CN109735624A - Application of the gene marker in diagnosis of thyroid cancer - Google Patents

Abstract

The application that the invention discloses gene markers in diagnosis of thyroid cancer, the gene marker are PLEKHN1.The invention discloses PLEKHN1 to express up-regulation in thyroid cancer patients, and the expression by detecting PLEKHN1 may determine that whether subject suffers from thyroid cancer.The present invention discloses application of the PLEKHN1 in the computation model of building prediction thyroid cancer.

Description

Application of the gene marker in diagnosis of thyroid cancer

Technical field

The invention belongs to biomedicine fields, are related to application of the gene marker in diagnosis of thyroid cancer, specifically relate to And application of the gene marker PLEKHN1 in follicular thyroid papillary carcinoma.

Background technique

Thyroid cancer is one of Chinese faster malignant tumour of growth rate in recent years, and is with thyroid papillary carcinoma It is main.Thyroid papillary carcinoma (papillary thyroid carcinoma, PTC) can learn feature according to different mirror undertissues It is subdivided into a variety of hypotypes wherein classic thyroid papillary carcinoma (classical of papillary thyroid Carcinoma, CPTC) and follicular thyroid papillary carcinoma (follicular variant of papillary thyroid Carcinoma, FVPTC) it is most common two kinds, FVPTC again can be according to whether there is or not coatings to be further divided into coating type (encapsulated follicular variant of papillary thyroid carcinoma, EFVPTC) and non-packet Membranous type (nonencapsulated follicular variant of papillary thyroid carcinoma, NEFVPTC).FVPTC accounts for about the 9%-41% of PTC.The diagnosis of FVPTC obviously rises in recent years, has caused clinical workers It to its attention and has carried out many researchs but has also still remained many yet unresolved issues, more such as FVPTC preoperative diagnosis Difficulty, as the diagnostic method that thyroid malignancy clinical guidelines are recommended, i.e. FNA, which is checked, to be asked there is also sensibility is lower Topic, remain to be discovered efficient preoperative diagnosis method；Secondly, currently there is an urgent need to the pathology of clear FVPTC and biological property, into The formulation of one step guiding clinical treatment scheme.

Thyroid cancer is a kind of multifactorial disease influenced by environment and gene, and the major influence factors being currently known include The variation of ionising radiation exposure, fat, gene and epigenetics.Different crowd, not agnate thyroid gland in world wide The disease incidence of cancer differs greatly, and prompts the generation of thyroid cancer related with gene genetic, social usage factor, but its pathogenic factor And there is also a large amount of problems to need to be solved for pathogenic mechanism.With the development of molecular biology, to the research of tumor pathogenesis Also it makes some progress.The abnormal expression and tumour of the difference of gene structure, the change of gene function and gene product Occurrence and development there are close connections.Find gene marker relevant to thyroid papillary carcinoma, especially follicular The gene marker of thyroid papillary carcinoma has the precision diagnosis and personalized treatment of realizing thyroid cancer important Meaning.

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 a kind of analysis marks of Diagnosis of Thyroid Carcinoma Object-PLEKHN1 gene.

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

The present invention provides application of the reagent of detection PLEKHN1 in the product for preparing Diagnosis of Thyroid Carcinoma.

Further, the reagent is selected from:

The probe of specific recognition PLEKHN1 gene；Or

The primer of specific amplification PLEKHN1 gene；Or

Specifically bind the bonding agent of the albumen of PLEKHN1 gene coding.

Probe in the present invention for PLEKHN1 gene can be DNA, RNA, DNA-RNA chimera, PNA or other spreads out Biology.There is no limit appoint as long as completing specific hybrid, specifically binding with purpose nucleotide sequence the length of the probe What length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe Degree can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Since different probe lengths is to hybridization Efficiency, signal specificity have different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than 30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary sequences Most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

In the present invention specifically bind PLEKHN1 gene coding albumen bonding agent such as protein PLEKHN1 by Body, the agglutinin of conjugated protein PLEKHN1, the antibody for protein PLEKHN1, the peptide for protein PLEKHN1 are anti- Body (peptidebody), the agent of bispecific dual combination or bispecific antibody form.The specific example of specific binding agent is Peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repeat protein, Kunitz type domain, antibody, single domain antibody And monovalent antibody fragments.In a specific embodiment of the present invention, the specific binding agent is PLEKHN1 specific antibody.

