CN107201411A - MYLK genes as diagnosis of endometrial carcinoma mark - Google Patents

Abstract

The invention discloses a diagnostic marker of endometrial cancer, the diagnostic marker is MYLK gene or MYLK protein, and the purpose of diagnosis is realized by detecting the expression level of MYLK gene. The research of the present invention proves that compared with normal endometrial tissue, the mRNA expression level of MYLK gene in endometrial cancer tissue is significantly decreased. According to the correlation between the MYLK gene and endometrial cancer, a test kit for diagnosing endometrial cancer can be prepared, and the test kit can be widely used clinically.

Description

MYLK gene as a marker for diagnosis of endometrial cancer

technical field

The invention belongs to the field of diagnosis, relates to a diagnostic tool for endometrial cancer, and also relates to the application of MYLK gene in preparing the diagnostic tool for endometrial cancer.

Background technique

Endometrial cancer (endometrial carcinoma or carcinoma of endometrium, EC) is a group of epithelial malignant tumors that occur in the endometrium, and adenocarcinoma derived from endometrial glands is the most common, and it is the three major malignant tumors of the female reproductive tract. One of them, it accounts for 7% of malignant tumors in women, and its incidence varies in different regions of the world. It is the most common gynecological malignant tumor in western industrialized countries, with the highest incidence in North America and Northern Europe, Asia, Japan, India, Central and South America, etc. The incidence rate is low in the area. my country still lacks more detailed epidemiological investigation data on endometrial cancer, and it is estimated that the incidence of endometrial cancer is similar to that in Japan. Developed countries in North America and Europe are higher than developing countries, the former is 10 times that of the latter, and its incidence rate is behind breast cancer, colorectal cancer, and lung cancer. one.

Although the symptoms of endometrial cancer appear earlier, the diagnosis is relatively easy. However, there are still some controversies about the diagnosis and treatment of endometrial cancer in actual clinical work. The degree of malignancy and extent of tumor, including surgical stage, histological type, tumor grade, depth of myometrial invasion, lymphatic metastasis and extrauterine metastasis, are related to the prognosis of endometrial cancer. Therefore, early diagnosis of endometrial cancer is very important. At present, the diagnosis of endometrial cancer is mainly based on medical history and clinical manifestations, imaging examinations such as B-ultrasound, CT and MRI, diagnostic curettage, hysteroscopy, tumor marker CA-125 and other auxiliary examinations. Among them, CA-125 can also be used as an indicator for curative effect observation. However, CA-125 lacks tissue sensitivity and specificity, and elevated levels of CA-125 can be detected in ovarian epithelial tumors, endometrial cancer, endometriosis, and digestive system tumors.

In recent years, genetic diagnosis has become a new development trend in the in vitro diagnostic industry. Gene diagnosis, also known as DNA diagnosis or molecular diagnosis, is a technology that uses molecular biology methods to detect changes in the structure or expression level of genetic material in patients, and then makes a diagnosis. and disease outcomes to provide more accurate information. In view of the above deficiencies in the current diagnostic methods for endometrial cancer, it is urgent to develop a method for genetic diagnosis of endometrial cancer that is cheap, easy to operate, and has high diagnostic sensitivity and specificity.

Contents of the invention

The present invention finds for the first time that the content of MYLK gene in endometrial cancer tissue is much lower than that in normal endometrial tissue, and the differential expression of MYLK gene can be used as a method for diagnosing endometrial cancer, which can be developed to diagnose endometrial cancer Tool of.

Specifically, the present invention provides the application of the product for detecting the expression of MYLK gene in the preparation of tools for diagnosing endometrial cancer.

Further, the detection products mentioned above include: detection of the expression level of MYLK gene by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform to diagnose endometrial cancer.

Further, the product for diagnosing endometrial cancer by RT-PCR includes at least a pair of primers for specifically amplifying the MYLK gene; the product for diagnosing endometrial cancer by real-time quantitative PCR includes at least a pair of primers for specifically amplifying the MYLK gene. Primers; the product for diagnosing endometrial cancer with immunoassay includes: an antibody that specifically binds to the MYLK protein; the product for diagnosing endometrial cancer with in situ hybridization includes: a probe that hybridizes with the nucleic acid sequence of the MYLK gene The product for diagnosing endometrial cancer with a chip includes: a protein chip and a gene chip; wherein, the protein chip includes an antibody that specifically binds to the MYLK protein, and the gene chip includes a probe that hybridizes to the nucleic acid sequence of the MYLK gene.

In a specific embodiment of the present invention, the product for diagnosing endometrial cancer by real-time quantitative PCR includes at least a pair of primers for specifically amplifying the MYLK gene, as shown in SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the diagnostic tools include chips, kits, test strips or high-throughput sequencing platforms. Among them, the high-throughput sequencing platform is a special diagnostic tool, and products for detecting MYLK gene expression can be applied to this platform to detect the expression of MYLK gene. With the development of high-throughput sequencing technology, the construction of a person's gene expression profile will become a very convenient task. By comparing the gene expression profiles of disease patients and normal people, it is easy to analyze which gene abnormality is related to the disease. Therefore, it is known in high-throughput sequencing that the abnormality of the MYLK gene is related to endometrial cancer, which also belongs to the use of the MYLK gene, and is also within the protection scope of the present invention.

The present invention also provides a tool for diagnosing endometrial cancer, and the product includes a chip, a kit, a test strip, or a high-throughput sequencing platform.

Wherein, the chip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, and the oligonucleotide probe includes a gene chip for detecting the MYLK gene transcription level. The oligonucleotide probe for the MYLK gene; the protein chip includes a solid phase carrier and the specific antibody of the MYLK protein immobilized on the solid phase carrier; the gene chip can be used to detect multiple genes including the MYLK gene (e.g., expression levels of multiple genes associated with endometrial cancer). The protein chip can be used to detect the expression levels of multiple proteins including MYLK protein (for example, multiple proteins related to endometrial cancer). By simultaneously detecting multiple endometrial cancer markers, the accuracy of endometrial cancer diagnosis can be greatly improved.

