WO2010091581A1

WO2010091581A1 - Detection of ptp1b gene mutations and the uses thereof in diagnosing cancers

Info

Publication number: WO2010091581A1
Application number: PCT/CN2009/075005
Authority: WO
Inventors: 郑新民; 梅文翰
Original assignee: 上海交通大学医学院
Priority date: 2009-02-13
Filing date: 2009-11-18
Publication date: 2010-08-19
Also published as: CN101487013A

Abstract

New mutant sites existed in PTP1B gene in Chinese population are provided, which are specifically associated with the occurrence of cancers and are significant useful in diagnosis in cancer individuals. Detection methods for diagnosing the causes and types of cancers by detecting whether there are mutant sites existed in PTP1B gene in the samples of individuals to be detected are also provided. The methods will be in favor of clinically screening the mutant sites existed in PTP1B gene in the individuals, and providing service for diagnosis and treatments for cancer individuals.

Description

Detection of PTP1B gene mutation and its application in cancer diagnosis

Technical field

The present invention relates to the field of diagnosis and detection of cancer. J=body involves a method for detecting a mutation in the PTP1B gene, a kit for detecting cancer, and its use in cancer diagnosis. Background technique

Protein tyrosine phosphorylation is an important cellular signal regulation mechanism that affects important physiological processes such as cell growth and differentiation, cell cycle regulation, apoptosis and migration. The tyrosine phosphorylation state in cells is a reversible dynamic balance that changes with cell function, and is maintained by protein tyrosine kinase (Protein Tyrosine Kinase, ΡΤΚ) and protein tyrosine phosphatase (Protein Tyrosine Phosphotase, ΡΤΡ). . ΡΤΚ, ΡΤΡ and their respective substrates together form an extensive signal transduction network. Once the genes of the key members of the above two enzyme families are mutated due to various reasons, abnormal knockdown or post-transcriptional modification of the protein can cause normal cell transformation or canceration.

PTP1B is a member of the ΡΤΡ gene family. Human PTP1B is a single-copy gene, which is encoded by PTPN1 gene and located at 20ql3.1-ql3.2. The gene is about 74 kb in length and contains 10 exons. Its cDNA is 3,318 bp in length and encodes 435 amino acids. It is 49,666 Da. PTP1B is widely expressed in vivo, and its substrate proteins include insulin receptor (IR) epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGFR), insulin growth factor I receptor (IGF-IR), and p210Bcr-Abl. Janus Kinase 2 ( JAK2 ) and TYK2 and other receptor-type PTK (RTK)o It is known that many PTK activity abnormalities are directly related to tumorigenesis. At least 20 PTKs have been identified as oncogenes. Recent studies have confirmed that guanidine can negatively regulate a variety of PTK signaling pathways by affecting the phosphorylation of PTK tyrosine residues, thereby negatively regulating various PTK signaling pathways and affecting important physiological functions of cells.

The nucleotide sequence of the PTP1B gene is described in the PTP1B file (3318 bp in total), which contains 10 exons, namely: Exonl: l-237 bp; Exon 2: 238-328 bp; Exon 3: 329-429 bp; Exon 4 : 430-528 bp; Exon 5: 529-666 bp; Exon 6: 667-876 bp; Exon 7: 877-1038 bp; Exon 8: 1039-1262 bp; Exon 9: 1263-1458 bp; Exon 10: 1459-3318 bp, Exonl Exon2 Exon3 Exon4 Exon5 Exon6 Exon7 Εχοηδ Exon9 ExonlO We first discovered the loss of PTPIB gene in a population of malignant tumors in China, that is, the different degrees of deletion of exons caused changes in the nucleotide sequence of PTP1B gene. The discovery of these PTP1B mutant genes will contribute to the accurate diagnosis of malignant tumors at an early stage and in different pathological stages. Summary of the invention

One of the objects of the present invention is to provide a PTP1B mutant gene related to cancer, which is characterized in that a mutation site exists in the PTP1B gene, and the mutation positions are 238-328 bp of the PTP1B gene, 309-328 bp of the PTPIB gene, and 529 of the PTPIB gene. -666 bp, PTPIB gene 667-876 bp, PTPIB gene 778-783 bp, PTPIB gene 730-953 bp, PTPIB gene 1037-1042 bp, PTPIB gene 1281-1384 bp, the sequences deleted at the above mutation positions are respectively SEQ ID: NoK SEQ ID : No. 2, SEQ ID: No3, SEQ ID: No4, SEQ ID: No5, SEQ ID: No. 6, SEQ ID: No. 7, SEQ ID: No. 8.

