum 37 DEG C, 5%CO2, relative humidity is cultivate in the incubator of 90%.Within 2-3 days, change liquid 1 time, use the 0.25% trypsinase conventional digestion containing EDTA to go down to posterity.
2, the process LAN of SHROOM4 gene
The structure of 2.1SHROOM4 expression vector
According to encoding sequence (as shown in the SEQIDNO.1) design of amplification primers of SHROOM4 gene, primer sequence is as follows:
Forward primer: 5 '-CCGAAGCTTGCCACCATGGAGAACCGGCCTG-3 ' (SEQIDNO.7)
Reverse primer: 5 '-CGGGCGGCCGCGAAATTGCTGGGCCCCA-3 ' (SEQIDNO.8)
From cDNA library (the clontech company becoming Human fetal spleen, article No.: the encoding sequence of the SHROOM4 gene of amplification total length 638831), above-mentioned cDNA sequence is inserted in the eukaryotic expression vector pcDNA3.1 of restriction enzyme HindIII and NotI double digestion after restriction enzyme HindIII and NotI double digestion, connects the recombinant vectors pcDNA3.1-SHROOM4 obtained and is used for subsequent experimental.
2.2 transfection
Rectal Adenocarcinoma Cells is divided into two groups, is respectively control group (transfection pcDNA3.1 empty carrier) and SHROOM4 process LAN group (transfection pcDNA3.1-SHROOM4).Use liposome 2000 to carry out the transfection of carrier, the instruction to specifications of concrete transfection method is carried out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-SHROOM4 is 0.5 μ g/ml.
2.3RT-PCR detect
Concrete steps are with embodiment 2.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
4, result
As shown in Figure 2, compared with the cell of transfection pcDNA3.1 empty carrier, in the cell of transfection pcDNA3.1-SHROOM4, the content of SHROOM4 significantly raises, and difference has statistical significance (P<0.05).
The impact of embodiment 4SHROOM4 gene pairs Rectal Adenocarcinoma Cells propagation
MTT experiment is adopted to detect the impact of SHROOM4 gene pairs Rectal Adenocarcinoma Cells multiplication capacity.
1, step: trysinization after each group cell transfecting 12h, make single cell suspension, be inoculated in 96 well culture plates with 6000, every hole cell, every component 7 time points, each time point establishes 6 multiple holes.After cell attachment, carry out the 1st time and detect: every hole adds the MTT liquid 20 μ l of 5g/L, after continuing to cultivate 4h, suck substratum, add DMSO150 μ l, careful piping and druming, hyacinthine is precipitated fully dissolve, survey absorbance (A value) by microplate reader at 490nm wavelength.Then every 12h detects 1 time, surveys 72h continuously, totally 7 times.This experiment repetition 3 times.
2, statistical method
Experiment has all come for 3 times according to repetition, adopts SPSS13.0 statistical software to carry out statistical study, and the difference between two groups adopts t inspection, thinks to have statistical significance as P<0.05.
3, result
Result display shown in Fig. 3: the vitro growth rates of transfection pcDNA3.1-SHROOM4 group is obviously butted on the vitro growth rates of transfection pcDNA3.1 empty carrier group, difference has statistical significance (P<0.05), and the above results shows that SHROOM4 expresses the growth that can suppress Rectal Adenocarcinoma Cells.
The impact of embodiment 5SHROOM4 gene pairs Rectal Adenocarcinoma Cells apoptosis
Use the apoptotic impact of flow cytomery SHROOM4 gene pairs.
1, cell culture step is with embodiment 3.
2, cell transfecting step is with embodiment 3.
3, step
After cell transfecting 72h, use precooling PBS washed cell, then use 0.25% trypsin digestion cell, stop digestion, using PBS resuspended in the cell of collected by centrifugation, is 1 × 10 by cell quantification 6individual/ml, gets the above-mentioned cell suspension of 200 μ l and is placed in Eppendorf pipe, add 10 μ lAnnexin-V-FITC and mix, and dyeing 15min is hatched in room temperature dark place, and before upper machine, 5min adds 10mg/L iodate third ingot (PI) and to dye 5 μ l.The cell of untransfected siRNA is used for standard quantitative with Annexin-V-FITC and PI dyeing respectively.Two Colour Fluorescence cell cytometry is carried out, observing apoptosis cell percentages with FACS flow cytometer.
3, statistical method
Experiment has all come for 3 times according to repetition, result data is all represent in the mode of mean+SD, adopt SPSS13.0 statistical software to carry out statistical study, the t inspection that difference between the two adopts, think to there is statistical significance as P<0.05.
4, result:
The apoptosis rate of transfection pcDNA3.1-SHROOM4 group is (25.67 ± 0.025) %, the apoptosis rate of transfection pcDNA3.1 empty carrier group is (8.91 ± 0.012) %, above-mentioned difference has statistical significance (P<0.05), the above results shows, the process LAN of SHROOM4 gene promotes the apoptosis of Rectal Adenocarcinoma Cells.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.

Claims (10)

1.SHROOM4 gene and the application of expression product in the product preparing Diagnosis of Rectal gland cancer thereof.
2. application according to claim 1, is characterized in that, described product comprises: by the expression level of RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip detection SHROOM4 gene and expression product thereof with the product of Diagnosis of Rectal gland cancer.
3. application according to claim 2, is characterized in that, the product of described RT-PCR Diagnosis of Rectal gland cancer at least comprises the primer of a pair specific amplified SHROOM4 gene; The product of described real-time quantitative PCR Diagnosis of Rectal gland cancer at least comprises the primer of a pair specific amplified SHROOM4 gene; The product of described immunodetection Diagnosis of Rectal gland cancer comprises: the antibody be combined with SHROOM4 protein-specific; The product of described in situ hybridization Diagnosis of Rectal gland cancer comprises: with the probe of the nucleic acid array hybridizing of SHROOM4 gene; The product of described chip Diagnosis of Rectal gland cancer comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with SHROOM4 protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of SHROOM4 gene.
4. a product for Diagnosis of Rectal gland cancer, is characterized in that, described product can carry out Diagnosis of Rectal gland cancer by the expression level detecting SHROOM4 gene in rectal tissue.
5. product according to claim 4, is characterized in that, described product comprises chip or test kit; Wherein, described chip comprises gene chip, protein chip; Described test kit comprises gene detecting kit, protein immunization detection kit.
The application in the pharmaceutical composition of preparation treatment rectal adenocarcinoma of 6.SHROOM4 gene and expression product thereof.
7. application according to claim 6, is characterized in that, described pharmaceutical composition comprises increases SHROOM4 genetic expression, strengthen the reagent of SHROOM4 expressive function and/or enhancing SHROOM4 expression product activity.
8. application according to claim 7, is characterized in that, described reagent comprises: reagent, the activator of SHROOM4 albumen, the reagent containing SHROOM4 protein of the nucleic acid containing energy encode functional SHROOM4 albumen.
9. the application according to claim 7 or 8, is characterized in that, described pharmaceutical composition also comprises pharmaceutically acceptable carrier.
10. treat a pharmaceutical composition for rectal adenocarcinoma, it is characterized in that, described pharmaceutical composition comprises increases SHROOM4 genetic expression, strengthen the reagent of SHROOM4 expressive function and/or enhancing SHROOM4 expression product activity.
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