CN102051408B - Kit for early diagnosis of metastasis of prostate cancer - Google Patents

Abstract

The present invention provides two pairs of primers for the early diagnosis of prostate cancer metastasis: the first pair of primers has the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the second pair of primers has the sequences shown in SEQ ID NO: 3 and SEQ ID NO :4 sequence shown. The present invention also provides a kit for early diagnosis of prostate cancer metastasis, comprising the above two pairs of primers. The kit can also include a pair of primers having the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 for nested PCR amplification. The invention detects the methylation status of the promotor region of the SNRP-4 gene in the prostate cancer tissue by methylation-specific polymerase chain reaction to determine whether there is metastasis of the prostate cancer. The method utilizes the principle of molecular biology, has high specificity and accuracy, and can clearly diagnose micrometastasis of prostate cancer at an early stage.

Description

Kit for early diagnosis of prostate cancer metastasis

technical field

The invention relates to a diagnostic kit for prostate cancer metastasis, especially a kit for early diagnosis of prostate cancer metastasis.

Background technique

Prostate cancer is the most common malignancy in men in many Western countries. Prostate cancer is the second leading cause of cancer death in men in the United States. In recent years, the incidence of prostate cancer in my country has increased significantly, and prostate cancer has become an increasingly important topic in the field of urology. The choice of treatment plan for prostate cancer is mainly based on the patient's age, general condition, tumor stage and grade. The specific situation of the tumor depends on the correct diagnosis. The current diagnosis of prostate cancer mainly has the following methods:

(1) Digital rectal examination: In physical examination, digital rectal examination is the most helpful first-line examination for discovering and diagnosing prostate cancer, but digital rectal examination is a non-specific examination, and when prostate cancer is found, it is often pathologically graded It has reached a higher level of malignancy, that is, it has reached the advanced stage of prostate cancer.

(2) Determination of prostate-specific antigen: prostate-specific antigen is a kind of protease, which is usually only detected in prostatic fluid and semen. If prostate-specific antigen is detected in blood, it can often be regarded as a patient with benign or malignant prostatic lesions. symbols of. As a single test, prostate specific antigen currently has a good positive diagnosis rate for prostate cancer. Similarly, it has also helped to improve the detection and diagnosis rates of many small lesions and low-grade prostate cancer. However, in nearly 30% of prostate cancer patients, the serum PSA level may not increase, but only fluctuates within the normal range (ie, PSA < 4.0 ng/ml). Even in patients with negative digital rectal examination and serum PSA < 4.0 ng/ml, there is still a 30% possibility of prostate biopsy to find prostate cancer.

(3) Transrectal ultrasonography and prostate biopsy: Transrectal ultrasonography is a very valuable method for diagnosing prostate cancer. It can help physicians examine the patient's prostate and surrounding tissue structures to find suspicious lesions and initially determine tumors volume size. also. It can also help guide physicians in performing needle biopsies of palpable or nonpalpable lesions of the prostate. Even under the guidance of ultrasound, the false positive rate of systematic six-point prostate biopsy can still be as high as 23% to 42%. Transrectal ultrasonography is also less specific in the diagnosis of prostate cancer, and the discovery of a hypoechoic lesion in the prostate must be differentiated from multiple diseases.

(4) Other imaging examinations: (a) Computed tomography (CT) examination: Since CT examination cannot display the three zones of normal prostate (peripheral zone, central zone and transition zone), and the density of most tumor tissues is similar to that of normal prostate Therefore, CT cannot be used to diagnose early prostate cancer and early metastasis of prostate cancer; (b) Magnetic resonance scan (MRI): MRI has good soft tissue resolution and can directly image multi-directional plane (three-dimensional) , Therefore, MRI examination of the prostate is superior to other imaging methods. However, only 60% of prostate cancers can be detected by abdominal MRI, and only 57% of prostate cancer patients can be accurately clinically staged. At the same time, the positive rate of diagnosis of early metastasis of prostate cancer is still low.