Wherein, the kit includes a gene detection kit and a protein immune detection kit; the gene detection kit includes reagents for detecting MYLK gene transcription level; the protein immune detection kit includes a specific antibody for the MYLK protein. Further, the reagents include the reagents required in the process of detecting the expression level of MYLK gene by using RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or chip method. Preferably, the reagents include primers and/or probes for the MYLK gene. Primers and probes that can be used to detect the expression level of the MYLK gene can be easily designed according to the nucleotide sequence information of the MYLK gene.

The high-throughput sequencing platform includes reagents for detecting the expression level of the MYLK gene.

The test paper includes a test paper carrier and an oligonucleotide fixed on the test paper carrier, and the oligonucleotide can detect the transcription level of the MYLK gene.

The probe hybridizing to the nucleic acid sequence of the MYLK gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, as long as it completes specific hybridization and specifically binds to the target nucleotide sequence, any length is acceptable. The probes can be as short as 25, 20, 15, 13 or 10 bases in length. Likewise, the probes can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs. The optimal length of complementarity is 15-25 base pairs. The self-complementary sequence of the probe is preferably less than 4 base pairs, so as not to affect the hybridization efficiency.

Further, the specific antibody of the MYLK protein includes monoclonal antibody and polyclonal antibody. The specific antibody of the MYLK protein includes a complete antibody molecule, any fragment or modification of the antibody (for example, chimeric antibody, scFv, Fab, F(ab') 2, Fv, etc. As long as the fragment can retain the MYLK Protein-binding ability is enough. Preparation of antibodies for protein level is well known to those skilled in the art, and any method can be used in the present invention to prepare the antibodies.

In a specific embodiment of the present invention, the primer sequence for the MYLK gene is as follows: the sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the reverse primer is shown in SEQ ID NO.4.

The sources of the MYLK gene and its expression products used for diagnosing endometrial cancer include but are not limited to tissues and body fluids, and body fluids include blood, interstitial fluid, and other liquid components in the body that contain DNA. In a specific embodiment of the present invention, the source of the MYLK gene and its expression product for diagnosing endometrial cancer is tissue.

The specific sequence of the MYLK gene (NC_000003.12 (123612296..123884302)) of the present invention can be found in the international public nucleic acid sequence database GeneBank.

The DNA sequence corresponding to the mRNA sequence of the MYLK gene of the present invention is the DNA sequence shown in SEQ ID NO.1 in the sequence listing.

"MYLK protein" of the present invention includes MYLK protein and any functional equivalent of MYLK protein. The functional equivalents include MYLK protein conservative variant proteins, or active fragments thereof, or active derivatives thereof, allelic variants, natural mutants, induced mutants, DNAs capable of binding to MYLK under high or low stringent conditions The protein encoded by the hybridized DNA.

Preferably, the MYLK protein is a protein having the following amino acid sequence:

(1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

(2) The amino acid sequence shown in SEQ ID NO.2 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as the amino acid sequence shown in SEQ ID NO.2. A protein derived from the amino acid sequence shown in ID NO.2. The number of substituted, deleted or added amino acids is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.

In a specific embodiment of the present invention, the MYLK protein is a protein having the amino acid sequence shown in SEQ ID NO.2.

In general, it is known that the modification of one or more amino acids in a protein does not affect the function of the protein. Those skilled in the art will recognize that changes to single amino acids or small percentages of amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications where changes to a protein result in a protein with similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.

An example of a protein modified by adding an amino acid or amino acid residues is a fusion protein of the MYLK protein. There is no limitation on the peptide or protein fused with the MYLK protein, as long as the resulting fusion protein retains the biological activity of the MYLK protein.

The MYLK protein of the present invention also includes non-conservative modifications to the amino acid sequence shown in SEQ ID NO.2, as long as the modified protein can still retain the biological activity of the MYLK protein. The number of amino acids mutated in such modified proteins is usually 10 or less, such as 6 or less, such as 3 or less.

In the context of the present invention, "diagnosing endometrial cancer" includes judging whether the subject has already suffered from endometrial cancer, also includes judging whether the subject has the risk of endometrial cancer, and also includes predicting whether the subject has endometrial cancer. The prognosis of patients with endometrial cancer also includes judging whether a subject with endometrial cancer has relapsed after treatment.

Description of drawings

Figure 1 shows the difference in expression of MYLK gene detected in endometrial cancer tissue and normal endometrial tissue by RNA-seq;

Figure 2 shows the difference in expression of MYLK gene detected in endometrial cancer tissue and normal endometrial tissue by QPCR.

specific implementation

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the embodiment, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1 Screening of differentially expressed genes

1. Sample collection:

Endometrial cancer tissue samples: Patients with endometrial cancer were collected, all of whom underwent surgical treatment, and 10 surgical paraffin specimens were collected. All patients were diagnosed with endometrial cancer by pathological examination. Other enrollment conditions are: all patients have not received any treatment before admission; no other malignant tumors; no other hormone-related diseases; complete clinical data.

10 cases of endometrial cancer patients, the average age of onset was 57 years old. The main clinical manifestations of patients are irregular vaginal bleeding, lower abdominal pain, menstrual disorders, vaginal discharge, etc. There are also some patients who have no obvious symptoms and are found in physical examination. The endometrial cancer specimens were sectioned by HE staining, and the histomorphological diagnosis was endometrial cancer.

Normal endometrial tissue samples: 10 normal endometrial surgical paraffin specimens. The diseases suffered by the patients from which the samples came from included: uterine fibroids, uterine prolapse, cystocele, and rectocele.

2. RNA extraction

Put the endometrial tissues in a ceramic mortar and grind them into powder under the low temperature environment of liquid nitrogen, add Trizol reagent to homogenate, centrifuge, take the supernatant, extract twice with 1:1 acidic phenol-chloroform, and then pass through acetic acid Extract with sodium and 5:1 acidic phenol-chloroform, precipitate with an equal volume of isopropanol, and dissolve the precipitate with Milli-Q water after centrifugation.

