CN109593771B

CN109593771B - 1100 th base mutant gene of human MAP2K5 and detection kit thereof

Info

Publication number: CN109593771B
Application number: CN201910095564.0A
Authority: CN
Inventors: 叶丰; 肖林; 姜勇; 步宏
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2018-07-27
Filing date: 2019-01-30
Publication date: 2022-03-29
Anticipated expiration: 2039-01-30
Also published as: CN109593771A

Abstract

The invention discloses a variant human MAP2K5 gene, which is a variant human MAP2K5 gene with the 1100 th base mutated into A. The invention also discloses application of the reagent for detecting the base variation in preparing a non-medullary thyroid cancer screening reagent and a non-medullary thyroid cancer screening kit. The kit can be used for auxiliary diagnosis of thyroid cancer and has a good application prospect.

Description

1100 th base mutant gene of human MAP2K5 and detection kit thereof

Technical Field

The present invention relates to the field of SNPs, in particular SNPs associated with non-medullary thyroid carcinoma.

Background

The incidence rate of thyroid cancer of Chinese females is increased by about 5 times in the last decade, and the thyroid cancer becomes the malignant tumor with the fastest speed increase of females. The incidence rates were ranked first in women younger than 30 years of age, with the sixth in the full age group. Non-myeloid carcinomas (NMTCs) originating from Thyroid Follicular cells (folliculus epidermis cells) account for approximately 95% of the total, with papillary Thyroid carcinomas being the most common type of Non-myeloid carcinoma; medullary carcinoma (MTC) originated from parathyroid C cells accounts for about 5%. Both types can be inherited.

Although the concept of NMTC has been widely accepted, its susceptibility genes and corresponding functions have not been elucidated and generally recognized, so that at present there is no recognized susceptibility gene variation as a target for NMTC control or therapy.

Disclosure of Invention

In order to solve the above problems, the present invention provides a kit for detecting NMTC.

The invention firstly provides a variant human MAP2K5 gene, which is a variant human MAP2K5 gene with the 1100 th base mutated into C, namely c.T1100C (p.M367T) (NM _ 145160).

The invention also provides a variant human MAP2K5 protein, which is a human MAP2K5 variant protein with the mutation of amino acid 367 to threonine.

The invention also provides application of a related reagent for detecting 1100 th base variation of the human MAP2K5 gene in preparation of a non-medullary thyroid cancer screening reagent.

Wherein the screening reagent is a screening reagent for detecting papillary thyroid carcinoma.

Wherein, the reagent is a related reagent for detecting the 1100 th T → C variation of the human MAP2K5 gene.

Wherein the related reagent for detecting the 1100 th T → C variation of the human MAP2K5 gene is a reagent for sequencing, a Snapshot reagent, a reagent for a restriction fragment length polymorphism method or a reagent for single-strand conformation polymorphism analysis.

Wherein the sequencing reagent comprises a reagent for amplifying the 1100 th site of the MAP2K5 gene by PCR.

Preferably, the reagent for amplifying the 1100 th site of the MAP2K5 gene comprises the following primer pairs:

the upstream primer is as follows: 5'-TCATAATGTGTCCAAGTGAGTC-3', the downstream primer is: 5'-TTTACAGTGGAGTGGAAAGAAA-3' are provided.

The invention also provides a screening kit for non-medullary thyroid cancer, which is characterized in that: it includes optional related reagent for detecting 1100 th base variation of human MAP2K5 gene.

The invention also provides a pair of DNA primers, which is characterized in that: the sequence is the sequence shown in SEQ ID NO. 1-2.

The invention discovers the mutant genes and specific sites of the mutant genes, namely MAP2K5 c.G961A and MAP2K5 c.T1100C, which are highly developed with thyroid cancer through the research of papillary thyroid cancer families.

The former site is that the 961 th base of MAP2K5 gene coding region is mutated from G to A, and the 321 nd amino acid of corresponding MAP2K5 protein is mutated from alanine to threonine. The latter site is that the 1100 th base of MAP2K5 gene coding region is mutated from T to C, and the 367 th amino acid of MAP2K5 protein is mutated from methionine to threonine. Through further experimental verification, the mutation site is found to cause the change of subsequent cell pathways, so that cancer-related genes are greatly up-regulated, and the malignant transformation of the thyroid gland follicular epithelial cells is promoted.