To sum up, there is still no effective method for the diagnosis of prostate cancer, especially the diagnosis of early metastasis of prostate cancer. The methods currently used to help clinical staging of prostate cancer all have the disadvantage of not being able to distinguish whether the tumor is an early organ-confined tumor or a locally advanced cancer that has already undergone micrometastasis. This differential diagnosis is very important for the patient's prognosis, because if it has developed locally advanced cancer, the possibility of surgical cure is very small. According to Chapter 50 "Prostate Tumors" of "Wu Jieping Urology" Volume 1, it is pointed out that nearly 50% of patients diagnosed as organ-confined tumors before surgery were locally advanced cancers confirmed by postoperative pathology. If it can be determined before surgery that prostate cancer patients suffer from organ-confined tumors or locally advanced cancers, and then choose an appropriate treatment plan, it will greatly benefit the improvement of treatment effects and patients' quality of life.

Therefore, it is necessary to provide a new diagnostic method for prostate cancer, especially a diagnostic method for early metastasis of prostate cancer to overcome the defects in the prior art.

Contents of the invention

In order to overcome the deficiency that the existing inspection methods cannot diagnose the metastasis of prostate cancer early, one of the purposes of the present invention is to provide a method for the early diagnosis of prostate cancer metastasis, which can clearly diagnose the metastasis of prostate cancer by using the principle of molecular biology. More importantly, the micrometastasis of prostate cancer can also be diagnosed early.

Another object of the present invention is to provide a kit for early diagnosis of prostate cancer metastasis, which can clearly diagnose micro-metastasis of prostate cancer with very high accuracy.

To achieve the above object, the present invention provides a method for early diagnosis of prostate cancer metastasis on the one hand, the method comprising: (a) extracting the genomic DNA of prostate cancer tissue; (b) using SEQ ID NO: 1, SEQ ID NO: 2 respectively The M primer pair shown and the U primer pair shown in SEQ ID NO: 3, SEQ ID NO: 4, use methylation-specific polymerase chain reaction (Methylation specific PCR, MSP) to carry out PCR amplification; (c ) electrophoresis of the above-mentioned PCR amplification products; (d) judging whether transfer occurs according to the presence or absence of specific bands.

In another embodiment of the present invention, the outer primer pair shown in SEQ ID NO: 5 and SEQ ID NO: 6 is also used to amplify the genomic DNA before step (b), followed by step (b) , that is, the genomic DNA is amplified by nested PCR, so as to increase the sensitivity of the method of the present invention and make the result more stable and reliable.

In another aspect, the present invention provides a kit for early diagnosis of prostate cancer metastasis, which comprises the M primer pair shown in SEQ ID NO: 1, SEQ ID NO: 2; and SEQ ID NO: 3, SEQ ID NO : U primer pair shown in 4. In a preferred embodiment, the kit also includes a pair of outer primers shown in SEQ ID NO: 5 and SEQ ID NO: 6. In practical applications, the kit of the present invention also includes one or more of PCR reaction solution and genomic DNA extraction reagent for convenient operation. The PCR reaction solution has a broad meaning, and usually includes other components in the PCR reaction system except primer pairs, such as dNTP, PCR buffer, polymerase, etc.; the genomic DNA extraction reagent has a broad meaning, and usually includes extraction Various reagents required for genomic DNA, such as phenol, chloroform, isopropanol, etc.

The present invention detects the methylation status of the promotor region of CRMP4 (Collapsin response mediator protein-4, CRMP4) gene in prostate cancer tissue by methylation-specific polymerase chain reaction to determine the presence or absence of Metastasis of prostate cancer. The method utilizes the principle of molecular biology, has high specificity and accuracy, and can clearly diagnose micrometastasis of prostate cancer at an early stage.

Description of drawings

Fig. 1 is a diagram showing the electrophoresis results of PCR products processed by the method of the present invention for each sample.

Detailed ways

The present invention will be described in detail below in conjunction with experiments and accompanying drawings.

Principle of invention

Deoxyribonucleic acid (DNA) methylation means that cytosine (C) in the CpG dinucleotide on the DNA molecule is in a methylated state, that is, a methyl group is bound to the CpG dinucleotide in the form of a covalent bond. On the 5-carbon atom of pyrimidine, 5-methylcytosine is formed. In the transcribed region of the genome, CpGs gather with each other, which are called CpG islands. CpG islands are generally 0.5-2.0kb in length and are often located in the 5' end gene promoter region. In normal cells, the 5'CpG islands of most housekeeping genes are in an unmethylated state; while in tumor cells, the CpG islands of many genes are abnormally hypermethylated. DNA methylation does not change the nucleotide sequence, but indirectly leads to the repression of gene transcription by changing the chromosome structure and the level of histone acetylation, that is, downregulating its expression.