3. Purity analysis of RNA samples (NanoDrop1000 spectrophotometer)

NanoDrop1000 spectrophotometer detects RNA samples, and the sample requirements for RNA-seq sequencing: OD260/OD280 is 1.8-2.2.

4. Quality analysis of RNA samples (Agilent Technologies 2100Bioanalyzer)

Agilent Technologies 2100Bioanalyzer detects the quality of RNA samples, and observes that the main bands of 28S rRNA and 18SrRNA are obvious, there is no degradation, the RNA integrity index is qualified, and the concentration meets the requirements, which meet the requirements of RNA-seq sequencing cDNA library construction, and can be used for library construction and sequencing.

5. High-throughput transcriptome sequencing

(1) RNA-seq read mapping

First remove the low-quality reads to get clean reads, and then use TopHat v1.3.1 to match the clean fragments with the UCSC H. sapiens reference genome (hg19), the pre-built index of H. sapiens UCSC hg19 version is downloaded from the TopHat homepage , and as a reference genome, when using TopHat to match the genome, each read (default to 20) is allowed to have multiple matching sites and a maximum of 2 mismatches. TopHat builds a library of possible splicing sites based on exon regions and GT-AG splicing signals, and maps reads that have not mapped to the genome to the genome based on these splicing site libraries. We use the system default parameters of the TopHat method.

(2) Transcript abundance assessment

The matched read files were processed by Cufflinks v1.0.3, and Cufflinks v1.0.3 normalized the number of RNA-seq fragments to calculate the relative abundance of transcripts. The FPKM value refers to the number of fragments matching the 1 kb exon region of a specific gene per million sequenced fragments. Confidence intervals for FPKM estimates were computed by Bayesian inference methods. The reference GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo_sapiens.GRCh37.63.gtf).

(3) Detection of differentially expressed genes

Transfer the downloaded Ensembl GTF file and the original file matched by TopHat to Cuffdiff, and Cuffdiff uses the original matched file to re-estimate the expression abundance of the transcripts listed in the GTF file and detect differential expression. Only q-values < 0.01 in the Cuffidff output where the test showed successful comparisons were considered differentially expressed.

6. Results

RNA-seq results showed that there were differentially expressed genes between normal endometrial tissue and endometrial cancer tissue, among which 374 genes were up-regulated and 268 genes were down-regulated. Among them, the mRNA level of MYLK gene in endometrial cancer tissue was significantly decreased, and the difference was statistically significant (P<0.05).

Example 2 Verification of Differentially Expressed Genes in Large Samples

1. Sample collection

According to the method of Example 1, 50 cases of endometrial cancer tissues and 45 cases of normal endometrial tissues were collected.

2. Validation at the mRNA level

2.1 Extract tissue RNA

Step is with embodiment 1.

2.2 Reverse transcription

Using a reverse transcription kit, 1 μg of total RNA was reverse-transcribed with reverse transcription buffer to synthesize cDNA. Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, and add the following components in PCR tubes: DEPC water, 5× reverse transcription buffer, 10mmol/l dNTP, 0.1mmol/l DTT, 30μmmol/l Oligo dT, 200 U/μl MMLVRT, template RNA. Incubate for 1 hour at 42°C, 10 minutes at 72°C, and centrifuge briefly.

2.3PCR

Use the primer design software Primer Premier 5.0 to design mRNA fluorescent quantitative upstream and downstream PCR primers, synthesize primer sequences, and use the SYBR Green PCR Master Mix kit to operate. The specific steps are operated according to the instructions. A 25 μl reaction system is used, and 3 parallel tubes are set for each sample. , all amplification reactions were repeated more than three times to ensure the reliability of the results. The following reaction system (as shown in Table 1) was prepared, and all operations were carried out on ice:

Table 1 Fluorescence quantitative PCR components and corresponding volumes

Using GAPDH as an internal reference and SYBR Green I as a fluorescent marker, the PCR reaction was carried out on a Light Cycler fluorescent quantitative PCR instrument, the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was carried out by the ΔΔCT method.

The primer sequences of the MYLK gene are as follows:

Upstream primer: 5'-AAGTGGAGGTGTCAGATG-3' (SEQ ID NO.3);

Downstream primer: 5'-TCAATGTCGTAGAAGTCAGA-3' (SEQ ID NO.4).

GAPDH gene primer sequences are as follows:

Upstream primer: 5'-AACTCTGGTAAAGTGGATATTG-3' (SEQ ID NO.5);

Downstream primer: 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO.6).

2.4 Results

The results are shown in Figure 2. Compared with normal endometrial tissue, the mRNA level of MYLK gene in endometrial cancer tissue was significantly down-regulated, and the difference was statistically significant (P<0.05). The results were the same as RNA-seq.

Preparation of Example 3 Endometrial Cancer Diagnostic Kit

According to the correlation between MYLK gene and endometrial cancer, whether endometrial cancer occurs can be diagnosed by detecting the expression of MYLK gene. Accordingly, the present invention provides a reagent for diagnosing endometrial cancer based on detecting MYLK gene expression box, the components in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system; primer pair for amplifying MYLK gene and GAPDH gene. The forward sequence of the amplified MYLK gene is 5'-AAGTGGAGGTGTCAGATG-3', the reverse sequence is 5'-TCAATGTCGTAGAAGTCAGA-3'; the forward primer sequence for amplifying GAPDH is 5'-AACTCTGGTAAAGTGGATATTG-3', and the reverse primer sequence is 5''-GGTGGAATCATATTGGAACA-3'. The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, and SYBR Green fluorescent dye. The composition of the PCR buffer solution is: 25mM KCL, 2.5mM MgCL ₂ , 200mM (NH ₄ ) ₂ SO ₄ .

