CN108950000A - Application of the lncRNA in rectal adenocarcinoma diagnosis and treatment - Google Patents

Abstract

The invention discloses application of the lncRNA in rectal adenocarcinoma diagnosis and treatment, the lncRNA is LOC105374343.Present invention firstly discovers that LOC105374343 expresses in rectal adenocarcinoma significant up-regulation, and being further demonstrated by large sample experiment can clinical diagnosis using LOC105374343 as potential molecular target applied to rectal adenocarcinoma.It is related to the proliferation of Rectal Adenocarcinoma Cells, migration and invasion that the present invention discloses LOC105374343, and LOC105374343 is prompted to can be used as potential molecular target using the clinical treatment with rectal adenocarcinoma.

Description

Application of the lncRNA in rectal adenocarcinoma diagnosis and treatment

Technical field

The invention belongs to biomedicine fields, are related to application of the lncRNA in rectal adenocarcinoma diagnosis and treatment, are specifically related to Application of the LOC105374343 in rectal adenocarcinoma diagnosis and treatment.

Background technique

The carcinoma of the rectum seriously affects the health and quality of life of the mankind.In world wide, colorectal cancer accounts for tumor mortality reason The 3rd (Tome LA, Bray F, Siegel RL, et al.Global cancer statistics, 2012 [J] .CA Cancer J Clin,2015,65(2):87-108.).Transfer and recurrence are the main reason for colorectal cancer patients are dead (Xiao YC,Yang ZB,Cheng XS,et al.CXCLB,overexpressed in colorectal cancer,enhances the resistance of colorectal cancer cells to anoikis[J].Cancer Lett 2015,361 (I):22-32.).The data that american cancer federation (American Joint Committee on Cancer, AJCC) provides 5 years survival rates for showing I phase rectal cancer patient are 93.2%, and the IV phase is only 8.1%, the early diagnosis of this prompt carcinoma of the rectum Be conducive to improve survival.Therefore, the key factor for studying rectum carcinogenesis and transfer illustrates the molecule machine of its effect System is of great significance for carcinoma of the rectum early diagnosis, Index for diagnosis and treatment.

Long-chain non-coding RNA (lncRNA) be a kind of newfound non-coding RNA molecule (Wilusz JE, Sunwoo H, Spector DL.Long noncoding RNAs:functional surprises from the RNAworld[J] .Genes Dev,2009,23(13):1494-504.).In recent years, research shows that 1ncRNA is the key that tumour occurrence and development One of regulatory factor.LncRNA expresses imbalance in tumour, and the malignant behaviors of tumour are participated in by number of mechanisms, LncRNA has great potential (Wang KC, Chang as diagnosing tumor and prognosis prediction index or therapy target HY.Molecular mechanisms of long noncoding RNAs [moon .Mol Cell, 2011,43 (6): 904- 14.).There is also the lncRNA of great expression imbalance in rectum cancer tissue, with the relationship of metastases and its molecule of effect Mechanism needs further to study.

Currently, application study of the 1ncRNA in rectal adenocarcinoma early diagnosis and prognosis prediction is also fewer.Although LncRNA plays a significant role during rectal adenocarcinoma occurrence and development, and part lncRNA can be used as rectal adenocarcinoma diagnosis And prognosis prediction index, but still have a large amount of lncRNA undiscovered, it is still desirable to further research 1ncRNA is in the carcinoma of the rectum Mechanism in expression and its modulate tumor occurrence and development.

Summary of the invention

In order to make up for the deficiencies of the prior art, one of the objects of the present invention is to provide a kind of and rectal adenocarcinoma occurrence and development Relevant lncRNA biomarker and its application in rectal adenocarcinoma diagnosing and treating.

The second object of the present invention is to provide a kind of method of the drug candidate of screening treatment rectal adenocarcinoma.

The third object of the present invention is to provide a kind of products of Diagnosis of Rectal gland cancer.

The fourth object of the present invention is to provide a kind of pharmaceutical composition for treating rectal adenocarcinoma.

To achieve the goals above, the present invention adopts the following technical scheme:

The first aspect of the present invention provides a kind of detection reagent, and the reagent is the examination for detecting LOC105374343 level Agent.

Further, the reagent is selected from:

The probe of specific recognition LOC105374343；Or

The primer of specific amplification LOC105374343.

Further, the primer sequence of the specific amplification LOC105374343 is as shown in NO.1~2 SEQ ID.

The second aspect of the present invention provides a kind of product, the reagent of the product testing LOC105374343 level.

Further, the reagent is selected from:

The probe of specific recognition LOC105374343；Or

The primer of specific amplification LOC105374343.

Further, the primer sequence of the specific amplification LOC105374343 is as shown in NO.1~2 SEQ ID.

Further, product described in second aspect of the present invention includes kit, chip, nucleic acid film item.