As a member of the CRMP family, CRMP4 gene is an important molecule involved in the regulation of neuron differentiation and synaptic reorganization. However, studies have found that CRMP4 is a tumor suppressor gene that inhibits prostate cancer metastasis in prostate cancer, and its expression in metastatic prostate cancer is significantly down-regulated compared with benign prostatic hyperplasia and localized prostate cancer. One of the reasons for the down-regulation of expression is that CRMP4 activates There is an obvious hypermethylation state in the subregion. Therefore, detecting the methylation status of the CRMP4 promoter region can determine whether prostate cancer has metastasis, that is, high methylation in the CRMP4 promoter region indicates that prostate cancer has metastasis, and low or no methylation in the CRMP4 promoter region indicates that prostate cancer does not. transfer. The key CpG sites that affect the expression of CRMP4 gene are the following 10: -848, -841, -690, -680, -678, -674, -671, -665, -660, -658. Therefore, detecting the degree of methylation of these 10 sites can determine whether prostate cancer has metastasized, and the detection method is the MSP method.

The principle of MSP: After the double-stranded DNA is denatured and unzipped, the conversion of cytosine (C)→uracil (U) occurs under the action of bisulfite (HSO ₃ ^- ), and there is no such change if C has been methylated. However, the methylation modification only occurs on the C of the CG (cytosine-guanine) link structure in the 5'→3' direction. Therefore, after the HSO ₃ ^-action , if the DNA CpG site is not methylated, the sequence change is C→U, CG→UG; if there is methylation, it is C→C, CG→CG. Therefore, this difference can be detected by using different primers for PCR, so as to determine whether the gene has CpG island methylation. According to the changes before and after the modification of the target gene, M (methylated) and U (unmethylated) primers are designed accordingly. If the M primer can amplify, it is judged that there is methylation; if the U primer can amplify, it is judged to be Non-methylation; if the M and U primers are amplified at the same time, it is determined that these CpG sites have both methylation and non-methylation states.

Electrophoresis of the amplified product can determine whether the corresponding primers (M primer and U primer) have amplified specific fragments, and judge whether the corresponding gene is methylated according to the presence or absence of fragments. In the presence of methylation, it can be determined that prostate cancer has metastasized.

Example

1. Primers

In an embodiment of the present invention, the primers designed according to the above principles are as follows:

M primer pair (SEQ ID NO: 1 and SEQ ID NO: 2):

Sense primer: TTTTTGTAGTTTTTTGAGAGCGAGTTAC;

Antisense primer: CTCTACCATAACGCGAATCGAATACGACG;

The amplified fragment was 222bp.

U primer pair (SEQ ID NO: 3 and SEQ ID NO: 4):

Sense primer: GTTTTTTGTAGTTTTTTGAGAGTGAGTTAT;

Antisense primer: CTCTCCTCTACCATAACACAAATCAAATACAACA;

The amplified fragment was 226bp.

Outer primer pair (SEQ ID NO: 5 and SEQ ID NO: 6)

Sense primer: TGATTTTGAGATTTAGTTAGGTAGTG;

Antisense primer: TTTCCTCTTTTACATCTACCTCAC;

The amplified fragment is 612bp.

2. Method steps

1. Genomic DNA Extraction

In the present invention, the phenol-chloroform method is used for the extraction of genomic DNA. Of course, those skilled in the art can expect that other methods known in the art can also be used to extract genomic DNA. In addition, the reagents and the amounts of the reagents used in this embodiment are only exemplary, and those skilled in the art can make corresponding adjustments according to actual conditions. Unless otherwise specified, all double-distilled water (DDW) and EP tubes used were sterilized by high-pressure steam.