Preparation of Example 4 Endometrial Cancer Diagnostic Kit

According to the correlation between MYLK gene and endometrial cancer, whether endometrial cancer occurs can be diagnosed by detecting the expression of MYLK gene. Accordingly, the present invention provides a reagent for diagnosing endometrial cancer based on detecting MYLK gene expression The components in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system, primer pair for amplifying MYLK gene and GAPDH gene, and M-MLV reverse transcription system. The forward sequence of the amplified MYLK gene is 5'-AAGTGGAGGTGTCAGATG-3', the reverse sequence is 5'-TCAATGTCGTAGAAGTCAGA-3'; the forward primer sequence for amplifying GAPDH is 5'-AACTCTGGTAAAGTGGATATTG-3', and the reverse primer sequence is 5''-GGTGGAATCATATTGGAACA-3'. The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, and SYBR Green fluorescent dye. The PCR buffer components are: 25mM KCL, 2.5mM MgCL ₂ , 200mM (NH ₄ ) ₂ SO ₄ . The components of the M-MLV reverse transcription system are: T repeat oligonucleotide Oligo (dT), reverse transcription reaction solution, M-MLV reverse transcriptase, RNase inhibitor, dNTPs. The components of the reverse transcription reaction solution are: 250mM Tris-HCL (pH8.3), 375mM KCL, 15mM MgCL ₂ , and 50mM DTT. RNase inhibitors are recombinant proteases expressed in Escherichia coli that non-competitively inhibit RNases.

Preparation of Example 5 Endometrial Cancer Diagnostic Kit

According to the correlation between MYLK gene and endometrial cancer, whether endometrial cancer occurs can be diagnosed by detecting the expression of MYLK gene. Accordingly, the present invention provides a reagent for diagnosing endometrial cancer based on detecting MYLK gene expression The components in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system, primer pair for amplifying MYLK gene and GAPDH gene, and RNA extraction reagent. The forward sequence of the amplified MYLK gene is 5'-AAGTGGAGGTGTCAGATG-3', the reverse sequence is 5'-TCAATGTCGTAGAAGTCAGA-3'; the forward primer sequence for amplifying GAPDH is 5'-AACTCTGGTAAAGTGGATATTG-3', and the reverse primer sequence is 5''-GGTGGAATCATATTGGAACA-3'. The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, and SYBR Green fluorescent dye. The PCR buffer components are: 25mM KCL, 2.5mM MgCL ₂ , 200mM (NH ₄ ) ₂ SO ₄ . RNA extraction reagents include Trizol, chloroform, isopropanol, and 75% ethanol.

Embodiment 6 Preparation of Endometrial Cancer Diagnostic Kit

According to the correlation between MYLK gene and endometrial cancer, whether endometrial cancer occurs can be diagnosed by detecting the expression of MYLK gene. Accordingly, the present invention provides a reagent for diagnosing endometrial cancer based on detecting MYLK gene expression The components in the diagnostic kit are as follows: SYBR Green polymerase chain reaction system, primer pair for amplifying MYLK gene and GAPDH gene, M-MLV reverse transcription system, RNA extraction reagent. The forward sequence of the amplified MYLK gene is 5'-AAGTGGAGGTGTCAGATG-3', the reverse sequence is 5'-TCAATGTCGTAGAAGTCAGA-3'; the forward primer sequence for amplifying GAPDH is 5'-AACTCTGGTAAAGTGGATATTG-3', and the reverse primer sequence is 5''-GGTGGAATCATATTGGAACA-3'. The SYBR Green polymerase chain reaction system includes PCR buffer, dNTPs, and SYBR Green fluorescent dye. The PCR buffer components are: 25mM KCL, 2.5mM MgCL ₂ , 200mM (NH ₄ ) ₂ SO ₄ . The components of the M-MLV reverse transcription system are: T repeat oligonucleotide Oligo (dT), reverse transcription reaction solution, M-MLV reverse transcriptase, RNase inhibitor, dNTPs. The components of the reverse transcription reaction solution are: 250mM Tris-HCL (pH8.3), 375mM KCL, 15mM MgCL ₂ , 50mM DTT. RNase inhibitors are recombinant proteases expressed in Escherichia coli that non-competitively inhibit RNases. RNA extraction reagents include Trizol, chloroform, isopropanol, and 75% ethanol.

The description of the above embodiments is only for understanding the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

SEQUENCE LISTING

<110> Shanghai Changning District Maternal and Child Health Hospital

<120> MYLK gene as a marker for diagnosis of endometrial cancer

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 7644

<212>DNA

<213> human source

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ctgagagtga tgcgactgtg aaaaagaaac ctgcccccaa gacacctccg aaggcagcaa 3780