The assay method of the present invention determines human-derived genomic DNA, and samples such as body fluids (e.g., blood, ascites and urine), tissue cells (e.g., liver tissue), etc., without limitation, can be prepared by extracting and purifying these samples.

The kit provided by the invention can effectively screen the possibility of the population to be detected on the thyroid cancer, and has a good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to specific embodiments in the form of examples and experimental examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic diagram showing the profiles of candidate mutations and MAP2K5 mutations in subjects: MAP2K5 c.g. g961a sequencing MAP, the arrow indicates the mutation site; b, MAP2K5 c, T1100C sequencing diagram, wherein an arrow head shows a mutation site; c, domain information of a321T and M367T in the protein MAP2K 5; d, conservative analysis of MAP2K 5A 321T and M367T among different species.

FIG. 2 shows the expression of ERK5 gene and MAP2K5-ERK5 target genes of blood leukocytes of FNMTC patients (F1-F4) and SNMTC patients (S1-S4): a, ERK5 expression; b, mRNA heat MAP of MAP2K5-ERK5 pathway target genes; detailed expression levels of C-H, target genes FOSB, MEF2, PPARG, JUN, CDK4 and CCND 1.

FIG. 3 shows the expression of MAP2K5, ERK5, FRA1, MEF2 genes in the parathyroid and tumor tissues of FNMTC patients (F1-F4) and SNMTC patients (S1-S4): A-H, MAP2K5 expression between FNMTC and SNMTC patients; I-K, ERK5, FRA1 and MEF2 expression in paraneoplastic tissues, N on the abscissa label indicates "normal tissue"; L-N, ERK5, FRA1 and MEF2 expression in cancer tissues, and T on the abscissa label represents "cancer tissues".

FIG. 4 is a Ki67 staining and discrimination chart of normal follicular epithelial tissue and tumor tissue of SNMTC and FNMTC patients: A-B, normal tissue staining; C-D, cancer tissue staining; and E, interpretation of staining results by three clinical pathologists.

FIG. 5 is a graph of TUNEL fluorescein staining for apoptosis detection.

FIG. 6 shows the construction of transgenic cells: a, GV358 vector structure; B-C, recombinant vector Sanger sequencing verification map; d multi-gene site validated B-CPAP cell lines; green fluorescence microscopy images of E-H, WT group, Mu1 group, Mu2 group, and GFP group.

FIG. 7 is a MAP2K5 schematic and ERK5 detection MAP of transgenic cells: a, MAP2K5 protein tertiary structure and mutation site schematic diagram; b, phospho ERK5 protein detection map; c, control protein detection map; D-F, subcellular localization map of WT group ERK5 protein; subcellular localization map of group G-I, Mu1(A321T) ERK5 proteins.

FIG. 8 is the MAP2K5-ERK5 gene target gene expression MAP of each group of transgenic cells.

Detailed Description

The release is as follows:

MAP2K5 c.g 961a: the 961G → A variation of MAP2K5 gene;

MAP2K5 p.aj321t or MAP2K5 a 321T: the 321 th amino acid of MAP2K5 protein is mutated from A to T, and the mutation can be caused by MAP2K5 c.G961A;

MAP2K5 c, t 1100c: the 1100 th T → C variation of MAP2K5 gene;

MAP2K5 p.m367t or MAP2K 5M 367T: the 367 th amino acid of the MAP2K5 protein is mutated from M to T, and the mutation can be caused by MAP2K5 c.T 1100C;

FNMTC patients: familial follicular cell derived non-medullary thyroid cancer patients; at least two first-degree relatives are diagnosed as familial patients with follicular cell-derived non-medullary thyroid carcinoma without other familial diseases;

SNMTC patients: sporadic follicular cell-derived patients with non-medullary thyroid cancer, the MAP2K5 gene was not mutated.

