CN109628594A

CN109628594A - One kind long-chain non-coding RNA relevant to liver cancer and its application

Info

Publication number: CN109628594A
Application number: CN201811607572.0A
Authority: CN
Inventors: 陈圣雄; 徐晓云; 胡静
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2019-04-16
Anticipated expiration: 2038-12-27
Also published as: CN109628594B

Abstract

The invention discloses a kind of long-chain non-coding RNA relevant to liver cancer and its applications, are specifically related to LINC01311 or LINC00189.The present invention has found that differential expression is presented in LINC01311 or LINC00189 in liver cancer tissue by high-flux sequence combination bioinformatic analysis for the first time, and further enlarged sample amount confirms that LINC01311 or the LINC00189 conspicuousness in liver cancer tissue raise.The present invention passes through cell experiment simultaneously and demonstrates the horizontal proliferation that can influence liver cancer cells for changing LINC01311 or LINC00189, illustrates the diagnosing and treating that LINC01311 or LINC00189 can be used as possible molecular marker and target is applied to liver cancer.

Description

One kind long-chain non-coding RNA relevant to liver cancer and its application

Technical field

The invention belongs to biomedicine fields, are related to one kind long-chain non-coding RNA relevant to liver cancer and its application, described Long-chain non-coding RNA is LINC01311 or LINC00189.

Background technique

Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of most common malignant tumour in China, Its death rate ranks cancer-associated tumor death rate third position.There is HCC onset concealment, grade malignancy height, progress rapidly, easily to turn The features such as shifting, bad high, the overall prognosis of case fatality rate, seriously endanger human health.Therefore, it explores liver cancer generation and its early stage turns The specific mechanism moved is for hepatocarcinoma early diagnosis and improves patient's prognosis with important value.Previously it is directed to liver cancer genesis and development Mechanism Study focus mostly in traditional protein coding gene, relate generally to science of heredity, epigenetics and coherent signal are logical The regulation on road.As high throughput sequencing technologies develop, researcher has revealed that the non-coding RNA (Non- in human genome Coding RNA, ncRNA) ratio occupies 95% or more, between encoding histone RNA and functional non-coding RNA it is complicated mutually It acts in the occurrence and development of cancer and plays an important role.Long-chain non-coding RNA (long non-coding RNA, 1ncRNA) as one of important composition ingredient of ncRNA, also increasingly it is taken seriously.

LncRNAs is that molecule amount is greater than 200 nucleotide, the very poor ncRNAs of conservative, and quantity is much larger than MiRNA, lncRNA can adjust gene expression in transcriptional level and post-transcriptional level, and formal and trans- regulation is its two kinds main Transcriptional control mode can target current and distant place gene with this lncRNA respectively.And the mechanism of post-transcriptional level is then main It including the montage to mRNAs, edits, transhipment, translation and degradation.Furthermore genomic imprinting and chromatin remodeling are also that it regulates and controls base The mode of cause.LncRNA point is five classes, just (sense) or antisense (antisense), two-way (bidirectional), is included Between gene.

LncRNA takes part in biological growth, development, the processes such as aging and death, while having also assisted in the generation of multiclass disease Development, but most noticeable or itself and various cancers close relation.More and more related experiments prove special in recent years There is very specific relationship in fixed lncRNA and specific cancer, perhaps the different expressions of these 1ncRNA can be used as The mark of metastases and prognosis.And the HCC as high-incidence tumour is naturally also found that there are a large amount of 1ncRNA is associated. The a large amount of 1ncRNA for testing the expression that notes abnormalities is found the generation with liver cancer in the presence of being associated with, in the transfer and prognosis of liver cancer On also play crucial role (CN201710522694.9, CN201710522693.4).Therefore, liver cancer is actively developed The research for developing related mechanism, finds new molecular marker and therapy target and carries out early intervention and individualized treatment, mention High survival has important clinical value and social effect.

Summary of the invention

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide relevant to hepatocellular carcinoma occurrence and development point Sub- marker by the expression of detection molecules marker and changes its related levels, realize the early diagnosis of liver cancer with Targeted therapy；Molecular marker can also be used for the drug candidate of screening treatment liver cancer simultaneously.

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

The present invention provides application of the reagent of detection gene expression dose in the product for preparing diagnosing hepatocellular carcinoma, institutes State the one or two that gene is selected from LINC01311 or LINC00189.