1) Take 0.1g of fresh or frozen prostate cancer tissue, cut it into pieces as much as possible, put it in a glass homogenizer, add 1ml of cell lysis buffer to homogenize until no tissue pieces are seen, transfer it to a 1.5ml centrifuge tube, and add proteinase K (500μg/ml) 20μl, mix well. Bathe in a constant temperature water bath at 65°C for 30 minutes, or transfer to a water bath at 37°C for 12-24 hours, and oscillate the centrifuge tube several times intermittently. Centrifuge at 12,000 rpm for 5 minutes in a tabletop centrifuge, and transfer the supernatant to another centrifuge tube;

2) Add 2 times the volume of isopropanol, invert and mix well, you can see filaments, pick out with a 100μl tip, dry, and redissolve with 200μl TE;

3) Add the same amount of phenol, chloroform, and isoamyl alcohol, shake and mix, and centrifuge at 12000rpm for 5min;

4) Take the upper layer solution to another tube, add an equal volume of chloroform:isoamyl alcohol, shake and mix, and centrifuge at 12000rpm for 5min;

5) Take the upper layer solution to another tube, add 1/2 volume of 7.5mol/L ammonium acetate, and then add 2 times the volume of absolute ethanol, mix well, settle at room temperature for 2min, and centrifuge at 12000rpm for 10min;

6) Pour off the supernatant carefully, put the centrifuge tube upside down on absorbent paper, and remove the residual liquid attached to the tube wall;

7) Wash the precipitate once with 1ml 70% ethanol, and centrifuge at 12000rpm for 5min;

8) Pour off the supernatant carefully, put the centrifuge tube upside down on absorbent paper, remove the residual liquid droplets attached to the tube wall, and dry at room temperature;

9) Add 200 μl TE to redissolve the precipitate, and then store it at -20°C for later use.

2. Sodium bisulfite modification of genomic DNA

1) Dilute about 2μg of DNA in a 1.5ml EP tube to 50ul with DDW;

2) Add 5.5 μl of freshly prepared 3M NaOH;

3) 42°C water bath for 30 minutes;

4) Add 30 μl 10mM hydroquinone (hydroquinone) to the above water-bathed mixture (the solution turns light yellow);

5) Add 520μl 3.6M sodium bisulfite (preparation method: 1.88g sodium bisulfite is diluted with DDW, and the solution is titrated with 3M NaOH to pH5.0 (accurate), the final volume is 5ml; large concentration of sulfurous acid Sodium hydrogen is difficult to dissolve, but will slowly dissolve after adding NaOH) into the solution after the above-mentioned water bath;

6) Wrap the EP tube with aluminum foil (protect from light), and gently invert the solution to mix;

7) Add 200 μl of paraffin oil (to prevent water evaporation and limit oxidation), and keep in a dark water bath at 50° C. for 16 hours.

3. Purification and recovery of modified DNA

According to the present invention, any suitable method known in the art can be used to purify and recover the modified DNA, for example, a commercially available suitable recovery kit can be used to complete this step. The following is an example of DNA purification and recovery after modification using the commercially available Promega Wizard Cleanup DNA Purification and Recovery System (Promega, A7280). The specific steps are as follows.

1) Insert the tip of the pipette under the paraffin oil layer, pressurize gently to discharge a small part of the paraffin oil, and then draw the mixture into a clean 1.5ml EP tube;

2) The following uses Promega A7280 DNA purification and recovery system

a) Preheat DDW in a water bath at 70°C; prepare 80% isopropanol;

b) Add 1ml Promega Wizard DNA Clean-up resin, and gently invert and mix well to fully combine the DNA with the resin;

c) After tightly connecting the syringe barrel with the recovery column provided in the kit, transfer the above mixture into the barrel with a pipette, and place an EP tube of more than 2ml under the column to receive the waste liquid. Add the needle plug, gently pressurize, and squeeze out the liquid. At this time, white resin deposits can be seen in the small column;

d) After separating the syringe from the small column, pull out the needle plug, then connect the syringe to the small column, add 2ml of 80% isopropanol into the syringe, insert the needle plug, press gently, squeeze the isopropanol out;

e) Separate the syringe from the small column, place the small column on a clean 1.5ml clean EP tube, centrifuge at 12000rpm for 2min, to shake off the residual isopropanol, and dry the resin (at this time, the modified DNA is in the state of binding to the resin status);

f) Remove the small column and put it on another clean 1.5ml EP tube, add 50ul preheated DDW to the pipette, and let it stand at room temperature for 5min; centrifuge at 12000rpm for 20s (this is the elution step), and the liquid in the EP tube For the eluted modified DNA solution, the final volume is 50ul;

3) Add 5.5 μl of freshly prepared 3M NaOH, and place at room temperature for 15 minutes;

4) Add 33 μl of 10M ammonium acetate to neutralize NaOH and make the pH of the solution around 7.0.