Another object of the present invention is to provide a method for detecting cancer, which is to detect whether a PTP1B gene in a sample from an individual to be tested has a mutation site, and the mutation positions are 238-328 bp of PTP1B gene and 309 bp of PTPIB gene, respectively. -328 bp, PTPIB gene 529-666 bp, PTPIB gene 667-876 bp, PTPIB gene 778-783 bp, PTPIB gene 730-953 bp, PTPIB gene 1037-1042 bp, PTPIB gene 1281-1384 bp, sequences deleted at the above mutation positions, respectively, SEQ ID: Nol, SEQ ID: No2, SEQ ID: No3, SEQ ID: No4, SEQ ID: No5, SEQ ID: No. 6, SEQ ID: No. 7, SEQ ID: No. 8.

The detection method includes the following steps:

1) collecting blood, body fluid or tissue samples of the individual to be tested, and extracting DNA;

2) using the DNA as a template, PCR reaction designed by PCR primers designed for the coding region near the mutation site to obtain a PCR reaction product;

3) The obtained PCR reaction product is subjected to direct sequencing analysis, and the obtained sequence is compared with the standard sequence of the PTP1B normal gene to determine whether or not the above-mentioned mutation site exists. 4) Based on the above results, it is judged whether the test subject has a cancer-associated PTP1B gene mutation.

The PCR primer used in the above detection method can be designed according to the known PTP1B gene sequence, usually 15-30 bases, and the GC content is about 45.50%, and specifically binds to the template at an appropriate temperature, which can be utilized. Specialized computer programming. In a specific embodiment of the invention, a set of PCR primers is designed using primer design software, the sequence of which is:

1) Paired primers: upstream 5,-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3,

A further object of the present invention is to provide a kit for detecting a mutation in the PTP1B gene, which kit may include one or a combination of the following reagents:

1) reagent for extracting DNA from the sample to be tested;

2) PCR primers for amplifying the PTP1B gene in the sample DNA and corresponding PCR reaction reagents; as well as

3) Reagents for sequencing PCR amplification products.

For example, in one embodiment of the present invention, a kit for detecting a mutation in the PTP1B gene is provided, which comprises one or more containers containing one or more components for detecting a mutation in the PTP1B gene, which are simultaneously provided It is information about the manufacture, use and sale of pharmaceuticals or biological products that has been reviewed by government drug regulatory agencies. For example, a kit for directly detecting a PTP1B gene mutation site in a sample after PCR amplification. It may contain one or more of amplification primers, dNTPs, DNA polymerases for PCR reactions, and buffers thereof. It is known to those skilled in the art that the above components are only illustrative, for example, the primers described above may employ a pair of primers, and the DNA polymerase for PCR reaction can be used. PCR amplified enzyme. For the method of use, please refer to the embodiment in the specific embodiment.

According to still another aspect of the present invention, the use of the PTP1B mutant gene for diagnosing a cancer disease is provided, and the cause and type of cancer of the individual are determined by detecting whether a PTP1B gene-specific mutation site is present in the test subject.

The present invention provides for the first time a novel mutation site of the PTP1B gene existing in the Chinese population, and proves that this type of mutant gene is specifically associated with the occurrence of cancer, which is of great significance for the diagnosis of cancer patients. The present invention also proposes a method for detecting the cause and type of cancer occurrence by detecting the presence or absence of a mutation in the PTP1B gene in a patient. This will facilitate the clinical screening of PTP1B gene mutations in cancer patients and provide services for the diagnosis and treatment of cancer patients. DRAWINGS

Figure 1A and Figure 1B are RT-PCR results of different types of tumor specimens;

Figure 2 is a diagram showing the results of immunoblotting for detecting wild-type and mutant PTP1B fusion proteins; Figure 3 is a graph showing RT-PCR results of partial mutation sites of PTP1B gene. detailed description

The invention will now be further described with reference to the drawings in conjunction with the embodiments.

The PTP1B gene was selected as a research object, and a new mutation site of a cancer-associated PTP1B gene was found by screening a sample of 105 cancer patients. These mutation sites are at home and abroad. None of them were reported. The specific results of the mutations are summarized in the table below.