Further, the primer sequence of the specific amplification PLEKHN1 gene such as SEQ ID NO.3 and SEQ ID NO.4 It is shown.

Further, the thyroid cancer is follicular thyroid papillary carcinoma.

The present invention provides a kind of product of PLEKHN1 expression in vitro detection sample, the product include chip, Kit or film item.

Further, the chip includes genetic chip, protein chip, and the genetic chip includes for detecting PLEKHN1 The oligonucleotide probe for PLEKHN1 gene of gene transcription level, the protein chip include the special of PLEKHN1 albumen Property antibody or ligand；The kit includes gene detecting kit, protein detection kit, the gene detecting kit packet The reagent or chip for detecting PLEKHN1 gene transcription level are included, the protein detection kit includes for detecting The reagent or chip of PLEKHN1 protein expression level；The film item includes that substrate and the specificity being fixed in the substrate are known The probe of other PLEKHN1；The substrate can be any substrate suitable for immobilized oligonucleotide probe, such as nylon membrane, nitric acid Cellulose membrane, polypropylene screen, sheet glass, silica gel chip, miniature magnetic bead etc..

Further, the kit include by RT-PCR method, qRT-PCR method, biochip test method, southern blotting technique method, The reagent of hybridization in situ, Western blot detection PLEKHN1 gene or protein expression level.

Further, the reagent with qRT-PCR method detection PLEKHN1 gene expression dose includes such as SEQ ID NO.3 With the primer sequence of specific amplification PLEKHN1 gene shown in SEQ ID NO.4.

Heretofore described chip, kit or film item can be used for detecting multiple bases including PLEKHN1 gene The expression of cause and its expression product (for example, multiple genes relevant to thyroid cancer).By multiple marks of thyroid cancer Object is detected simultaneously, is greatly improved the accuracy rate of diagnosis of thyroid cancer.

The present invention provides application of the above-mentioned product in the tool for preparing Diagnosis of Thyroid Carcinoma.

The present invention provides application of the PLEKHN1 in the computation model of building prediction thyroid cancer.

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 thyroid cancer or with the other intentional diagnostic uses for helping to assess thyroid cancer patients.With one Kind preferred mode, such logarithmic function obtain as follows: individual segregation a) being entered group, such as normal person, has thyroid cancer The individual of risk, the patient with thyroid cancer 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 thyroid cancer 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).

The present invention provides application of the PLEKHN1 in the pharmaceutical composition of preparation treatment thyroid cancer.

Further, described pharmaceutical composition includes the inhibitor of PLEKHN1 functional expression.The inhibitor refers to any The activity that PLEKHN1 albumen can be reduced, the stability for reducing PLEKHN1 gene or albumen, the expression for lowering PLEKHN1 albumen, It reduces PLEKHN1 albumen effective acting time or inhibits the substance of the transcription and translation of PLEKHN1 gene, these substances For the present invention, as the substance useful for downward PLEKHN1, so as to for preventing or treating thyroid cancer.Such as institute The inhibitor stated includes nucleic acid inhibitor, protein inhibitor, proteolytic enzyme, protein binding molecule.Wherein nucleic acid inhibitor selects From: using PLEKHN1 or its transcript as target sequence and it is able to suppress the disturbing molecule of PLEKHN1 gene expression or genetic transcription, Include: shRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or be formed The shRNA, siRNA, dsRNA, Microrna, antisense nucleic acid construction.Protein binding molecule is selected from: with PLEKHN1 The substance that protein-specific combines, is such as able to suppress the antibody or ligand of PLEKHN1 protein active.