The third aspect of the present invention provides a kind of composition, and the composition includes a effective amount of LOC105374343 Inhibitor.The inhibitor is selected from: as target sequence and being able to suppress LOC105374343 using LOC105374343 or its transcript The disturbing molecule of gene expression or genetic transcription, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, micro- Tiny RNA, antisense nucleic acid, or can express or be formed the building of the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid Object.

Further, the inhibitor is siRNA.

Further, the sequence of the siRNA is as shown in NO.7~14 SEQ ID.

Preferably, the sequence of the siRNA is as shown in NO.9~10 SEQ ID.

The fourth aspect of the present invention provides a kind of method of the drug candidate of screening treatment rectal adenocarcinoma, the method packet It includes:

The system expressed or containing LOC105374343 gene is handled with substance to be screened；With

Detect the expression of LOC105374343 gene in the system；

Wherein, if the substance to be screened can inhibit the level of LOC105374343 gene, show that this is to be screened Substance is the drug candidate for treating rectal adenocarcinoma.

The system is selected from: cell system, subcellular system, solution system, organizational framework, organ systems or animal body System.

The candidate substances include but is not limited to: for LOC105374343 gene or its upstream or downstream gene design Disturbing molecule, nucleic acid inhibitor, small molecule compound etc..

The fifth aspect of the present invention provides any one of following application:

A. application of the reagent described in first aspect present invention in the tool for preparing Diagnosis of Rectal gland cancer；

B. application of the product described in second aspect of the present invention in the tool for preparing Diagnosis of Rectal gland cancer；

Application of the c.LOC105374343 in the computation model of building Diagnosis of Rectal gland cancer；

D.LOC105374343 is in preparation treatment rectal adenocarcinoma or the drug of rectal adenocarcinoma invasion or glandula rectalis transfer Application；

E. composition described in third aspect present invention is in preparation treatment rectal adenocarcinoma or rectal adenocarcinoma invasion or rectum Application in the drug of gland cancer transfer；

Application of the f.LOC105374343 in the drug candidate of screening treatment rectal adenocarcinoma.

G. application of the method described in fourth aspect present invention in the drug candidate of screening treatment rectal adenocarcinoma.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection LOC105374343 gene in rectal adenocarcinoma tissue；

Fig. 2 is to detect siRNA to the silencing efficiency figure of LOC105374343；

Fig. 3 is the influence diagram that CCK8 method detection LOC105374343 is proliferated Rectal Adenocarcinoma Cells；

Fig. 4 is the influence diagram migrated using scratch experiment detection LOC105374343 to rectal adenocarcinoma；

Fig. 5 is the influence diagram invaded using the cell Transwell detection LOC105374343 to Rectal Adenocarcinoma Cells.

Specific embodiment

The present invention after extensive and in-depth study, passes through high-flux sequence and bioinformatic analysis method, detection LncRNA has found wherein there is obvious differential expression in the expression of tumor tissues and cancer beside organism in rectal adenocarcinoma sample LncRNA inquires into its relationship between the occurrence and development of rectal adenocarcinoma, so that the diagnosis and targeted therapy for rectal adenocarcinoma are sought Look for better approaches and methods.By screening, present invention firstly discovers that LOC105374343 conspicuousness up-regulation in rectal adenocarcinoma, And be experimentally confirmed that LOC105374343 is related to the occurrence and development of rectal adenocarcinoma, for rectal adenocarcinoma early diagnosis and control Treatment provides new tumor markers and therapy target.

LOC105374343

The gene of transcription LOC105374343 is to be located at No. 41 areas 6 of dye galianconism of people to take, in the present invention LOC105374343 includes wild type, saltant type or its segment.In an embodiment of the present invention, a kind of representative transcription LOC105374343 base in the nucleotide sequence of LOC105374343 gene such as at present international public nucleic acid database GeneBank Because shown in (XR_001741546.1).

The present invention can use the expression of any method known in the art measurement gene.Those skilled in the art It should be appreciated that the means of measurement gene expression are not importances of the invention.Biological marker can be detected on transcriptional level The expression of object.

Some detections of lncRNA level or quantitative approach are known in the art and are suitable for provided herein Method is to measure the level of biomarker.Illustrative method includes but is not limited to RNA trace (northern blots), core The method of ribonuclease T. protection test and based on PCR.When biomarker is lncRNA molecule, lncRNA sequence or its segment It can be used for preparing the probe of at least partial complementarity.Then using the method for such as based on PCR, RNA blotting (Northern Blotting) or test strips detect any suitable measuring methods such as (dipstick assay), can be in probe in detecting sample LncRNA sequence.

The measuring method can change according to the type of required lncRNA information.Illustrative method includes but unlimited In the method (for example, qRT-PCR) of RNA trace (Northern blots) and based on PCR.The methods of qRT-PCR can also be right The amount of lncRNA carries out accurate quantitative analysis in sample.