tgccccctca gatcatccag ttccctgagg accagaaggt acgcgcagga gagtcagtgg 3840

agctgtttgg caaagtgaca ggcactcagc ccatcacctg tacctggatg aagttccgaa 3900

agcagatcca ggaaagcgag cacatgaagg tggagaacag cgagaatggc agcaagctca 3960

ccatcctggc cgcgcgccag gagcactgcg gctgctacac actgctggtg gagaacaagc 4020

tgggcagcag gcaggcccag gtcaacctca ctgtcgtgga taagccagac cccccagctg 4080

gcacaccttg tgcctctgac attcggagct cctcactgac cctgtcctgg tatggctcct 4140

catatgatgg gggcagtgct gtacagtcct acagcatcga gatctgggac tcagccaaca 4200

agacgtggaa ggaactagcc acatgccgca gcacctcttt caacgtccag gacctgctgc 4260

ctgaccacga atataagttc cgtgtacgtg caatcaacgt gtatggaacc agtgagccaa 4320

gccaggagtc tgaactcaca acggtaggag agaaacctga agagccgaag gatgaagtgg 4380

aggtgtcaga tgatgatgag aaggagcccg aggttgatta ccggacagtg acaatcaata 4440

ctgaacaaaa agtatctgac ttctacgaca ttgaggagag attaggatct gggaaatttg 4500

gacaggtctt tcgacttgta gaaaagaaaa ctcgaaaagt ctgggcaggg aagttcttca 4560

aggcatattc agcaaaagag aaagagaata tccggcagga gattagcatc atgaactgcc 4620

tccaccaccc taagctggtc cagtgtgtgg atgcctttga agaaaaggcc aacatcgtca 4680

tggtcctgga gatcgtgtca ggaggggagc tgtttgagcg catcattgac gaggactttg 4740

agctgacgga gcgtgagtgc atcaagtaca tgcggcagat ctcggaggga gtggagtaca 4800

tccacaagca gggcatcgtg cacctggacc tcaagccgga gaacatcatg tgtgtcaaca 4860

agacgggcac caggatcaag ctcatcgact ttggtctggc caggaggctg gagaatgcgg 4920

ggtctctgaa ggtcctcttt ggcaccccag aatttgtggc tcctgaagtg atcaactatg 4980

agcccatcgg ctacgccaca gacatgtgga gcatcggggt catctgctac atcctagtca 5040

gtggcctttc ccccttcatg ggagacaacg ataacgaaac cttggccaac gttacctcag 5100

ccacctggga cttcgacgac gaggcattcg atgagatctc cgacgatgcc aaggatttca 5160

tcagcaatct gctgaagaaa gatatgaaaa accgcctgga ctgcacgcag tgccttcagc 5220

atccatggct aatgaaagat accaagaaca tggaggccaa gaaactctcc aaggaccgga 5280

tgaagaagta catggcaaga aggaaatggc agaaaacggg caatgctgtg agagccattg 5340

gaagactgtc ctctatggca atgatctcag ggctcagtgg caggaaatcc tcaacagggt 5400

caccaaccag cccgctcaat gcagaaaaac tagaatctga agaagatgtg tcccaagctt 5460

tccttgaggc tgttgctgag gaaaagcctc atgtaaaacc ctatttctct aagaccattc 5520

gcgattaga agttgtggag ggaagtgctg ctagatttga ctgcaagatt gaaggatacc 5580

cagaccccga ggttgtctgg ttcaaagatg accagtcaat cagggagtcc cgccacttcc 5640

agatagacta cgatgaggac gggaactgct ctttaattat tagtgatgtt tgcggggatg 5700

acgatgccaa gtacacctgc aaggctgtca acagtcttgg agaagccacc tgcacagcag 5760

agctcattgt ggaaacgatg gaggaaggtg aaggggaagg ggaagaggaa gaagagtgaa 5820

acaaagccag agaaaagcag tttctaagtc atattaaaag gactatttct ctaaaactca 5880

aaaaaaaaaaaaaaactcaa gatagtaaaa gcacctagtg tgatagatta tcggttaggt 5940

catttgtggg ttgattcttc agaaacagca gttgatacct agcagcgtta ttgatgggca 6000

ttaatctatg ttagttggca ccttaagata ctagtgcagc tagatttcat ttagggaaat 6060

caccagtaac ttgactgacc aattgatttt agagagaaag taaccaaacc aaatatttat 6120

ctgggcaaag tcataaattc tccacttgaa tgcgctcatg aaaaataagg ccaaaacaag 6180

agttctgggc cacagctcag cccagagggt tcctggggat gggaggcctc tctctcccca 6240

ccccctgact ctagagaact gggttttctc ccagtactcc agcaattcat ttctgaaagc 6300

agttgagcca ctttattcca aagtacactg cagatgttca aactctccat ttctctttcc 6360

ccttccacct gccagttttg ctgactctca acttgtcatg agtgtaagca ttaaggacat 6420

tatgcttctt cgattctgaa gacagtccc tgctcatgga tgactctggc ttccttagga 6480

aaatattttt cttccaaaat cagtaggaaa tctaaactta tcccctcttt gcagatgtct 6540

agcagcttca gacatttggt taagaaccca tgggaaaaaa aaaatccttg ctaatgtggt 6600

ttcctttgta aaccaggatt cttatttgtg ctgttataga atatcagctc tgaacgtgtg 6660

gtaaagattt ttgtgtttga atataggaga aatcagtttg ctgaaaagtt agtcttaatt 6720

atctattggc cacgatgaaa cagatttcaa ctgataaaga gctggagaac tccatgtact 6780

ttggaatctc ctccaagata gccagagttt aatacatctt cattctcaac actctccaaa 6840

gaacttgacc taccttatgg gttccatatt tttcttctta aatgtgcatc aatcatgcct 6900

tgcccccaac ctttaaatat attcttagac ctggtaaatg cactcagact tgcgtcttta 6960

ggaattttta actttctttc actacattgg cacttaaatt ttttctttat aaagcttttt 7020

gaaggtcata aacaaagacc ataattgatg atagacctaa tacatttcct ctgtgtgtgt 7080

gtgtaacatt ccaaatactt tttttttctt ttccactgtt tgtaaggtgc aacaatttaa 7140

tatttttaag ggacttttta agagttcctt aagaaccaat ttaaaattac ttcagtgcaa 7200

tcctacacag tatcaacatt agaattttga tattagtctt atgttatctt ccattctatt 7260

tttatctgct ttttgctgct agtttcaaac tgccagtatt tttccttttg cttttaaaat 7320

agttacaata tttttcatga tagccacagt attgccacag tttattataa taaagggttt 7380