Example detection of mutant Gene MAP2K5 c. T1100C

DNA extraction

Taking 200 microliters of whole Blood sample, and extracting by using a New hundred-base UPure Blood DNA Extraction Kit (M2002-01) Kit according to the instruction of the New hundred-base UPure Blood DNA Extraction Kit (M2002-01) Kit.

PCR amplification

1) PCR primers:

the upstream primer is as follows: 5'-TCATAATGTGTCCAAGTGAGTC-3' (SEQ ID NO.1),

the downstream primer is: 5'-TTTACAGTGGAGTGGAAAGAAA-3' (SEQ ID NO.2),

concentration: 100. mu. mol/L.

2) Preparing a system:

DNA	2 microliter
		2X Taq PCR MasterMix(KT201)	12.5 microliter
Upstream of	1 microliter
		Downstream
	1 microliter
		Sterilized water	9.5 microliter

3) Reaction procedure:

step (ii) of	Temperature of	Time
			1	94℃	3min
2	94℃	30s
			3	60℃	30s
5	72℃	1min
			6	Returning to the step 2 for 30 times
7	72℃	5min

Sanger sequencing

And (4) carrying out agarose gel electrophoresis on the PCR product, identifying the size of the product, then, carrying out sample sequencing, and detecting whether the related site is mutated.

4. Interpretation of results

The sequencing result is aligned to a reference genome, and if the 68040594 th position of the 15 th chromosome is found to be C (the reference sequence is T), MAP2K5 c.T1100C is detected.

The method can effectively detect the mutation of the 1100 th base of the MAP2K5 gene from T to C.

In order to explain the effect of the MAP2K5 c.T1100C mutation on the promotion of non-medullary thyroid cancer, experimental examples are described below.

Experimental example correlation between MAP2K5 c.G961A/c.T1100C mutation and non-medullary thyroid cancer

1. Method of producing a composite material

1.1 ethics and patient information

The study was approved and performed by the ethical committee of the western hospital (2015-108). FNMTC was defined as a family patient without other familial diseases, at least two first-degree relatives being diagnosed with follicular cell-derived thyroid adenocarcinoma. In this study, a total of 77 FNMTC patients met these criteria. FNMTC patients were from 34 families (female to male ratio 3.53: 1), all diagnosed with thyroid papillary carcinoma.

1.2Sanger sequencing and Gene frequency query

The DNA of the aforementioned patients is extracted and flanking primers are designed to amplify the target region mutation sites. The purified PCR products were sequenced on an ABI 3730xL sequencer (Applied Biosystems, USA) using the BigDye 3.1 terminator sequencing kit (Applied Biosystems) according to the manufacturer's protocol.

The frequency of the mutant gene at the MAP2K5 c.G961A/c.T1100C site was searched in the Novo-Zhonghua genome project database.

1.3RT-qPCR assay

For fresh samples and cell samples, total RNA was extracted using Invitrogen TRIzol reagent (cat.15596026, Thermo Fisher, usa). For formalin fixed paraffin embedded samples, total RNA was extracted using Qiagen RNeasy FFPE kit (cat.73504, Qiagen Inc, germany). For reverse transcription, 1mg of mRNA was converted to cDNA in a 20ul reaction using the Qiagen Omniscript RT kit (Cat.205111, Qiagen) according to the manufacturer's instructions. And qPCR was performed using the Bio-Rad Real-Time PCR system (Bio-Rad Inc, USA). The relative mRNA expression of the target genes was compared after normalization with control genes (GAPDH, ACTIN and GFP).

1.4 immunohistochemistry

For human samples, immunohistochemical staining was performed on 4- μm thick paraffin sections. Slides were deparaffinized in toluene and rehydrated by a gradient ethanol solution. The endogenous' peroxidase was blocked with 3% hydrogen peroxide at room temperature for 15 minutes. The slides were washed with double distilled water and then incubated in a microwave oven for 15 minutes for antigen retrieval, followed by washing with PBS. The slides were then incubated with working dilutions of each monoclonal antibody at 4 ℃ overnight. Slides were washed with PBS and stained using DAKO EnVision + system (K5007, Denmark). Monoclonal antibodies against Ki-67 (1: 200 dilution; Cat. RM-9106-S0, Thermo Fisher) and polyclonal antibodies against MEK5 (1: 200 dilution; Cat. ab210748, Abcam, USA) were used. Finally, the slides were counterstained with hematoxylin.