Any suitable measurement platform is used equally for determining the presence of lncRNA in sample.For example, measurement can be in test paper Item (dipstick), film (membrane), chip (chip), disk (disk), test-strips (test strip), filter (filter), microballoon (microsphere), slide glass (slide), porous plate (multiwell plate) or optical fiber (optical Fiber form).Measurement system can have the solid support for being attached with nucleic acid corresponding with lncRNA thereon.It is described solid Phase support may include for example plastics, silicon wafer, metal, resin, glass, film, particle, sediment (precipitate), gel, Polymer, thin slice (sheet), ball, polysaccharide, capillary, film (film), plate or slide glass.After the measurement component can be prepared It is packaged together as the kit for detecting lncRNA.

Nucleic acid can be labeled if necessary to make labeled lncRNA group.In general, sample can use Method well-known in the art is marked.In certain embodiments, the sample is fluorescently labeled substance markers.It is exemplary Fluorescent dye include but is not limited to xanthene (xanthene) dyestuff, fluorescein(e) dye, rhodamine, fluorescein isothiocynate (FITC), 6- Fluoresceincarboxylic acid (FAM), 6- carboxyl -2', 4', 7', 4,7- chlordene fluorescein (HEX), 6- carboxyl -4', 5'- bis- Chloro- 2', 7'- dimethoxyfluorescein (JOE or J), N, N, N', N'- tetramethyl -6- carboxyrhodamine (TAMRA or T), 6- carboxylic Base-X- rhodamine (ROX or R), 5- carboxyrhodamine 6G (R6G5 or G5), 6- carboxyrhodamine 6G (R6G6 or G6) and rhodamine 110；Cyanine dye, such as Cy3, Cy5 and Cy7 dyestuff；Alexa dyestuff, such as Alexa-fluor-555；Cumarin, diethylamino Butylcoumariii, umbelliferone；Benzimide dyestuff, such as Hoechst 33258；Phenanthridines dyestuff, such as texas Red (Texas red)；Second ingot dyestuff；Acridine dye；Carbazole dye；Phenoxazine dye；Porphyrin dye；Polymethin dyes, BODIPY dyestuff, quinoline Quinoline dyestuff, pyrene (pyrene), fluorescein chlorotriazine base (fluorescein chlorotriazinyl), R110, Eosin, JOE, R6G, tetramethylrhodamine, lissamine, ROX and naphthofluorescein.

Nucleic acid may be present in the particular addressable position on solid support；Each position corresponds to biomarker LncRNA sequence at least part.

In certain embodiments, 1) lncRNA measurement is the following steps are included: obtain one or more biomarkers Surface-combination probe；2) make lncRNA group miscellaneous under conditions of being enough to provide specific binding with surface-combination probe It hands over；3) nucleic acid being not associated in hybridization step is removed；With the lncRNA of 4) detection hybridization.

Hybridization can carry out under suitable hybridization conditions, and the stringency of the condition can optionally change.Typical item Part is the complementary lncRNA on a solid surface between complementary binding members, i.e., in surface-combination probe and sample Between be enough to generate probe/target compound.

In certain embodiments, using stringent hybridization conditions.Including temperature, salinity, polynucleotides concentration, hybridization The selection of felicity condition including the stringency of time and wash conditions depends on experimental design, including sample source, capturing agent Type, expected complementary degree etc., and can be used as routine experiment means come by those of ordinary skill in the art it is true It is fixed.

After lncRNA hybridization procedures, the polynucleotides that surface is combined are washed to remove unbonded nucleic acid.It can be with It is washed using any convenient washing scheme.In certain embodiments, wash conditions are stringent.Then it can use Standard technique detection target lncRNA hybridizes with probe.

Kit, chip, nucleic acid film item

The present invention provides a kind of kit, the kit can be used for detecting the expression of LOC105374343.

As preferred embodiment, the kit includes the one kind specifically bound with the lncRNA of biomarker Or a variety of probes or primer.

As highly preferred embodiment, the kit also includes washing solution.

As highly preferred embodiment, the kit also includes the reagent for carrying out cross experiment, lncRNA separation Or tools for purification, detection instrument and the positive and negative control.

As further preferred embodiment, the kit also includes the specification using the kit.The examination Agent box can customize for use at home, clinical use or research use.

The kit includes the specific primer pair for expanding LOC105374343；Standard DNA template；PCR reaction Liquid.

Chip in the present invention includes: solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited Needle, the oligonucleotide probe some or all of specifically correspond to shown in LOC105374343 sequence.

The various common used materials in genetic chip field, such as, but not limited to nylon can be used in heretofore described solid phase carrier Film, slide or silicon wafer, unmodified slide, plastic sheet through active group (such as aldehyde radical, amino) modification etc..