ttatttgatt tagcgcattc aaagcttttt tctatcactt ttgtgttcag aatataacct 7440

ttgtgtgcgt gtatgttgtg tgtgtgcatg tgtggcgtat atgtgtgtta caggttaatg 7500

ccttcttgga attgtgttaa tgttctcttg gtttattatg ccatcagaat ggtaaatgag 7560

aacactacaa ctgtagtcag ctcacaattt ttaaataaag gataccacag tgcatgctgt 7620

ttgttcaaaaaaaaaaaaaaaaaa 7644

<210> 2

<211> 1738

<212> PRT

<213> human source

<400> 2

Met Gly Arg Phe Ser Cys Lys Ile Thr Gly Arg Pro Gln Pro Gln Val

1 5 10 15

Thr Trp Leu Lys Gly Asn Val Pro Leu Gln Pro Ser Ala Arg Val Ser

20 25 30

Val Ser Glu Lys Asn Gly Met Gln Val Leu Glu Ile His Gly Val Asn

35 40 45

Gln Asp Asp Val Gly Val Tyr Thr Cys Leu Val Val Asn Gly Ser Gly

50 55 60

Lys Ala Ser Met Ser Ala Glu Leu Ser Ile Gln Gly Leu Asp Ser Ala

65 70 75 80

Asn Arg Ser Phe Val Arg Glu Thr Lys Ala Thr Asn Ser Asp Val Arg

85 90 95

Lys Glu Val Thr Asn Val Ile Ser Lys Glu Ser Lys Leu Asp Ser Leu

100 105 110

Glu Ala Ala Ala Lys Ser Lys Asn Cys Ser Ser Pro Gln Arg Gly Gly

115 120 125

Ser Pro Pro Trp Ala Ala Asn Ser Gln Pro Gln Pro Pro Arg Glu Ser

130 135 140

Lys Leu Glu Ser Cys Lys Asp Ser Pro Arg Thr Ala Pro Gln Thr Pro

145 150 155 160

Val Leu Gln Lys Thr Ser Ser Ser Ser Ile Thr Leu Gln Ala Ala Arg Val

165 170 175

Gln Pro Glu Pro Arg Ala Pro Gly Leu Gly Val Leu Ser Pro Ser Gly

180 185 190

Glu Glu Arg Lys Arg Pro Ala Pro Pro Arg Pro Ala Thr Phe Pro Thr

195 200 205

Arg Gln Pro Gly Leu Gly Ser Gln Asp Val Val Ser Lys Ala Ala Asn

210 215 220

Arg Arg Ile Pro Met Glu Gly Gln Arg Asp Ser Ala Phe Pro Lys Phe

225 230 235 240

Glu Ser Lys Pro Gln Ser Gln Glu Val Lys Glu Asn Gln Thr Val Lys

245 250 255

Phe Arg Cys Glu Val Ser Gly Ile Pro Lys Pro Glu Val Ala Trp Phe

260 265 270

Leu Glu Gly Thr Pro Val Arg Arg Gln Glu Gly Ser Ile Glu Val Tyr

275 280 285

Glu Asp Ala Gly Ser His Tyr Leu Cys Leu Leu Lys Ala Arg Thr Arg

290 295 300

Asp Ser Gly Thr Tyr Ser Cys Thr Ala Ser Asn Ala Gln Gly Gln Leu

305 310 315 320

Ser Cys Ser Trp Thr Leu Gln Val Glu Arg Leu Ala Val Met Glu Val

325 330 335

Ala Pro Ser Phe Ser Ser Val Leu Lys Asp Cys Ala Val Ile Glu Gly

340 345 350

Gln Asp Phe Val Leu Gln Cys Ser Val Arg Gly Thr Pro Val Pro Arg

355 360 365

Ile Thr Trp Leu Leu Asn Gly Gln Pro Ile Gln Tyr Ala Arg Ser Thr

370 375 380

Cys Glu Ala Gly Val Ala Glu Leu His Ile Gln Asp Ala Leu Pro Glu

385 390 395 400

Asp His Gly Thr Tyr Thr Cys Leu Ala Glu Asn Ala Leu Gly Gln Val

405 410 415

Ser Cys Ser Ala Trp Val Thr Val His Glu Lys Lys Ser Ser Arg Lys

420 425 430

Ser Glu Tyr Leu Leu Pro Val Ala Pro Ser Lys Pro Thr Ala Pro Ile

435 440 445

Phe Leu Gln Gly Leu Ser Asp Leu Lys Val Met Asp Gly Ser Gln Val

450 455 460

Thr Met Thr Val Gln Val Ser Gly Asn Pro Pro Pro Glu Val Ile Trp

465 470 475 480

Leu His Asn Gly Asn Glu Ile Gln Glu Ser Glu Asp Phe His Phe Glu

485 490 495

Gln Arg Gly Thr Gln His Ser Leu Cys Ile Gln Glu Val Phe Pro Glu

500 505 510

Asp Thr Gly Thr Tyr Thr Cys Glu Ala Trp Asn Ser Ala Gly Glu Val

515 520 525

Arg Thr Gln Ala Val Leu Thr Val Gln Glu Pro His Asp Gly Thr Gln

530 535 540

Pro Trp Phe Ile Ser Lys Pro Arg Ser Val Thr Ala Ser Leu Gly Gln

545 550 555 560

Ser Val Leu Ile Ser Cys Ala Ile Ala Gly Asp Pro Phe Pro Thr Val

565 570 575

His Trp Leu Arg Asp Gly Lys Ala Leu Cys Lys Asp Thr Gly His Phe

580 585 590

Glu Val Leu Gln Asn Glu Asp Val Phe Thr Leu Val Leu Lys Lys Val

595 600 605

Gln Pro Trp His Ala Gly Gln Tyr Glu Ile Leu Leu Lys Asn Arg Val

610 615 620

Gly Glu Cys Ser Cys Gln Val Ser Leu Met Leu Gln Asn Ser Ser Ala

625 630 635 640

Arg Ala Leu Pro Arg Gly Arg Glu Pro Ala Ser Cys Glu Asp Leu Cys

645 650 655

Gly Gly Gly Val Gly Ala Asp Gly Gly Gly Ser Asp Arg Tyr Gly Ser

660 665 670

Leu Arg Pro Gly Trp Pro Ala Arg Gly Gln Gly Trp Leu Glu Glu Glu Glu

675 680 685

Asp Gly Glu Asp Val Arg Gly Val Leu Lys Arg Arg Val Glu Thr Arg

690 695 700

Gln His Thr Glu Glu Ala Ile Arg Gln Gln Glu Val Glu Gln Leu Asp

705 710 715 720

Phe Arg Asp Leu Leu Gly Lys Lys Val Ser Thr Lys Thr Leu Ser Glu

725 730 735

Asp Asp Leu Lys Glu Ile Pro Ala Glu Gln Met Asp Phe Arg Ala Asn

740 745 750

Leu Gln Arg Gln Val Lys Pro Lys Thr Val Ser Glu Glu Arg Lys

755 760 765

Val His Ser Pro Gln Gln Val Asp Phe Arg Ser Val Leu Ala Lys Lys

770 775 780

Gly Thr Ser Lys Thr Pro Val Pro Glu Lys Val Pro Pro Pro Lys Pro