1.5TUNEL analysis

The apoptosis detection system Fluorescein (Promega G3250) incorporates Fluorescein-12-dUTP at the 3' -OH DNA end to detect fragmented DNA from apoptotic cells by terminal deoxynucleotidyl transferase (TdT) action. Paraffin embedded tissues were processed into 4- μm paraffin sections for TUNEL apoptotic cell detection. The sections were dewaxed in xylene and rehydrated through a gradient ethanol solution. Sections were washed in 0.85% NaCl then PBS. Sections were incubated with 100. mu.120. mu.g/ml proteinase k for 10 min at 37 ℃. Sections were covered with 100. mu.l of equilibration buffer, then 50. mu.l of TdT incubation buffer. Negative controls were performed without TdT. Positive cells were detected by confocal microscopy.

1.6 mutant construction and lentivirus preparation

Total RNA was extracted using TRIzol reagent (Invitrogen, 15596-026, USA). The RNA was resuspended in ultrapure water and treated with DNAse I (Ambion, AM2222) for 30 minutes at 37 ℃ and RNA purified with RNeasy Midi kit (QIAGEN) according to the manufacturer's instructions. Complementary DNA (cDNA) was randomly primed from 2.0. mu.g total RNA using the Omniscript reverse transcription kit (205113, Qiagen). Normal PCR and overlap PCR methods were performed using primers designed for cDNA cloning. The PCR products were cloned into a TA vector (Invitrogen, K451020V, USA; for cloning and DNA sequencing) and sequenced. The gene fragment with correct sequencing is cloned to Age I site of GV358 vector (Jikai, China). Lentiviruses were produced by co-transfection of the above constructs with packaging plasmids into HEK293T cells, and at 48 hours post-transfection HEK293T cell culture medium was harvested for the next experiment.

1.7 cell culture and transfection

Human thyroid cancer cell line B-CPAP (DSMZ No. ACR 273) was cultured as described. Cell identity was verified by STR assay. The B-CPAP cell line was then transfected with Wild Type (WT) MAP2K5, MAP2K5 a321T and M367T overexpressing lentiviruses and empty lentivirus expressing GFP. Puromycin at a final concentration of 4 μ g/ml was used to screen cell lines stably infected with virus. Finally, four adenovirus-infected cell lines were obtained, which had stable target gene expression.

1.8 immunofluorescence staining

B-CPAP cell lines stably expressing MAP2K5, MAP2K 5A 321T and MAP2K 5M 367T were seeded and cultured in six-well plates. ERK5 (batch number: AP070721, Boke, China), phosphorylated ERK5(Thr218+ Tyr220) (batch number: AE050702, Boke, China), phosphorylated ERK5(Ser731+ Thr733) (batch number: AC09223656, Boke, China) and phosphorylated ERK5(Ser496) (batch number: AC11012356, Boke, China) were used as primary antibodies for target protein detection. The labeled secondary antibody was used for fluorescence detection. Nuclei were stained with DAPI. Finally, the images were analyzed by Nikon two-photon confocal microscopy.

1.9 phosphorylation assay

Five phosphorylation sites of ERK5 were examined in this study by phosphorylated ERK5(Thr218+ Tyr220) (batch No.: AE050702, bock, china), phosphorylated ERK5(Ser731+ Thr733) (batch No.: AC09223656, bock, china) and phosphorylated ERK5(Ser496) (batch No.: AC11012356, bock, china). HRP-conjugated anti-secondary antibodies were used for further studies, and then either phosphorylated ERK5(Thr218+ Tyr220), phosphorylated ERK5(Ser731+ Thr733) and phosphorylated ERK5(Ser496) were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit) or for GAPDH and GFP secondary antibodies (anti-mouse). Protein bands were visualized using ECL Plus Western blot detection reagents (WBULS0500, Millipore Inc, USA) and detected and quantified on Storm860 phophorimager using Image Quant TL version 2003.02 software.