The conventional manufacturing method of biochip known in the art can be used in the preparation of the LOC105374343 chip. It, can will be few for example, 5 ' ends of probe are gone here and there containing amido modified poly- dT if solid phase carrier is using modification slide or silicon wafer Nucleotide probe is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence Or array, it is then fixed by standing overnight, so that it may obtain lncRNA chip of the invention.

In the present invention, nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate；The substrate Can be any substrate suitable for immobilized oligonucleotide probe, for example, nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, Silica gel chip, miniature magnetic bead etc..

It includes LOC105374343 that gene detecting kit or genetic chip or nucleic acid film item, which can be used for detecting, in the present invention The expression of multiple genes (for example, multiple genes relevant to rectal adenocarcinoma) including gene, by the multiple of rectal adenocarcinoma Marker is detected simultaneously, is greatly improved the accuracy rate of rectal adenocarcinoma diagnosis.

In the present invention, as those of skill in the art know, it can be implemented in various ways marker water and realize Flat the step of getting up with certain possibility or risk association.Preferably, mathematically composite marker object and one or more other The measurement concentration of marker, and combined value is associated with basic diagnosis problem.Any suitable existing skill can be passed through Art mathematical method combines the measurement of marker levels.

Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a Body associates about the risk of rectal adenocarcinoma or with the other intentional diagnostic uses for helping to assess rectal adenocarcinoma patient.With one Kind preferred mode, such logarithmic function obtain as follows: individual segregation a) being entered group, such as normal person, has rectal adenocarcinoma The individual of risk, the patient with rectal adenocarcinoma etc., b) significant difference between these groups is identified by univariate analysis Marker, c) logarithmic regressions analysis to be to assess the independent difference values that can be used for assessing these difference groups of marker, and d) structure It builds logarithmic function and carrys out composition independency difference value.In such analysis, marker is no longer independent, but represents one Marker combination.

Logarithmic function for marker combination to be got up with disease association is preferably developed using by applied statistical method With the algorithm of acquisition.For example, suitable statistical method is discriminant analysis (DA) (i.e. linear, secondary, regular DA), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (the i.e. logic based on tree Recurrence, CART, random forest method, boosting/bagging method), generalized linear model (i.e. logarithm regression), the side based on principal component Method (i.e. SIMCA), broad sense Additive Model, the method based on fuzzy logic, the method based on artificial neural network and genetic algorithms.Skillfully Technical staff merges in the suitable statistical method of selection to assess marker group of the invention thus to obtain suitable mathematical algorithm Aspect will not be problematic.In one embodiment, for obtaining the statistics side of mathematical algorithm used in assessment rectal adenocarcinoma Method is selected from DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), nonparametric technique (i.e. k- nearest-neighbors Classifier), PLS (partial least square), method (i.e. logistic regression, CART, random forest method, boosting side based on tree Method) or generalized linear model (i.e. logarithm regression).

Inhibitor and pharmaceutical composition

Discovery based on inventor, the present invention provides the inhibitor of LOC105374343 a kind of, the property of the inhibitor Matter has no importance for the present invention, as long as it inhibits the functional expression of LOC105374343 gene, for example, this The inhibitor of invention can be using LOC105374343 gene as target sequence and be able to suppress the interference of LOC105374343 gene Molecule, comprising: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or Form the construction of the shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid.These inhibitor are used as lowering LOC105374343 useful substance can be used for treating rectal adenocarcinoma.

As a kind of preferred embodiment of the invention, the inhibitor of the LOC105374343 is that a kind of LOC105374343 is special Anisotropic siRNA molecule.As used herein, " siRNA " refers to a kind of short-movie section double stranded rna molecule, energy Enough using the lncRNA of homologous complementary sequence as the target specific lncRNA of degradation, this process is exactly RNA interference (RNA Interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, it contains a positive-sense strand and one anti- Adopted chain, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by the positive-sense strand that is separated from each other It is prepared with antisense strand.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, and can pass through annealing thereafter Hybridization, generates the double-stranded RNA compound of synthesis.

When screening effective siRNA sequence, the present inventor is by largely comparing analysis, to find out optimal effective Segment.The present inventor's design has synthesized a variety of siRNA sequences, and they are passed through transfection reagent respectively and transfects Rectal Adenocarcinoma Cells System is verified, and the optimal siRNA of interference effect is selected, in a specific embodiment of the present invention, the sequence of the siRNA such as SEQ Shown in NO.9~10 ID, is further tested in cellular level, as a result prove effectively inhibit cell for the siRNA The expression of middle LOC105374343 gene and the proliferation of Rectal Adenocarcinoma Cells.