Example 1 PCR amplification of each exon of PTP1B gene

(1). Extracting RNA

The tumor tissue of the patient was removed by surgery. The RNA was extracted with 1 ml of TRIzol reagent (Invitrogen), added with 0.2 ml of chloroform, vigorously shaken for 15 seconds, allowed to stand at room temperature for 5 minutes, centrifuged at 12,000 rpm for 15 minutes at 4 ° C, and the supernatant was aspirated and added. 0.5 ml of isopropanol was mixed, allowed to stand for 10 minutes, centrifuged at 12,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed with 75% ethanol and then dissolved with 20 ul of DEPC-H ₂ O. 2 ul was diluted and the absorbance was measured by an ultraviolet spectrophotometer. The RNA concentration was calculated according to the formula RNA (ug/ml) = OD _26Qnm x 40x dilution factor ÷ cuvette thickness (cm). (2) ■ T-PC

Using the Invitrogen reverse transcription kit, the above RNAlug was taken, random primers lul and dNTP lul were added, and 10 μl was made up with DEPC-H ₂ 0, placed in a water bath at 65 ° C for 5 minutes, and immediately placed in an ice bath. Add the cDNA synthesis mixture lOul, leave it at 25 ° C for 10 minutes, then place it at 50 ° C for 50 minutes, then place it at 85 ° C for 5 minutes, then place it in an ice bath, add lul RNase H and place it in a 37 ° C water bath for 20 minutes. The cDNA was stored at -20 °C.

The PTP1B gene was amplified by polymerase chain reaction (PC) of the sample DNA. The amplified fragment was the 175-1482 reading frame of the PTP1B gene, which was 1308 bp long and encompassed 9 complete exons. An upstream primer sequence designed to amplify the PTP1B gene: 5,-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3,, downstream primer sequence: 5'-CTATGTGTTGCT GTTGAACAGGAACCTGTAG-3. The amplification system is: 10x buffer 5ul 4NTP2ul, 10umol L upstream primer and downstream primer 0.5ul gCl ₂ 2ul, sample cDNA 3ul, supplemented with double distilled water volume 48ul ₀ 95. After C inactivation for 5 minutes, 1 U (diluted to 1 U / 2 ul before use) Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with high fidelity was added. The amplification conditions were 95 ° C for 40 seconds, 55 ° C for 40 seconds, and 68 ° C for 120 seconds for a total of 25 cycles. Example 2 PCR product purification and sequencing

(1) . Connection and transformation of PCR products

The above PCR product was first separated by 1% agarose gel electrophoresis to determine whether the size of the amplified fragment was consistent with the expected 1212 bp, and then the correct PCR product was ligated to the 3.9 kb pCR2.1-TOPO vector and transformed into In E. coli cells (TOPO TA Cloning Kits, Invitrogen products, the specific steps are: take 4 ul of PCR product, add salt solution and TOPO carrier M, mix gently, let stand for 5 minutes at room temperature, then wait at 30 ° C for 10 minutes. Then connect 2ul to the One Shot ^R E.coli E. coli solution, gently mix and place on ice for 10 minutes to wait for transformation, then place a 42 ° C water bath for 30 seconds for heat shock, immediately place the ice bath. Add at room temperature The SOC culture solution was 250 ul, and it was shaken and shaken at 37 ° C for 1 hour under a constant temperature shaker.

(2). Screening, identification and sequencing

Pipette lOOul of transformed bacteria solution onto a 1.5% LB agar plate containing ampicillin 100u _g / ml, add 40ul X-gal, immediately spread evenly with a glass rod, and then place the plate at 37 ° C constant temperature culture Incubate for 16-18 hours and observe the bacterial growth on the plate (with blue colonies and white colonies A single white colony was taken into 3 ml of LB liquid medium, and cultured overnight at 37 ° C under a constant temperature shaker, and a small amount of amplification was performed. The next day, 1 ml of the bacterial solution was taken to the EP tube, and the bacterial plasmid DNA was extracted using Geneaid's rapid small-volume plasmid extraction cassette. First centrifuge the bacteria solution at 12000 rpm for 1 minute, then discard the supernatant. Add 200 ul of RNaseA-containing solution I to the pellet, resuspend the bacteria, add 200 ul of solution II, gently invert and mix for 5 minutes to lyse the bacteria, then add 300 ul of solution III. Mix gently by inverting, centrifuge at full speed for 3 minutes, pipette the supernatant to the spin column, centrifuge the spin column at 13000 rpm for 30 seconds to bind the DNA, then add the ethanol-containing washing buffer to the spin column and centrifuge again for 30 seconds at OOO rpm to discard the buffer. Add 50 μl of 0 mmol/LTris-HCl buffer (ρΗ8·5) to the spin column to dissolve the DNA. After standing for 2 minutes, centrifuge at full speed for 2 minutes. Collect the effluent as bacterial plasmid DNA. Take 5ul of DNA, add 2ull0x buffer and 10U restriction enzyme EcoR I, make up 20ul volume with double distilled water, cut into the water bath at 37 °C for 2 hours, and separate it by 1% agarose gel electrophoresis. The external fragment, which is about 1.3 kb in size (Fig. 1), was sent to Yingjun for DNA sequencing.