The inhibitor of PLEKHN1 can be given by liposome in the present invention, and the effect of the liposome is by medicine target To in specific tissue, and the half-life period of increase drug.Liposome includes emulsifier, foaming agent, liquid fatty substance, solid-state fat Matter, insoluble monolayer, phospholipid dispersions, surfactant etc..Can also include in the liposome can be with the cell of targeting In acceptor molecule combine or other therapeutic or immunogenic composition.

Drug of the invention can also can be with master with the drug combination of other treatment thyroid cancer, other therapeutic compound The active constituent (for example, inhibitor of PLEKHN1) wanted is administered simultaneously, or even is administered simultaneously in same composition.It can be with Other therapeutic compounds are individually given with individual composition or the dosage form different from main active constituent.Mainly at Dividing the Fractional of (such as PLEKHN1 inhibitor) can be administered simultaneously with other therapeutic compounds, and other dosage can be single Solely administration.

The gene I/D of PLEKHN1 is 84069, and in the present invention, PLEKHN1 includes wild type, saltant type or its segment.This Field technical staff knows, when carrying out sequencing analysis, can compare primitive sequencer result onto the reference genome of people, therefore PLEKHN1 in the selection result may include different transcripts, as long as the PLEKHN1 on reference genome can be compared (gene I/D: 84069).There are three hypotypes by the PLEKHN1 having disclosed at present: PLEKHN1 hypotype b (NM_ 001160184.2), PLEKHN1 hypotype c (NM_001367552.1), PLEKHN1 hypotype a (NM_032129.3).In the present invention In, a kind of representative PLEKHN1 gene order is as shown in NM_001160184.2.

The advantages of the present invention:

Present invention firstly discovers that the new molecular marker-PLEKHN1 gene of thyroid cancer a kind of, tested by detecting The expression of PLEKHN1 in person's parathyroid tissue, it can be determined that whether subject suffers from thyroid cancer, to instruct clinical doctor Teacher provides prevention scheme or therapeutic scheme to subject；The diagnosing and treating that disease is realized using molecular marked compound, is compared Traditional means have higher specificity and sensitivity.

Detailed description of the invention

Fig. 1 shows the expression figure using QPCR detection PLEKHN1 gene in human thyroid carcinoma.

Specific embodiment

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 thyroid cancer

1, sample collection

4 follicular Papillary Thyroid Carcinomas and cancer beside organism's sample are respectively collected, other malignant tumor patients are excluded, The acquirement of above-mentioned all samples passes through the agreement of the committee, organizational ethics.

2, the preparation of RNA sample (utilizesMiRNA kit is operated)

The tissue of above-mentioned acquisition shred after put into liquid nitrogen in and be ground to it is powdered, according in kit specification extract Separate RNA.Specific step is as follows:

1) it is added in tissue homogenate or cellReagent II 1ml；

2) it is placed at room temperature for 3min, is aggressively shaken 15s after 0.2ml chloroform is added；

3) be placed in prevents 10min on ice；

4) 12000g, 4 DEG C of centrifugation 15min；

5) water phase of transfer 80% enters in new 2ml EP pipe, and the dehydrated alcohol of 1/2 amount, shaking is added；

6) aforesaid liquid less than 700 μ l is transferred toRNA Mini column, 10000g room temperature after shaking It is centrifuged 60s.

7) toRNA Mini column is added 500 μ l RWC Wash Buffer, 10000g and is centrifuged 30s；

8) 500 μ l RWB Wash Buffer, 10000g centrifugation 30s are added, take maximum centrifugal complete after being repeated twice It is dryRNA Mini column；

9) the DEPC water that 15 μ l are preheated to 70 DEG C is added to pillar, is centrifuged at full speed after being placed at room temperature for 2min.