785 790 795 800

Ala Thr Pro Asp Phe Arg Ser Val Leu Gly Gly Lys Lys Lys Leu Pro

805 810 815

Ala Glu Asn Gly Ser Ser Ser Ala Glu Thr Leu Asn Ala Lys Ala Val

820 825 830

Glu Ser Ser Lys Pro Leu Ser Asn Ala Gln Pro Ser Gly Pro Leu Lys

835 840 845

Pro Val Gly Asn Ala Lys Pro Ala Glu Thr Leu Lys Pro Met Gly Asn

850 855 860

Ala Lys Pro Ala Glu Thr Leu Lys Pro Met Gly Asn Ala Lys Pro Asp

865 870 875 880

Glu Asn Leu Lys Ser Ala Ser Lys Glu Glu Leu Lys Lys Asp Val Lys

885 890 895

Asn Asp Val Asn Cys Lys Arg Gly His Ala Gly Thr Thr Asp Asn Glu

900 905 910

Lys Arg Ser Glu Ser Gln Gly Thr Ala Pro Ala Phe Lys Gln Lys Leu

915 920 925

Gln Asp Val His Val Ala Glu Gly Lys Lys Leu Leu Leu Gln Cys Gln

930 935 940

Val Ser Ser Asp Pro Pro Ala Thr Ile Ile Trp Thr Leu Asn Gly Lys

945 950 955 960

Thr Leu Lys Thr Thr Lys Phe Ile Ile Leu Ser Gln Glu Gly Ser Leu

965 970 975

Cys Ser Val Ser Ile Glu Lys Ala Leu Pro Glu Asp Arg Gly Leu Tyr

980 985 990

Lys Cys Val Ala Lys Asn Asp Ala Gly Gln Ala Glu Cys Ser Cys Gln

995 1000 1005

Val Thr Val Asp Asp Ala Pro Ala Ser Glu Asn Thr Lys Ala Pro

1010 1015 1020

Glu Met Lys Ser Arg Arg Pro Lys Ser Ser Leu Pro Pro Val Leu

1025 1030 1035

Gly Thr Glu Ser Asp Ala Thr Val Lys Lys Lys Pro Ala Pro Lys

1040 1045 1050

Thr Pro Pro Lys Ala Ala Met Pro Pro Gln Ile Ile Gln Phe Pro

1055 1060 1065

Glu Asp Gln Lys Val Arg Ala Gly Glu Ser Val Glu Leu Phe Gly

1070 1075 1080

Lys Val Thr Gly Thr Gln Pro Ile Thr Cys Thr Trp Met Lys Phe

1085 1090 1095

Arg Lys Gln Ile Gln Glu Ser Glu His Met Lys Val Glu Asn Ser

1100 1105 1110

Glu Asn Gly Ser Lys Leu Thr Ile Leu Ala Ala Arg Gln Glu His

1115 1120 1125

Cys Gly Cys Tyr Thr Leu Leu Val Glu Asn Lys Leu Gly Ser Arg

1130 1135 1140

Gln Ala Gln Val Asn Leu Thr Val Val Asp Lys Pro Asp Pro Pro

1145 1150 1155

Ala Gly Thr Pro Cys Ala Ser Asp Ile Arg Ser Ser Ser Leu Thr

1160 1165 1170

Leu Ser Trp Tyr Gly Ser Ser Tyr Asp Gly Gly Ser Ala Val Gln

1175 1180 1185

Ser Tyr Ser Ile Glu Ile Trp Asp Ser Ala Asn Lys Thr Trp Lys

1190 1195 1200

Glu Leu Ala Thr Cys Arg Ser Thr Ser Phe Asn Val Gln Asp Leu

1205 1210 1215

Leu Pro Asp His Glu Tyr Lys Phe Arg Val Arg Ala Ile Asn Val

1220 1225 1230

Tyr Gly Thr Ser Glu Pro Ser Gln Glu Ser Glu Leu Thr Thr Val

1235 1240 1245

Gly Glu Lys Pro Glu Glu Pro Lys Asp Glu Val Glu Val Ser Asp

1250 1255 1260

Asp Asp Glu Lys Glu Pro Glu Val Asp Tyr Arg Thr Val Thr Ile

1265 1270 1275

Asn Thr Glu Gln Lys Val Ser Asp Phe Tyr Asp Ile Glu Glu Arg

1280 1285 1290

Leu Gly Ser Gly Lys Phe Gly Gln Val Phe Arg Leu Val Glu Lys

1295 1300 1305

Lys Thr Arg Lys Val Trp Ala Gly Lys Phe Phe Lys Ala Tyr Ser

1310 1315 1320

Ala Lys Glu Lys Glu Asn Ile Arg Gln Glu Ile Ser Ile Met Asn

1325 1330 1335

Cys Leu His His Pro Lys Leu Val Gln Cys Val Asp Ala Phe Glu

1340 1345 1350

Glu Lys Ala Asn Ile Val Met Val Leu Glu Ile Val Ser Gly Gly

1355 1360 1365

Glu Leu Phe Glu Arg Ile Ile Asp Glu Asp Phe Glu Leu Thr Glu

1370 1375 1380

Arg Glu Cys Ile Lys Tyr Met Arg Gln Ile Ser Glu Gly Val Glu

1385 1390 1395

Tyr Ile His Lys Gln Gly Ile Val His Leu Asp Leu Lys Pro Glu

1400 1405 1410

Asn Ile Met Cys Val Asn Lys Thr Gly Thr Arg Ile Lys Leu Ile

1415 1420 1425

Asp Phe Gly Leu Ala Arg Arg Leu Glu Asn Ala Gly Ser Leu Lys

1430 1435 1440

Val Leu Phe Gly Thr Pro Glu Phe Val Ala Pro Glu Val Ile Asn

1445 1450 1455

Tyr Glu Pro Ile Gly Tyr Ala Thr Asp Met Trp Ser Ile Gly Val

1460 1465 1470

Ile Cys Tyr Ile Leu Val Ser Gly Leu Ser Pro Phe Met Gly Asp

1475 1480 1485

Asn Asp Asn Glu Thr Leu Ala Asn Val Thr Ser Ala Thr Trp Asp

1490 1495 1500

Phe Asp Asp Glu Ala Phe Asp Glu Ile Ser Asp Asp Ala Lys Asp

1505 1510 1515

Phe Ile Ser Asn Leu Leu Lys Lys Asp Met Lys Asn Arg Leu Asp

1520 1525 1530

Cys Thr Gln Cys Leu Gln His Pro Trp Leu Met Lys Asp Thr Lys

1535 1540 1545

Asn Met Glu Ala Lys Lys Leu Ser Lys Asp Arg Met Lys Lys Tyr

1550 1555 1560

Met Ala Arg Arg Lys Trp Gln Lys Thr Gly Asn Ala Val Arg Ala

1565 1570 1575

Ile Gly Arg Leu Ser Ser Met Ala Met Ile Ser Gly Leu Ser Gly

1580 1585 1590

Arg Lys Ser Ser Thr Gly Ser Pro Thr Ser Pro Leu Asn Ala Glu

1595 1600 1605

Lys Leu Glu Ser Glu Glu Asp Val Ser Gln Ala Phe Leu Glu Ala

1610 1615 1620

Val Ala Glu Glu Lys Pro His Val Lys Pro Tyr Phe Ser Lys Thr

1625 1630 1635

Ile Arg Asp Leu Glu Val Val Glu Gly Ser Ala Ala Arg Phe Asp