The results of RT-PCR for different types of tumor specimens are shown in Figure 1A and Figure 1B. In Figure 1A, from left to right: l.lkb DNA Ladder; 2. normal thyroid tissue; 3. thyroid cancer 62; 4. thyroid cancer 186; 5. thyroid cancer 234; 6. thyroid cancer 330; 7. normal stomach Organization 227; 8. Gastric cancer 4. In Fig. 1B, from the left, in order: l.lkb DNA Ladder; 2. normal colon tissue; 3. intestinal cancer 8, 4. intestinal cancer 36, 5. intestinal cancer 10, 6. intestinal cancer 13, 7. Cancer 93, 8. Intestinal cancer 69; 9. Intestinal cancer 45.

The RT-PCR results are analyzed as shown in the following table:

Example 3 Gene transfection, protein expression and immunoblotting

3ug pTetsplice-PTP 1 B WT-HA and 3ug pTetsplice-ΡΤΡΙΒ E6-HA were co-transfected with REF ( rat embryo fibroblast ) cells with 3ugpTet-tTAK and 0.3ugpSV2neo respectively. After 48 hours of transfection, G418 0.5mg/ml was screened for two. Week, pick up stable transfected cell clones. After stable cells were induced by doxycycline ( _Dox ) for 16 hours, they were collected and made into whole cell suspensions. 30 ug of protein was taken from each whole cell suspension for PAGE electrophoresis, and then transferred to NC membrane. 5% skim milk powder TBS was blocked for 2 hours, and anti-HA antibody (diluted 1:200) was placed at 4 °C for 12-14 hours, TBS (including 0.1% Tween-20) Wash the NC membrane 3 times at room temperature for 10 minutes each time, then add secondary antibody (1: 10000 dilution), and finally develop. The results are shown in Figure 2. In the figure, wild-type (mutant-type) and mutant (mutants) PTP1B fusion proteins (anti-HA antibodies) were detected by Western blot, wherein 1. REF cells were not transfected; 2. pTetsplice-PTPlB ^ E6-HA transfected REF cells, doxycycline (Dox); 3. pTetsplice-PTPlBA E6-HA transfected REF cells, do not add doxycycline 16 small days; 4. pTetsplice-PTPlBWT-HA transfected EF Cells, doxycycline was added; 5. pTetsplice-PTP 1 B WT-HA was transfected into REF cells without the addition of doxycycline for 16 hours. Example 4 PCR reaction primer design

Based on the multiple PTP1B mutation sites found in the experiment, the inventors designed a series of downstream primers to pair with the upstream and downstream primers for amplifying the PTP1B gene for detection of clinical pathological tissue samples, which were specific for the mutant gene. PCR amplification can obtain the corresponding mutant gene amplification product, thereby confirming that the sample has a mutation at the corresponding site. This mutant gene-specific PCR method cannot amplify PTP1B in para-tumor tissues, ie, normal tissues, as shown in the table below.

1) Paired primers: upstream 5,-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3,

2) Paired primers: downstream 5,-CTATGTGTTGCT GTTGAACAGGAACCTGTAG-3,

Detection of mutation site primer sequence amplified fragment length deletion sequence 238-328 Upstream: 5,- CATTTACCAGTTGACCAT -3' 329bp

Downstream: 5,- AGCTGTCGCACTGTATAA -3'

Missing sequence 309-328 Upstream: 5'-CCGAAATAGGTTGACCAT-3, 329bp

Downstream: 5'- AGCTGTCGCACTGTATAA -3'

The RT-PCR results for some of the mutation sites are shown in Figure 3. In the figure, from the left are: l.lkb DNA Ladder; 2. Para-tumor tissue; 3. Missing sequence 667-876 (500 bp); 4. Para-neural tissue; 5. Missing sequence 529-666 (362 bp); 6. Para-neural tissue; 7. Missing sequence 730-953 (563 bp); 8. Para-tumor tissue; 9. Missing sequence 778-783 (610 bp) 10. Para-neoplastic tissue; 11. Missing sequence 1281-1384 (108 bp); 12. Para-neural tissue; 13. Missing sequence 1037-1042 (448 bp)

In the above examples, PTP1B gene analysis was performed on various types of tumor tissues, and several PTP1B mutant genes found in the experiment have not been reported at home and abroad so far. The invention provides a specific detection method and clinical application of PTP1B mutation gene in Chinese population, and the above detection method can judge and track important molecular indexes in the early occurrence, development and metastasis of malignant tumors, from molecular level to malignant tumors. Early diagnosis and observation of effective predictions after anti-tumor drug therapy are instructive.