1.10 computational model of A321T and M367T in MAP2K5

Both the natural and mutant models of MAP2K5 residue 146-438, A321T and M367T, were generated by Swiss-Model Server from the protein crystal structure of human Mek-1 kinase (PDB: 3SLS) (Nucleic Acids Res.2014.7; 42[ web service No.: W252-8 ]). The models shown herein were generated using PyMOL software (Delano WL.2002; PyMOL molecular graphics system, Delano Scientific, Palo Alto, CA. http:// www.pymol.org).

1.11 statistical analysis

Statistical analysis was performed using SPSS (version 22.0; SPSS inc., Chicago, USA). Two groups of student t tests show that the statistical difference is significant when P is less than 0.05, and a plurality of groups of single-factor analysis of variance (analysis of variance) analysis. All tests were two-sided, and P < 0.05 was considered statistically significant.

2. Results

2.1 MAP2K5 mutation in the investigator

MAP2K5 c.g961a (p.a321t) was found in three first-degree relatives in a family. MAP2K5 c.t1100c (p.m367t) was found in two primary relatives in another family. In terms of gene frequency, the gene frequency of a321T and M367T in FNMTC patients was 0.0390 and 0.0259, respectively; while the healthy chinese control (norstanding-chinese genome project, n 2200, P < 0.001) has gene frequencies of 0 and 0.00022523 for the two sites, respectively. It can be seen that MAP2K5 a321T and M367T have very significant correlation with FNMTC.

The results of Sanger sequencing of MAP2K5 c.G961A (p.A321T) and MAP2K5 c.T1100C (p.M367T) are shown in FIG. 1A, B.

FIG. 1C shows the positions of the two aforementioned mutation sites in MAP2K 5.

FIG. 1D shows that the corresponding wild-type bases of the two mutation sites are well conserved, and have high homology in human, rat, cow, etc.

2.2 post-mutation MAP2K5 activates the MAPK-ERK5 signaling pathway

The inventor uses RT-qPCR to detect the expression of the downstream gene ERK5 of MAP2K5 and the downstream gene of MAP2K5-ERK5 pathway in MAP2K5 mutant population.

In blood leukocytes, the FNMTC population had higher expression levels of FOSB, MEF2, PPARG, CDK4, and CCND genes than the SNMTC population (fig. 2).

In thyroid tissue, whether malignant tissue or healthy tissue, there was no significant difference in the aforementioned gene expression between FNMTC population and SNMTC population, but the expression of downstream target genes ERK5, FRA1, MEF2, etc. was more highly expressed in MAP2K5 mutant NMTC population (fig. 3).

Further, Ki67 staining (fig. 4) and TUNEL assay (fig. 5) showed no significant difference in cell proliferation and apoptosis levels between SNMTC and FNMTC populations.

2.3MAP2K5 mutations leading to Ser496 and Ser731+ Thr733 phosphorylation of the ERK5 protein

The MAP2K5 protein structure diagram and mutation sites are shown in fig. 7A.

Integrating three genes of MAP2K5 wild type (WT group), MAP2K 5A 321T (Mu1 group) and MAP2K 5M 367T (Mu2 group) into a GV358 vector (with a CMV promoter and an EGFP label), and transferring the integrated gene into a human thyroid cancer cell strain B-CPAP (DSMZ No. ACC 273) through adenovirus; the empty GV358 vector was transferred by adenovirus into B-CPAP as a control (GFP panel) (FIG. 6).

The phosphorylation sites of ERK5, corresponding to Ser496, Ser731+ Thr733, and Thr218+ Tyr220, respectively, were then detected (by western blotting) using commercial antibodies. For B-CPAP, GAPDH and GFP overexpressing GFP, WT, Mu1 and Mu2 were used as references to ensure that the total amount of endogenous and exogenous proteins was approximately the same (FIGS. 7B and C).