Further, the reagent includes:

The probe of specific recognition LINC01311 or LINC00189；Or

The primer of specific amplification LINC01311 or LINC00189.

Further, the primer sequence of specific amplification LINC01311 is as shown in NO.1~2 SEQ ID, specific amplification The primer sequence of LINC00189 is as shown in NO.3~4 SEQ ID.

The present invention provides it is a kind of diagnose early hepatocyte cancer product, the product include detection LINC01311 or The reagent of LINC00189 expression, the product include but is not limited to chip, nucleic acid film item, kit.

Further, the reagent includes the probe of specific recognition LINC01311 or LINC00189；Or specific amplification The primer of LINC01311 or LINC00189.

The present invention provides application of the gene in the drug candidate of screening treatment hepatocellular carcinoma, the gene is LINC01311 or LINC00189.

Further, the step of drug candidate of screening treatment hepatocellular carcinoma is as follows:

The cultivating system expressed or containing LINC01311 or LINC00189 is handled with substance to be screened；With

Detect the expression of LINC01311 or LINC00189 gene in the system；

Wherein, if the substance to be screened can inhibit the level of LINC01311 or LINC00189 gene (preferably aobvious Writing reduces, and such as low 20% or more, preferably low 50% or more；More preferably low 80% or more) then shows that the substance to be screened is to control Treat the drug candidate of hepatocellular carcinoma.

Further, the step further include: it is further tested to the drug candidate that previous step obtains and inhibits liver cancer Effect, if test compound have significant inhibitory effect to liver cancer, illustrate the compound be treatment liver cancer drug candidate.

The cultivating system includes but is not limited to cell system, subcellular system, solution system, organizational framework, organ System or animal system (such as animal model, the preferably animal model of non-human mammal, such as mouse, rabbit, sheep, monkey) etc..

The present invention provides application of the gene in the pharmaceutical composition of preparation treatment hepatocellular carcinoma, the gene is selected from The one or two of LINC01311 or LINC00189.

Further, described pharmaceutical composition includes the inhibitor of LINC01311 or LINC00189.

Wherein, the inhibitor of the LINC01311 or LINC00189 is selected from: with LINC01311 or LINC00189 or its Transcript is target sequence and the disturbing molecule for being able to suppress LINC01311 or LINC00189 gene expression or genetic transcription, packet It includes: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or be formed institute State the construction of shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid.

Preferably, the inhibitor is siRNA.

The sequence of the more preferably described siRNA is as shown in NO.9~20 SEQ ID.

The present invention provides a kind of pharmaceutical compositions, which is characterized in that described pharmaceutical composition include LINC01311 or The inhibitor of LINC00189.

Preferably, the inhibitor is siRNA.

More preferably, the siRNA has the sequence as shown in NO.9~20 SEQ ID.

Further, the composition further includes its other medicine class compatible with the inhibitor and pharmaceutically acceptable Carrier and/or auxiliary material.

Pharmaceutically acceptable carrier and/or auxiliary material include but is not limited to) diluent, adhesive, surfactant, cause Humectant, absorption carrier, lubricant, filler, disintegrating agent.

Detailed description of the invention

Fig. 1 is the expression figure using QPCR detection LINC01311 or LINC00189 in patients with hepatocellular carcinoma；Its In, figure A is LINC01311, and figure B is LINC00189.

Fig. 2 is expression influence diagram of the detection transfection siRNA to LINC01311 in hepatocellular carcinoma cells or LINC00189；Its In, figure A is LINC01311, and figure B is LINC00189.

Fig. 3 is the influence diagram using CCK8 detection LINC01311 or LINC00189 to hepatoma cell proliferation；Wherein, scheme A It is LINC01311, figure B is LINC00189.

Specific embodiment

The present invention after extensive and in-depth study, by high-flux sequence method, detects by Tissues of Hepatocellular Carcinoma and cancer The expression of lncRNA in tissue, discovery wherein with the lncRNA segment of obvious differential expression, inquire into itself and hepatocellular carcinoma Generation between relationship, so that the early detection and targeted therapy for hepatocellular carcinoma find better approaches and methods.Pass through Screening, present invention firstly discovers that LINC01311 or LINC00189 conspicuousness up-regulation in hepatocellular carcinoma, and large sample is into one Step demonstrates the above results；Meanwhile cell experiment proves, the expression for changing LINC01311 or LINC00189 can influence The proliferative capacity of hepatocellular carcinoma cells；Prompt LINC01311 or LINC00189 can be used as the morning that target is applied to hepatocellular carcinoma Phase diagnosis and precisely medical treatment.