Claims

Claim

1. A class of PTP1B mutant genes involved in cancer, characterized in that there are mutation sites in the PTP1B gene, and the mutation positions are 238-328 bp of PTP1B gene, 309-328 bp of PTP1B gene, 529-666 bp of PTP1B gene, PTP1B gene. 667-876 bp, PTP1B gene 778-783 bp, PTP1B gene 730-953 bp, PTP1B gene 1037-1042 bp PTP1B gene 1281-1384 bp, the sequence deleted at the above mutation position are as shown in the sequence listing SEQ ID: Nol, SEQ ID: No 2, SEQ ID : No3, SEQ ID: No4, SEQ ID: No5, SEQ ID: No. 6, SEQ ID: No. 7, SEQ ID: No. 8.

2. A method for detecting cancer, which is to detect whether a PTP1B gene has a mutation site in a sample from an individual to be tested, and the mutation positions are 238-328 bp of PTP1B gene, 309-328 bp of PTP1B gene, and 529 of PTP1B gene. -666 bp, PTP1B gene 667-876 bp, PTP1B gene 778-783 bp, PTP1B gene 730-953 bp, PTP1B gene 1037-1042 bp, PTP1B gene 1281-1384 bp, the sequences deleted at the above mutation positions are as shown in the sequence listing SEQ ID: Nol, SEQ, respectively. ID: No. 2, SEQ ID: No3, SEQ ID: No. 4, SEQ ID: No. 5, SEQ ID: No. 6, SEQ ID: No. 7, SEQ ID: No. 8.

3. The detecting method according to claim 2, comprising the following steps:

2) using the DNA as a template, PCR reaction designed by PCR primers designed for the coding region near the mutation position to obtain a PCR reaction product;

3) The obtained PCR reaction product is subjected to direct sequencing analysis, and the obtained sequence is compared with the standard sequence of the normal gene of PTP1B to determine whether or not the above-mentioned mutation site exists.

4) Based on the above results, it is judged whether the test subject has a cancer-associated PTP1B gene mutation.

4. The detection method according to claim 3, wherein the sequence of the PCR primers designed is: 1) paired primer: upstream 5'-ATGGAGATGGAAAAGGAGTTCGAGCAGATC-3'

2) Paired primers: downstream 5 ' -CTATGTGTTGCT GTTGAACAGGAACCTGTAG-3 ' Detection of mutation site primer sequence amplified fragment length

Missing sequence 1281-1384 Upstream: 5'-AAGACACTGAGTTAC-3, 108bp

Missing sequence 1037-1042 Upstream: 5'-TTCCGTGCAGTGGAA-3' 448bp

3)

5. A kit for detecting a mutation in a PTP1B gene, which kit may comprise one or a combination of the following reagents:

1) a reagent for extracting DNA from a sample to be tested;

2) PCR primers for amplifying the PTP1B mutant gene in the sample DNA and corresponding PCR reaction reagents;

3) Reagents for sequencing PCR amplification products.

The kit according to claim 5, wherein the PTP1B mutant gene has a mutation position of 238-328 bp of PTP1B gene, 309-328 bp of PTPIB gene, 529-666 bp of PTPIB gene, and 667-876 bp of PTPIB gene. The PTPIB gene is 778-783 bp, the PTPIB gene is 730-953 bp, the PTPIB gene is 1037-1042 bp, and the PTPIB gene is 1281-1384 bp. The sequences deleted at the above mutation positions are as shown in the sequence listings SEQ ID: Nol, SEQ ID: No 2, SEQ ID: No3, SEQID, respectively. : No4, SEQID: No5, SEQID: No6, SEQID: No7, SEQID: No8.

7. The application of the PTPIB mutant gene in the diagnosis of cancer diseases, and determining the cause and type of cancer in the individual by detecting whether a PTP1B gene-specific mutation site exists in the test subject.

8. The use according to claim 7, wherein the positions of the mutation sites are 238-328 bp of PTP1B gene, 309-328 bp of PTPIB gene, 529-666 bp of PTPIB gene, 667-876 bp of PTPIB gene, PTPIB. The gene is 778-783 bp, the PTPIB gene is 730-953 bp, the PTPIB gene is 1037-1042 bp, and the PTPIB gene is 1281-1384 bp. The sequences deleted at the above mutation positions are respectively SEQ ID: Nol, SEQ ID: No 2, SEQ ID: No. 3, SEQ. ID: No4, SEQID: No5, SEQID: No6, SEQID: No7, SEQID: No8 shown _c