As a result, B-CPAP overexpressing WT, Mu1 and Mu2 was found to significantly increase the total amount of downstream ERK5 with GFP as a reference (FIG. 7C). Further detection of ERK5 phosphorylation site: ser496, Ser731+ Thr733 and Thr218+ Tyr 220. The phosphorylation levels of Ser496 ERK5 were significantly increased in the Mu1 and Mu2 groups compared to the GFP and WT groups (FIG. 7B). Furthermore, ERK5 Ser731+ Thr733 phosphorylation was significantly increased in the Mu1 group compared to the GFP and WT groups (FIG. 7B).

2.4 transport of phosphorylated ERK5 to the nucleus to regulate downstream gene expression

The inventors used immunofluorescence staining to detect the subcellular localization of ERK5 in the above transgenic cells. It was found that in the MAP2K5 overexpression panel, p-ERK5 Ser731+ Thr733 was located throughout the cytoplasm (FIGS. 7D-7F); however, in the MAPK2K 5A 321T overexpression group, the downstream p-ERK5 Ser731+ Thr733 was located only in the nucleus (FIGS. 7G-7I). It was shown that MAPK2K5 a321T mutation caused ERK5 to be transported to the nucleus.

The inventors further analyzed the expression of the target gene in the above transgenic cells. The results show that most MAPK-ERK5 signaling targets were upregulated in the a321T and M367T groups, such as ERK5, FOSB, MEF2, CDK4, CCND1, and FRA1, with JUN and PPARG being upregulated only in one group (fig. 8).

The results of experimental examples show that MAP2K5 mutation causes ERK5 phosphorylation, further causes ERK5 to be transported to cell nucleus, further regulates and controls downstream genes, enables cancer-related genes to be greatly up-regulated, promotes malignant transformation of thyroid follicular epithelial cells, and causes the incidence of thyroid cancer to be increased.

In conclusion, the invention clarifies the relevance of the variation of the 1100 th base of the MAP2K5 gene from T to C and thyroid cancer; the kit provided by the invention is used for detecting human tissues, and if the mutation is detected, the risk of thyroid cancer in the detected population can be judged. The invention can be used for the auxiliary diagnosis of thyroid cancer.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

<120> human MAP2K5 1100 th base mutant gene and detection kit thereof

<130> GY026-2018P012679CC

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<400> 1

tcataatgtg tccaagtgag tc 22

<210> 2

<211> 22

<212> DNA

<213> Artificial sequence

<400> 2

tttacagtgg agtggaaaga aa 22

Claims

1. The application of a related reagent for detecting 1100 th base variation of human MAP2K5 gene in preparing a familial non-medullary thyroid cancer screening reagent is characterized in that: the related reagent is used for detecting the T → C variation at the 1100 th site of the human MAP2K5 gene;

the human MAP2K5 gene has accession number NM-145160 in NCBI.

2. Use according to claim 1, characterized in that: the screening reagent is used for detecting familial papillary thyroid carcinoma.

3. Use according to claim 1, characterized in that: the related reagent for detecting the 1100 th T → C variation of the human MAP2K5 gene is a reagent for sequencing, a Snapshot reagent, a reagent for a restriction fragment length polymorphism method or a reagent for single-strand conformation polymorphism analysis.

4. Use according to claim 3, characterized in that: the sequencing reagent comprises a reagent for amplifying the 1100 th site of the MAP2K5 gene by PCR.

5. A familial non-medullary thyroid cancer screening kit, comprising: the kit comprises a related reagent for detecting 1100 th base variation of a human MAP2K5 gene, wherein the related reagent is a related reagent for detecting 1100 th T → C variation of a human MAP2K5 gene;

the human MAP2K5 gene has accession number NM-145160 in NCBI.

6. The kit of claim 5, wherein: the related reagent for detecting the 1100 th T → C variation of the human MAP2K5 gene is a reagent for sequencing, a Snapshot reagent, a reagent for a restriction fragment length polymorphism method or a reagent for single-strand conformation polymorphism analysis.

7. The kit of claim 6, wherein: the sequencing reagent comprises a reagent for amplifying the 1100 th site of the MAP2K5 gene by PCR.