LINC01311 or LINC00189 gene

LINC01311 or LINC00189 gene be located at 2 area 1 of No. 22 chromosome long arms take it is long with No. 21 chromosomes 2 area 1 of arm takes, and the ID number in international public nucleic acid database GeneBank is respectively 100652736 and 193629.The present invention In LINC01311 or LINC00189 include wild type, saltant type or its segment.In an embodiment of the present invention, a kind of representative Property people's LINC01311 or LINC00189 gene nucleotide sequence respectively such as public nucleic acid database international at present In GeneBank shown in LINC01311 (NR_103767.1) or LINC00189 (NR_027072.2).Of the invention LINC01311 or LINC00189 nucleotide full length sequence or its segment can usually use PCR amplification method, recombination method or artificial conjunction At method obtain.

In the present invention, it can use any method known in the art measurement gene expression.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 table of LINC01311 or LINC00189 It reaches.

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 LINC01311 or LINC00189；Standard DNA template； PCR reaction solution.

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 LINC01311 or LINC00189 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 routine of biochip known in the art can be used in the preparation of the LINC01311 or LINC00189 chip Manufacturing method.For example, amido modified poly- dT is contained at 5 ' ends of probe if solid phase carrier is using modification slide or silicon wafer Oligonucleotide probe, can be configured to solution by string, then point sample instrument be used on modification slide or silicon wafer, to be arranged in its point pre- Fixed sequence or array, 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..

In the present invention gene detecting kit or genetic chip or nucleic acid film item can be used for detect include LINC01311 or The expression of multiple genes (for example, multiple genes relevant to liver cancer) including LINC00189 gene, by the multiple of liver cancer Marker is detected simultaneously, is greatly improved the accuracy rate of diagnosing cancer of liver.

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 liver cancer or with the other intentional diagnostic uses for helping to assess liver cancer patient.With a kind of preferred Mode, such logarithmic function obtain as follows: individual segregation a) being entered group, such as normal person, the individual for having liver cancer risk, tool Have the patient etc. of liver cancer, b) marker of the significant difference between these groups, c are identified by univariate analysis) logarithm returns Return analysis to assess the independent difference value that can be used for assessing these difference groups of marker, and d) building logarithmic function is only to combine Vertical difference value.In such analysis, marker is no longer independent, but represents a 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 statistical method choosing of mathematical algorithm used in assessment liver cancer From DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), (i.e. k- nearest-neighbors are classified for nonparametric technique Device), PLS (partial least square), the method (i.e. logistic regression, CART, random forest method, propelled method) based on tree or Generalized linear model (i.e. logarithm regression).

Inhibitor and pharmaceutical composition

Discovery based on inventor, the present invention provides the inhibitor of LINC01311 or LINC00189 a kind of, the suppression The property of preparation has no importance for the present invention, as long as it inhibits the functional expression of LINC01311 or LINC00189 gene , for example, inhibitor of the invention can be using LINC01311 or LINC00189 gene as target sequence and can press down The disturbing molecule of LINC01311 or LINC00189 gene processed, comprising: shRNA (children purpura nephritis), siRNA (siRNA), DsRNA, Microrna, antisense nucleic acid, or can express or be formed the shRNA, siRNA, dsRNA, Microrna, antisense core The construction of acid.These inhibitor can be used for treating liver as lowering the useful substance of LINC01311 or LINC00189 Cell cancer.

As a kind of preferred embodiment of the invention, the inhibitor of the LINC01311 or LINC00189 are a kind of The siRNA molecule of LINC01311 or LINC00189 specificity.As used herein, " siRNA " refers to one Kind short-movie section double stranded rna molecule, can be using the mRNA of homologous complementary sequence as the target specific mRNA of degradation, this process is just It is RNA interference (RNA interference) process.SiRNA can be prepared into the form of double-strandednucleic acid, it contains one Positive-sense strand and an antisense strand, this two chains only form double-strand under conditions of hybridization.One double-stranded RNA compound can be by phase It is prepared by the positive-sense strand and antisense strand that mutually separate.Therefore, for example, complementary positive-sense strand and antisense strand are chemical synthesis, Thereafter the double-stranded RNA compound of synthesis can by anneal, be generated.