1640 1645 1650

Cys Lys Ile Glu Gly Tyr Pro Asp Pro Glu Val Val Trp Phe Lys

1655 1660 1665

Asp Asp Gln Ser Ile Arg Glu Ser Arg His Phe Gln Ile Asp Tyr

1670 1675 1680

Asp Glu Asp Gly Asn Cys Ser Leu Ile Ile Ser Asp Val Cys Gly

1685 1690 1695

Asp Asp Asp Ala Lys Tyr Thr Cys Lys Ala Val Asn Ser Leu Gly

1700 1705 1710

Glu Ala Thr Cys Thr Ala Glu Leu Ile Val Glu Thr Met Glu Glu

1715 1720 1725

Gly Glu Gly Glu Gly Glu Glu Glu Glu Glu Glu

1730 1735

<210> 3

<211> 18

<212>DNA

<213> Artificial sequence

<400> 3

aagtggaggt gtcagatg 18

<210> 4

<211> 20

<212>DNA

<213> Artificial sequence

<400> 4

tcaatgtcgt agaagtcaga 20

<210> 5

<211> 22

<212>DNA

<213> Artificial sequence

<400> 5

aactctggta aagtggatat tg 22

<210> 6

<211> 20

<212>DNA

<213> Artificial sequence

<400> 6

ggtggaatca tattggaaca 20

Claims (10)

1. detect application of the product of MYLK gene expressions in the instrument of diagnosis of endometrial carcinoma is prepared.

2. application according to claim 1, it is characterised in that the product includes：By RT-PCR, real-time quantitative PCR, Immune detection, in situ hybridization, the expression of chip or high-flux sequence detection of platform MYLK genes are with diagnosis of endometrial carcinoma Product.

3. application according to claim 2, it is characterised in that the product of the use RT-PCR diagnosis of endometrial carcinoma is at least Include the primer of a pair of specific amplified MYLK genes；The product of the use real-time quantitative PCR diagnosis of endometrial carcinoma at least includes The primer of a pair of specific amplified MYLK genes；The product of the use immune detection diagnosis of endometrial carcinoma includes：With MYLK albumen The antibody of specific binding；The product of the use in situ hybridization diagnosis of endometrial carcinoma includes：With the nucleotide sequence of MYLK genes The probe of hybridization；The product of the use chip diagnosis of endometrial carcinoma includes：Protein chip and genetic chip；Wherein, albumen core Piece includes the antibody combined with MYLK protein-specifics, and genetic chip includes the probe with the nucleic acid array hybridizing of MYLK genes.

4. application according to claim 3, it is characterised in that the production of the use real-time quantitative PCR diagnosis of endometrial carcinoma The primer for a pair of specific amplified MYLK genes that product at least include is as shown in SEQ ID NO.3 and SEQ ID NO.4.

5. a kind of instrument for diagnosis of endometrial carcinoma, it is characterised in that the instrument can be by detecting MYLK in sample The expression of gene carrys out diagnosis of endometrial carcinoma.

6. instrument according to claim 5, it is characterised in that the instrument includes chip, kit, test paper or high flux Microarray dataset.

7. instrument according to claim 6, it is characterised in that the chip includes genetic chip, protein-chip；It is described Genetic chip includes solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and the oligonucleotide probe includes being used for Detect the oligonucleotide probe for MYLK genes of MYLK gene transcription levels；The protein-chip include solid phase carrier with And it is fixed on the specific antibody of the MYLK albumen of solid phase carrier；The kit includes gene detecting kit and protein immunization Detection kit；The gene detecting kit includes the reagent for being used to detect MYLK gene transcription levels；The protein immunization Detection kit includes the specific antibody of MYLK albumen；The test paper includes the examination for being used to detect MYLK gene transcription levels Agent；The high-flux sequence platform includes the reagent for being used to detect MYLK gene transcription levels.

8. instrument according to claim 7, it is characterised in that the reagent of the detection MYLK gene transcription levels includes pin To the primer and/or probe of MYLK genes.

9. instrument according to claim 8, its spy is characterised by, the primer sequence for MYLK genes is as follows：Just To primer sequence as shown in SEQ ID NO.3, reverse primer is as shown in SEQ ID NO.4.

10. the instrument according to any one of claim 5-9, it is characterised in that the sample is tissue.