Nucleic acid inhibitor of the invention such as siRNA can be with chemical synthesis, can also be by a recombinant nucleic acid structure Expression cassette is prepared after being transcribed into single stranded RNA.The nucleic acid inhibitors such as siRNA, can be by using transfection reagent quilt appropriate It is transported into the cell, or multiple technologies known in the art also can be used and be transported into the cell.

In the present invention, " drug ", " pharmaceutical composition " can be general.As in selectable embodiment, medicine group Close the inhibitor and pharmaceutically acceptable carrier that object includes LOC105374343 gene.Pharmaceutically acceptable carrier includes (but being not limited to): diluent, excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch, sugarcane Sugar etc.；Adhesive such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyvinyl pyrrole Alkanone；Wetting agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and carbonic acid Hydrogen sodium；Sorbefacient quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan fat Acid esters, lauryl sodium sulfate, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier is such as Starch, lactose, bentonite, silica gel, kaolin and soap clay etc.；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol, Boric acid powder etc..

In the present invention, pharmaceutical composition can be used different additives and be prepared, for example, buffer, stabilizer, Bacteriostatic agent, isotonic agent, chelating agent, pH controlling agent and surfactant.

Pharmaceutical composition Orally-administrable of the present invention, parenteral administration are given by sucking spray delivery, part Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral administration or injection Administration.Pharmaceutical composition of the invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.In certain situations Under, medicinal acid, alkali or buffer can be used to adjust the pH of preparation to improve the stabilization of prepared compound or its form of administration Property.Terms used herein parenteral route include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, in breastbone, In bringing up, in damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention Receptor can be given by any approach.

Pharmaceutical composition of the present invention can be administered orally in the form of any peroral dosage form, including but not limited to capsule, piece Agent, emulsion and water slurry, dispersing agent and solution.For oral tablet, common carrier includes lactose and cornstarch.It is general to go back Lubricant such as magnesium stearate is added.In order to be administered with capsules per os, applicable diluent includes lactose and anhydrous corn Starch.When water slurry and/or lotion is administered orally, active component can be suspended or dissolved in oily phase, and and emulsifier And/or suspending agent merges.If necessary, some sweeteners and/or corrigent and/or colorant can be added.Where appropriate, can The dosage unit preparations packet micro-capsule that will be used to be administered orally.For example, by the way that particulate matter is coated or is wrapped in polymer, wax etc. It buries, the preparation can also be prepared and extended or maintained release.Pharmaceutical composition of the invention can be used for reducing endogenic LOC105374343 is overexpressed, by reducing the expression of LOC105374343, so that treatment is raised because LOC105374343 is expressed Caused rectal adenocarcinoma.

In the present invention, the compound of LOC105374343 expression can will be inhibited to act as naked RNA and delivery of agents one It is applied for nucleic acid (such as recombinant plasmid or viral vectors) to subject, the nucleic acid includes the sequence for inhibiting LOC105374343 expression Column.Delivery of agents can be lipophilic agent, polycation, liposome etc..

In the present invention, term " effective quantity " refers to that its dosage is enough to treat disease, to be suitable for any therapeutic treatment Reasonable interests/risk-ratio.The effective dose level of composition can according to the type of subject, the severity of disease, The age of subject and gender, pharmaceutical activity, the sensibility to drug, administration time, administration route, excretion rate, treatment time, With composition associated in drug and medical field other known facts determine.Pharmaceutical composition of the invention can be used alone Or it is administered in combination with other therapeutic agents, and can be sequentially or simultaneously administered with conventional therapeutic agent.It can be used one or more Composition is applied in dosage form.Consider all above-mentioned factors, minimum of the maximum efficiency without causing side effect can shown Lower application composition is most important, which can be readily determined by those skilled in the art.

Pharmaceutical composition of the invention can also be with the drug combination of other treatment rectal adenocarcinoma, and other therapeutic compound can To be administered simultaneously with main active constituent, or even it is administered simultaneously in same composition.

Statistical analysis

In a specific embodiment of the present invention, experiment be all to be completed according to being at least repeated 3 times, result data be all with The mode of mean+SD indicates, using SPSS18.0 statistical software come for statistical analysis, difference between the two It is different to be examined using t, it is believed that there is statistical significance as P < 0.05.

Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention, It is not intended to limit protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1 screens gene marker relevant to rectal adenocarcinoma

1, sample collection

Normal epithelial tissues and rectal adenocarcinoma tissue samples, all cases by 3 rectal adenocarcinoma cancers are collected respectively to perform the operation It is preceding not receive chemotherapy and radiation, without other tumor diseases, autoimmune disease and serious chronic disease, it is normal by cancer on Skin tissue is derived from all patients informed consent away from tumour upper limb 5cm, and passes through the agreement of the committee of organizational ethics.

2, the preparation of RNA sample

The extraction of tissue RNA is carried out using the tissue RNA extracts kit of QIAGEN, concrete operations by specification carries out.