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 respectively transfection reagent transfecting hepatoma cells system into Row verifying, selects the optimal siRNA of interference effect, in a specific embodiment of the present invention, the sequence of the siRNA such as SEQ ID Shown in NO.9~20, is further tested in cellular level, as a result prove effectively inhibit in cell the siRNA The expression of LINC01311 or LINC00189 gene and the proliferation of liver cancer 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.

Pharmaceutical composition

As in selectable embodiment, pharmaceutical composition includes the inhibitor of LINC01311 or LINC00189 gene And pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier includes but is not limited to diluent, adhesive, surface Activating agent, Humectant, absorption carrier, lubricant, filler, disintegrating agent.

Wherein, diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive such as starch, pregelatinated form sediment Powder, dextrin, maltodextrin, sucrose, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose, ethyl cellulose, poly- second Enol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methyl Cellulose etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, lauryl sodium sulfate, stearic acid list glycerol Ester, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier such as starch, lactose, bentonite, silica gel, kaolin and soap Clay etc.；Lubricant such as zinc stearate, glycerin monostearate, polyethylene glycol, talcum powder, calcium stearate and magnesium, polyethylene glycol, Boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, Dan Yuegui sucrose acid ester, laruyl alcohol sulfuric acid Sodium, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery), xylitol, sorbierite, wheat Bud sugar, erythrose, microcrystalline cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp Polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate etc.；Disintegrating agent such as cross-linked ethylene pyrrolidones, sodium carboxymethyl starch, low Replace hydroxypropyl methyl, croscarmellose sodium, soybean polyoses etc..

Pharmaceutical composition in the present invention can also include stabilizer, fungicide, buffer, isotonic agent, chelating agent, pH control The additives such as preparation and surfactant.Wherein, stabilizer includes that Human serum proteins, l-amino acid, sugar and cellulose are derivative Object.L-amino acid can also include any one in glycine, cysteine and glutamic acid.Carbohydrate includes monosaccharide, such as Portugal Grape sugar, mannose, galactolipin, fructose etc.；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, malt Sugar, lactose etc.；Polysaccharide, such as glucan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.It is fine Tieing up plain derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose And sodium cellulose glycolate.Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, mountain Pears glycan monoacyl ester, fatty glyceride.Additive buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid And corresponding salt (their alkali metal or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes Potassium chloride, sodium chloride, sugar and glycerol.Chelating agent includes sodium ethylene diamine tetracetate and citric acid.

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

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens gene marker relevant to hepatocellular carcinoma

1, sample collection

The cancerous tissue and corresponding cancer beside organism's sample for collecting 35 patients with hepatocellular carcinoma, therefrom randomly select 5 into Row high-flux sequence, the equal informed consent of patient, the acquirement of above-mentioned all samples pass through the agreement of the committee, organizational ethics.

2, the preparation of RNA sample

The extraction of tissue RNA is carried out using the tissue RNA extracts kit of QIAGEN, the specific steps of by specification carry out Operation.

3, the quality analysis of RNA sample

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, rRNA is removed

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

5, construction cDNA library

The building of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit By specification carries out.

6, upper machine sequencing

CDNA library is sequenced using Illumina X-Ten microarray dataset, concrete operations by specification carries out.

7, high-throughput transcript profile sequencing data analysis

The lncRNA for being not easy to detect is deleted, carries out bioinformatic analysis using the DESeq2 in R-3.3.3 kit, According to reference genome hg38, Ensembl Gene ID is switched into Gene Symbol, carries out the screening of differential expression lncRNA, The screening criteria of differential expression lncRNA: FDR < 0.05, abs (log₂FC)>1。

8, result

RNA-seq is the results show that expression quantity of the LINC01311 or LINC00189 gene in Tissues of Hepatocellular Carcinoma is significant Higher than control group (cancer beside organism), LINC01311 or LINC00189 is prompted to can be used as possible detection target thin applied to liver The diagnosis of born of the same parents' cancer.

The differential expression of 2 QPCR sequence verification LINC01311 or LINC00189 gene of embodiment

1, to the 35 patient's cancerous tissue samples and cancer beside organism's sample of collection to LINC01311 or LINC00189 gene Differential expression carries out large sample QPCR verifying.