10) quantitative and purity analysis is carried out to total serum IgE

3, construction cDNA library

The rRNA in total serum IgE is removed using Ribo-Zero kit；It is using metal ion that complete RNA is random It is broken into the small fragment of 200bp or so；CDNA text is carried out using the TruseqTM RNA sample Prep Kit of Illumina The building in library.

4, it is sequenced

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

5, high-throughput transcript profile sequencing data analysis

Bioinformatic analysis is carried out to sequencing result, is carried out using reads number of the DESeq packet in R software to gene Analysis, sets screening criteria as P < 0.05.

6, result

RNA-seq is the results show that expression quantity of the PLEKHN1 gene in follicular Papillary Thyroid Carcinoma is significantly high Expression quantity in control.

The differential expression of embodiment 2QPCR sequence verification PLEKHN1 gene

1, large sample QPCR verifying is carried out to PLEKHN1 gene differential expression.According to the sample collection mode in embodiment 1 29 cancerous tissue samples and cancer beside organism's sample are collected respectively.

2, RNA extraction step is the same as embodiment 1

3、QPCR

1) reverse transcription reaction

LncRNA reverse transcription, reactant are carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106) System and reaction condition are as shown in table 1.

1 reverse transcription reaction system of table and reaction condition

2) design of primers

QPCR amplimer is designed according to the coded sequence of PLEKHN1 gene and GAPDH gene in Genebank, wherein Design of primers is carried out to the common region of PLEKHN1 different subtype, specific primer sequence is as follows:

GAPDH gene:

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

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

PLEKHN1 gene:

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

Reverse primer is 5 '-CCCTTCTTGAACACACTC-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, reagent and shown in reaction system such as table 2.

2 QPCR amplification reaction system of table and reaction condition

4) 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

For QPCR result as shown in Figure 1, compared with cancer beside organism, PLEKHN1 expresses significant up-regulation in human thyroid carcinoma, Difference has statistical significance (P < 0.05), consistent with high-flux sequence result；Wherein expression up-regulation has 29, prompts PLEKHN1 application value with higher in the diagnosis of thyroid 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.

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Claims (10)

1. detecting application of the reagent of PLEKHN1 in the product for preparing Diagnosis of Thyroid Carcinoma.

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

The probe of specific recognition PLEKHN1 gene；Or

The primer of specific amplification PLEKHN1 gene；Or

Specifically bind the bonding agent of the albumen of PLEKHN1 gene coding.

3. application according to claim 2, which is characterized in that the primer sequence of the specific amplification PLEKHN1 gene As shown in SEQ ID NO.3 and SEQ ID NO.4.

4. application according to claim 1-3, which is characterized in that the thyroid cancer is follicular thyroid gland cream Head cancer.

5. the product of PLEKHN1 expression in a kind of vitro detection sample, which is characterized in that the product includes chip, examination Agent box or nucleic acid film item.

6. product according to claim 5, which is characterized in that the chip includes genetic chip, protein chip, the base Because chip includes the oligonucleotide probe for PLEKHN1 gene for detecting PLEKHN1 gene transcription level, the albumen Chip includes the specific antibody or ligand of PLEKHN1 albumen；The kit includes gene detecting kit, Protein Detection examination Agent box, the gene detecting kit include the reagent or chip for detecting PLEKHN1 gene transcription level, the albumen Detection kit includes the reagent or chip for detecting PLEKHN1 protein expression level.

7. product according to claim 6, which is characterized in that the kit includes passing through RT-PCR method, qRT-PCR Method, biochip test method, southern blotting technique method, hybridization in situ, Western blot detection PLEKHN1 gene or protein expression water Flat reagent.

8. product according to claim 7, which is characterized in that described to detect PLEKHN1 gene expression water with qRT-PCR method Flat reagent includes the primer sequence of the specific amplification PLEKHN1 gene as shown in SEQ ID NO.3 and SEQ ID NO.4.

9. application of the described in any item products of claim 5-8 in the tool for preparing Diagnosis of Thyroid Carcinoma.

Application of the 10.PLEKHN1 in the computation model of building prediction thyroid cancer.