3, total serum IgE is quantitative and purity analysis

The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total 5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

4, construction cDNA library

1) rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kit；

2) fragmentation RNA

To complete RNA sequence, interrupted at random using metal ion, by RNA random fracture at the small of 200bp or so Segment.

3) reversion synthesis cDNA

The building that cDNA library is carried out using Illumina TruseqTM RNA sample Prep Kit, in reverse transcription Under the action of enzyme, using random primer, one chain cDNA of synthesis is inverted by template of lncRNA, when carrying out the synthesis of two chains, dNTPs examination DTTP is replaced with dUTP in agent, making base in the second chain of cDNA includes A/U/C/G.

4) adaptor is connected

End Repair Mix is added to mend the cohesive end of double-strand cDNA at flat end, then adds one in 3 ' ends A base, for connecting the connector of Y-shaped.

5) bis- chain of UNG enzymic digestion cDNA

The second chain of cDNA is digested with UNG enzyme, to make in library only comprising the first chain of cDNA.

5, it is sequenced

Using Illumina X-Ten microarray dataset, 2*150bp sequencing is carried out.

6, high-throughput transcript profile sequencing data analysis

1) trim is carried out to the 5 ' of reads and 3 ' sections with cutadapt, trim falls the base of quality < 20, and it is big to delete N In 10% reads；

2) tophat is compared onto reference genome.Reference genome version used is GRCh38.p7, fasta and gff File download is from NCBI；

3) cuffquant quantifies the expression quantity and normalization output of lncRNA；Cuffquant quantifies the expression quantity of lncRNA And normalization output；

4) cuffdiff compares control group with the differential expression of disease group lncRNA, the screening mark of differential expression lncRNA It is quasi-: p_value < 0.05, | log₂FC|>1。

7, result

The results show that compared with normal epithelial tissues by cancer, expression water of the LOC105374343 in rectal adenocarcinoma tissue Head up display writes up-regulation.

The differential expression of 2 QPCR sequence verification LOC105374343 gene of embodiment

1, large sample QPCR verifying is carried out to LOC105374343 gene differential expression.It is received according to the sample in embodiment 1 Mode set selects rectal adenocarcinoma Carcinoma side normal tissue and each 45, rectal adenocarcinoma tissue.

2, RNA is extracted

RNA sample is extracted using the tissue RNA extracts kit of QIAGEN, concrete operations are detailed in specification.

3、QPCR

1) reverse transcription reaction

LncRNA reverse transcription is carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106), is gone first Except genomic DNA reacts, 5 × gDNA B μ ffer, 2.0 μ l is added in test tube, 1 μ g of total serum IgE adds Rnase Free ddH₂O Make total volume to 10 μ l, 42 DEG C of heating 3min in water-bath, then by 10 × Fast RT B μ ffer, 2.0 μ l, RT Enzyme 1.0 μ l, FQ-RT Primer Mix of Mix, 2.0 μ l, RNase Free ddH₂5.0 μ l of O, is added in above-mentioned test tube after mixing It is mixed together totally 20 μ l, 42 DEG C of heating 15min, 95 DEG C of heating 3min in water-bath.

2) design of primers

QPCR amplimer is designed according to the coded sequence of LOC105374343 gene and GAPDH gene in Genebank, It is synthesized by Bo Maide biotech firm.Specific primer sequence is as follows:

LOC105374343 gene:

Forward primer is 5 '-AGGAGAACAGCATTATTAG-3 ' (SEQ ID NO.1)；

Reverse primer is 5 '-GTTCACTGGACTGATTAG-3 ' (SEQ ID NO.2).

GAPDH gene:

Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；

Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).

3) QPCR amplification is examined

It with SuperReal PreMix Plus (SYBR Green) (article No.: FP205), is expanded, experimental implementation is by production Product specification carries out.

Using 20 μ l reaction systems: 2 × SuperReal PreMix Plus 10 μ l, each 0.6 μ of forward and reverse primer (10 μM) L, 5 × ROX Reference Dye^△2 μ l, 2 μ l of DNA profiling, 4.8 μ l of sterile purified water.3 parallel pipes are arranged in each sample, All amplified reactions are repeated three times the above reliability to guarantee result.

Amplification program are as follows: 95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C 60s, 95 DEG C of 15s).

4) screening of cDNA template concentrations

After each sample cDNA is mixed, 10 times of gradients (10,100,1000,10000,100000 times) are carried out as template Dilution, sample respectively takes 2 μ l to make template after dilution, is expanded respectively with target gene primer and reference gene primer, while 60-95 DEG C of progress melt curve analysis analysis carries out the screening of template concentrations according to amplification efficiency height and the unimodal principle of solubility curve.