2, RNA is extracted

3, reverse transcription:

1) by the total serum IgE template of 1 μ g and 2 10 × buffers of μ l, 2 μ l dATP (10mM), 0.5 μ l polyA polymerase, 0.5 μ l ribalgilase (RNase) inhibitor and deoxyribonuclease water (RNase free water) mixing, volume are finally 20 μ l, 37 DEG C of incubation 1h；

2) it is added 1 μ l 0.5 μ g/ μ l Oligo (dT) specific RT primer into reaction tube, 70 DEG C of incubation 5min, immediately It sets and is incubated at least 2min on ice；

3) by reaction mixture and 45 × buffers of μ l, 1 μ l dNTP (10mM), 0.5 μ l M-MLV reverse transcriptase, 0.5 μ L ribalgilase (RNase) inhibitor, 10 μ l polyA reaction mixtures and 4 μ l deoxyribonuclease water (RNase free Water it) mixes, 42 DEG C of incubation 1h.

4, QPCR amplification is examined

1) design of primers

Expanded according to the coded sequence of LINC01311 or LINC00189 gene and GAPDH gene design QPCR in Genebank Increase primer, is synthesized by Bo Maide biotech firm.Specific primer sequence is as follows:

The primer sequence of LINC01311:

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

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

The primer sequence of LINC00189:

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

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

The primer sequence of GAPDH:

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

Reverse primer is 5 '-TTAATCCACTGGTTATCA-3 ' (SEQ ID NO.6).

2) reaction system

25 μ l reaction systems: 12.5 μ l of SYBR Green polymerase chain reaction system are prepared, forward and reverse primer (5 μM) is each 1 μ l, template cDNA 2.0 μ l, ddH₂O 8.5μl.Operations are carried out on ice.

3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times the above reliability to guarantee result.

3) reaction condition

95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 45 circulations.Using SYBR Green as fluorescent marker, On Light Cycler fluorescence quantitative PCR instrument carry out PCR reaction, using GAPDH as reference gene, by melt curve analysis analysis with Electrophoresis determines that purpose band, Δ Δ CT method carry out relative quantification.

5, result

As a result as shown in Figure 1, compared with cancer beside organism, LINC01311 or LINC00189 gene is in Tissues of Hepatocellular Carcinoma There is statistical significance (P < 0.05) prompt LINC01311 or LINC00189 can be used as Testing index for expression up-regulation, difference Auxiliary diagnosis applied to hepatocellular carcinoma.

The silencing of 3 LINC01311 or LINC00189 gene of embodiment

1, cell culture

Human hepatocellular carcinoma Cell Line HepG2, with the culture medium DMEM containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5% CO₂, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is conventional Had digestive transfer culture.

2, siRNA is designed

For the sequence design siRNA of LINC01311 or LINC00189 gene, design negative control siRNA-NC, The sequence of the siRNA of LINC01311 and LINC00189, siRNA-NC interfere LINC01311 as shown in NO.7~8 SEQ ID SiRNA sequence as shown in NO.9~14 SEQ ID, interfere the sequence such as SEQ ID NO.15 of the siRNA of LINC00189~ Shown in 20.Particular sequence information is as shown in the table:

The siRNA sequence of table 1 LINC01311 or LINC00189

3, it transfects

Cell is pressed 2 × 10⁵/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO₂Cell culture in incubator 24h；In DMEM culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 3000 Invitrogen company) specification transfection.

Experiment be divided into blank control group (HepG2), negative control group (siRNA-NC) and experimental group (20nM) (siRNA1~ 6), wherein the sequence of negative control group siRNA-NC and LINC01311 and LINC00189 gene without homology, concentration 20nM/ Hole, while being transfected respectively.

4, QPCR detects the expression of LINC01311 or LINC00189 gene

1) extraction of cell total rna

Cell total rna is extracted using the cell RNA extracts kit of QIAGEN, concrete operations are referring to 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 control group HepG2, transfection zero load siRNA-NC group, experimental group (siRNA1~6) Can reduce the expression of LINC01311 or LINC00189, wherein siRNA1 and siRNA4 interference LINC01311 or The effect of LINC00189 is the most significant, therefore selects siRNA1 and siRNA4 respectively as LINC01311's and LINC00189 Inhibitor carries out subsequent experiment.