According to solubility curve, it can be seen that when 10 times of dilutions of carry out of cDNA, the amplification efficiency of PCR is higher, and dissolution is bent Line is unimodal relatively good.

5) sample RealTime PCR is detected

2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progresss solubility curve analysis, purpose band is determined by melt curve analysis analysis and electrophoresis, 2^-ΔΔCT Method carries out relative quantification.

4, result

QPCR result is as shown in Figure 1, compared with rectal adenocarcinoma Carcinoma side normal tissue, and LOC105374343 is in rectal adenocarcinoma group Middle expression up-regulation is knitted, difference has statistical significance (P < 0.05)；Positive rate=up-regulated expression number of cases/always detect number of cases × 100%=44/45=97.8% prompts LOC105374343 application value with higher in the diagnosis of rectal adenocarcinoma.

The silencing of 3 LOC105374343 gene of embodiment

1, cell culture

Human rectal adenocarcinoma cell strain HRC-99, with the RPMI1640 culture medium containing 10% calf serum and 1%P/S 37 DEG C, 5%CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, uses 0.25% tryptose containing EDTA The passage of enzyme conventional digestion, takes the cell in logarithmic growth phase for testing.

2, the design of siRNA

For the sequence design siRNA of LOC105374343 gene, the siRNA sequence of design is as follows:

The sequence of negative control siRNA-NC:

Positive-sense strand: 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.5),

Antisense strand: 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.6)；

SiRNA1:

Positive-sense strand: 5 '-UUUUGUUUGCUUCAAGAUGUU-3 ' (SEQ ID NO.7),

Antisense strand: 5 '-CAUCUUGAAGCAAACAAAAUG-3 ' (SEQ ID NO.8)；

SiRNA2:

Positive-sense strand: 5 '-AACUAAUGUUUAUAGACAGAU-3 ' (SEQ ID NO.9),

Antisense strand: 5 '-CUGUCUAUAAACAUUAGUUUU-3 ' (SEQ ID NO.10)；

SiRNA3:

Positive-sense strand is 5 '-AAAACUAAUGUUUAUAGACAG-3 ' (SEQ ID NO.11),

Antisense strand is 5 '-GUCUAUAAACAUUAGUUUUAG-3 ' (SEQ ID NO.12)

SiRNA4:

Positive-sense strand is 5 '-UUGUGAUUUUUAUUUUCUCAC-3 ' (SEQ ID NO.13),

Antisense strand is 5 '-GAGAAAAUAAAAAUCACAAUG-3 ' (SEQ ID NO.14)

3, it transfects

Cell is pressed 1 × 10⁴/ hole is inoculated into 24 porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator For 24 hours, in RPMI1640 culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000 Invitrogen company) specification transfection.

Experiment be divided into blank control group (HRC-99), negative control group (siRNA-NC) and experimental group (siRNA1, SiRNA2, siRNA3, siRNA4), wherein the sequence of negative control group siRNA and LOC105374343 gene is dense without homology Degree is the hole 20nM/, while being transfected respectively.

4, QPCR detects the transcriptional level of LOC105374343 gene

1) extraction of cell total rna

The total serum IgE in cell is extracted using QIAGEN cell RNA extracts kit, specific steps are detailed in specification.

2) reverse transcription step is the same as embodiment 2.

3) QPCR amplification step is the same as embodiment 2.

5, result

As a result such as Fig. 2 is shown, compared with HRC-99 and transfection zero load siRNA-NC group, experimental group (siRNA1~4) can drop The level of low LOC105374343, wherein the effect of siRNA2 is the most significant, therefore siRNA2 is selected to carry out subsequent experimental.

The influence that 4 LOC105374343 of embodiment is proliferated Rectal Adenocarcinoma Cells

1, Rectal Adenocarcinoma Cells HRC-99 inoculation is cultured in 6 orifice plates, and is reached 85%-90% to cell density, is used rouge Plastid 2000 transfects siRNA1.More renew culture medium after serum free medium culture 4-6h.

2, it is cultivated for 24 hours after transfecting siRNA1, interference group cell and cellular control unit is digested, transfection is inoculated in 96 orifice plates HRC-99 cell suspension and each control group (100 hole μ l/) afterwards, inoculum density are 5 × 10⁴/L.Culture plate is placed on culture Preculture in case (37 DEG C, 5%CO₂)。

3,10 μ l CCK8 solution are added to every hole.

4, culture plate is placed in incubator and cultivates 1-4h.

5, the absorbance at 490nm is measured using microplate reader.

6, result

As a result as shown in figure 3, compared with the control group, with the extension of growth time, siRNA2 transfection group cell Proliferation subtracts Slow, difference tool is statistically significant (P < 0.05), prompts LOC105374343 related to the proliferation of Rectal Adenocarcinoma Cells, can As possible potential target apply in the treatment of rectal adenocarcinoma.

Rectal Adenocarcinoma Cells proliferation, transfer ability after 5 scratch experiment of embodiment detection transfection siRNA

1, by HRC-99 plating cells in six orifice plates, reach 85%-90% to cell density, transfected using Lipo2000 Culture medium is replaced after siRNA1, free serum culture 4-6h.

2, Rectal Adenocarcinoma Cells culture plate bottom is uniformly covered it is full after, with 1ml pipette tips alignment orifice plate keep vertical angle into Row cell scratch, makes the of same size of each scratch as far as possible.

3, cell original fluid is removed, orifice plate is rinsed with phosphate buffer, by what is formed due to cell scratch Fragment washes away, and serum free medium is added, takes pictures, leaves and takes data.

4, culture plate is placed in cell incubator and is cultivated, and takes out culture plate after cultivating 4-6h, takes pictures again, records number According to calculating scratch healing rate.

5, result

As a result as shown in figure 4, with incubation time growth, the scratch healing rate of siRNA2 group is significantly lower than siRNA-NC Group and blank control group, difference are statistically significant (P < 0.05).This result shows that, inhibit the expression of LOC105374343 can be with The migration of Rectal Adenocarcinoma Cells is reduced, the transfer that can be applied to rectal adenocarcinoma using LOC105374343 as possible target is prompted Treatment.

6 Matrigel Matrigel of embodiment

1, colorectal cancer HRC-99 cell inoculation is cultured in 6 orifice plates, and reaches 85%-90% to cell density, is used Lipo 2000 transfects siRNA1.Culture medium is replaced after serum free medium culture 4-6h.

2, after interference group and control group culture for 24 hours, pancreatin digestion centrifugation is resuspended using serum free medium, is adjusted Cell density is 5 × l0⁵A/ml, the basal medium cell suspension addition by 200 μ l without fetal calf serum have been completed The culture medium that 500 μ l contain 10% fetal calf serum is added in the cell Transwell of Matrigel matrigel, the lower room of 24 orifice plates, carefully Born of the same parents cultivate for 24 hours.

3, cell is dyed after culture using DAPI.Small ventricular cell is first rinsed 2 times with PBS, DAPI work is put into Room temperature dyes 5-20min in liquid.It is rinsed 2 times, be put into fluorescence microscopy under the microscope and counted with PBS.

4, result

As a result as shown in figure 5, compared with the control group, experimental group wears the reduction of theca cell number, illustrate that cell invasion ability is bright Aobvious decline (P < 0.05) prompts that controlling for rectal adenocarcinoma invasion can be applied to using LOC105374343 as possible molecular target It treats.

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

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Claims (10)

1. a kind of detection reagent, which is characterized in that the reagent is the reagent for detecting LOC105374343 level.

2. reagent according to claim 1, which is characterized in that the reagent is selected from:

The probe of specific recognition LOC105374343；Or

The primer of specific amplification LOC105374343.

3. application according to claim 2, which is characterized in that the primer sequence of the specific amplification LOC105374343 As shown in NO.1~2 SEQ ID.

4. a kind of product, which is characterized in that the product includes the described in any item reagents of claim 1-3.

5. product according to claim 4, which is characterized in that the product includes kit, chip, nucleic acid film item.

6. a kind of composition, which is characterized in that the composition includes the inhibitor of a effective amount of LOC105374343.

7. composition according to claim 6, which is characterized in that the inhibitor is siRNA.

8. composition according to claim 7, which is characterized in that the sequence of siRNA is as shown in NO.9~10 SEQ ID.

9. a kind of method of the drug candidate of screening treatment rectal adenocarcinoma, which is characterized in that the described method includes:

The system expressed or containing LOC105374343 gene is handled with substance to be screened；With

Detect the expression of LOC105374343 gene in the system；

Wherein, if the substance to be screened can inhibit the level of LOC105374343 gene, show the substance to be screened It is the drug candidate for treating rectal adenocarcinoma.

10. any one of following application:

A. application of the described in any item reagents of claim 1-3 in the tool for preparing Diagnosis of Rectal gland cancer；

B. application of the product described in claim 4 or 5 in the tool for preparing Diagnosis of Rectal gland cancer；

Application of the c.LOC105374343 in the computation model of building Diagnosis of Rectal gland cancer；

D.LOC105374343 answering in preparation treatment rectal adenocarcinoma or the drug of rectal adenocarcinoma invasion or glandula rectalis transfer With；

E. the described in any item compositions of claim 6-8 are in preparation treatment rectal adenocarcinoma or rectal adenocarcinoma invasion or glandula rectalis Application in the drug of metastasis of cancer；

Application of the f.LOC105374343 in the drug candidate of screening treatment rectal adenocarcinoma.

G. application of the method as claimed in claim 9 in the drug candidate of screening treatment rectal adenocarcinoma.