4 CCK8 of embodiment detects cell proliferation experiment

1, cell culture and transfection procedure are the same as embodiment 4

2, CCK8 detects cell Proliferation

By the HepG2 cell inoculation of logarithmic proliferation phase in 96 orifice plates, every hole 2 × 10³Cell is divided into three groups by a cell, It is blank control group, transfection siRNA-NC group, transfection siRNA1 and transfection siRNA4 group respectively, every group sets 6 multiple holes；Exist respectively Transfection 0h, for 24 hours, 10 hole μ l/ CCK8 reagents are added after 48h, 72h, 96h, be put into incubator be incubated for 1h after examined using microplate reader Survey the light absorption value of A450.

3, 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.

4, result

It is shown in Fig. 3 as the result is shown: blank control group is transfected with unloaded group no significant difference (P > 0.05) of transfection The proliferative cell of siRNA1 and siRNA4 group be significantly lower than control group proliferative cell, difference have statistical significance (P < 0.05), the above results show that LINC01311 or LINC00189 are related with the proliferation of liver cancer cells, prompt to inhibit LINC01311 Or LINC00189 may inhibit the development of liver cancer.

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

Sequence table

<110>No.2 Hospital, Hebei Medical Univ.

<120>a kind of long-chain non-coding RNA relevant to liver cancer and its application

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

tggtcaatgg agaattaag 19

<210> 2

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gcccttatta cactgttg 18

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tctttgattg actgattctt t 21

<210> 4

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ttgaggaaca catctgaa 18

<210> 5

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ataggtctcg tattgtaat 19

<210> 6

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ttaatccact ggttatca 18

<210> 7

<211> 19

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 7

uucuccgaac gugucacgu 19

<210> 8

<211> 19

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 8

uucuccgaac gugucacgu 19

<210> 9

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 9

uuauuuccuc uguguuuuca c 21

<210> 10

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gaaaacacag aggaaauaaa a 21

<210> 11

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 11

acuuaauucu ccauugacca g 21

<210> 12

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ggucaaugga gaauuaagug g 21

<210> 13

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 13

uuuuuuuacu auuuuccuca c 21

<210> 14

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 14

gaggaaaaua guaaaaaaaa a 21

<210> 15

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 15

agacuaaggc aaguuucagc a 21

<210> 16

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cugaaacuug ccuuagucuu u 21

<210> 17

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 17

uguaaucuca gcacuuuggg a 21

<210> 18

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 18

ccaaagugcu gagauuacag a 21

<210> 19

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 19

aaacauguuc cuuuccuugc a 21

<210> 20

<211> 21

<212> RNA

<213>artificial sequence (Artificial Sequence)

<400> 20

caaggaaagg aacauguuug a 21

Claims

1. detecting application of the reagent of gene expression dose in the product for preparing diagnosing hepatocellular carcinoma, which is characterized in that described Gene is selected from the one or two of LINC01311 or LINC00189.

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

The probe of gene described in specific recognition；Or

The primer of gene described in specific amplification.

3. a kind of product for diagnosing early hepatocyte cancer, which is characterized in that the product includes detection base described in claim 1 The reagent of the expression of cause.

4. product according to claim 3, which is characterized in that the reagent includes the spy of gene described in specific recognition Needle；Or the primer of gene described in specific amplification.

5. product according to claim 4, which is characterized in that the primer sequence of the specific amplification LINC01311 is such as Shown in NO.1~2 SEQ ID, the primer sequence of specific amplification LINC00189 is as shown in NO.3~4 SEQ ID.

6. application of the gene in the drug candidate of screening treatment hepatocellular carcinoma, which is characterized in that the gene is LINC01311 Or LINC00189.

7. application of the gene in the pharmaceutical composition of preparation treatment hepatocellular carcinoma, which is characterized in that the gene is selected from The one or two of LINC01311 or LINC00189.

8. application according to claim 7, which is characterized in that described pharmaceutical composition include LINC01311 or The inhibitor of LINC00189.

9. application according to claim 8, which is characterized in that the inhibitor is siRNA.

10. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes the inhibition of LINC01311 or LINC00189 Agent, it is preferred that the inhibitor is siRNA；More preferably, the siRNA has the sequence as shown in NO.9~20 SEQ ID Column.