5) Add 4 μl 10mg/ml glycogen (as a precipitation indicator);

6) Add 270 μl of ice-free ethanol, place at -20°C, and precipitate overnight;

7) Centrifuge at 12000rpm at 4°C for 30min, pour off the supernatant, and collect the precipitate (no need to aspirate);

8) Add 500μl 70% ethanol (don’t blow the precipitate up, just add the ethanol), gently tilt the EP tube, rotate it once, centrifuge again, 4°C 12000rpm, 5min; pour off the supernatant after centrifugation, and add Do it again with the same amount of ethanol (this is the washing step, 2 times in total);

9) Pour off the supernatant and centrifuge briefly at room temperature, then centrifuge the ethanol attached to the wall to the bottom of the EP tube, carefully absorb the residual liquid with a pipette, and dry at room temperature for 5 minutes, or when the precipitate changes from opaque to translucent or transparent, add 20-30μl DDW to dissolve the precipitate; store the DNA solution at -20°C.

4. Perform MSP on the modified DNA

Those skilled in the art can realize that suitable means (such as kits) can be used to carry out MSP, and the TaKaRa Taq ^TM (No. DR001A) kit is used as an example for illustration below. In the method of the present invention, two PCR methods can be used for MSP, namely (1) direct PCR method or (2) nested PCR method.

(1) Direct PCR method

Set up the reaction system as shown in Table 1 below, and carry out PCR amplification under the conditions shown in Table 2 below:

Table 1 reaction system

Table 2 Reaction Conditions

94°C 1min 1 cycle

98℃ 10sec

64℃ 30sec 30 cycles

72℃ 30sec

(2) In addition, in this step, the genomic DNA can be amplified using nested PCR, and the specific methods and conditions are as follows:

Set up the reaction system as shown in Table 3 below, and carry out the PCR amplification of the outer primers under the conditions shown in Table 4 below:

Table 3 reaction system

Table 4 reaction conditions

95℃ 1min 1 cycle

98℃ 10sec

55℃ 30sec 30 cycles

72℃ 30sec

After using the outer primer pair to amplify, take an appropriate amount of outer primer amplified product, use the above-mentioned M primer pair and U primer pair as inner primers for PCR amplification, establish a reaction system as shown in Table 5 below, and follow Table 6 Perform PCR amplification under the conditions indicated:

Table 5 reaction system

Table 6 Reaction Conditions

94°C 1min 1 cycle

94℃ 30sec

64℃ 30sec 30 cycles

72℃ 30sec

5. Gel electrophoresis of MSP products

1) Get 12 μl of PCR product (direct PCR product) and carry out 3% agarose gel electrophoresis;

2) Observe the electrophoresis bands under ultraviolet light to determine whether specific bands are obtained.

Utilizing the above method steps, experiments were carried out with tissues with metastatic properties and tissues without metastatic properties, and the electrophoresis results of each sample obtained are shown in FIG. 1 . Shown in Figure 1 are: localized prostate cancer (L1, L2, L3, L4, L5), metastatic prostate cancer (M1, M2, M3, M4, M5, M6, M7), prostate cancer cell lines (PC3 , PC3M, DU145), benign prostatic hyperplasia (BPH1, BPH2), normal prostate (N1, N2), localized prostate cancer lymph nodes (L3-LN, L4-LN, L5-LN) and metastatic prostate cancer lymph nodes (M5-LN , M6-LN, M7-LN). The results showed that all prostate cancer cell lines with metastatic properties, most metastatic prostate cancers and lymph nodes of metastatic prostate cancers showed methylated bands (ie, M bands). Using nested PCR products for gel electrophoresis of MSP products can get a better picture than the results in Figure 1, and the results are more reliable and stable.

The present invention has high specificity, because CRMP4 is only down-regulated in metastatic prostate cancer (one of the reasons is methylation), and this phenomenon has not been observed in other tumors (such as liver cancer, lung cancer, gastric cancer, etc.), thus CRMP4 methylation was present only in metastatic prostate cancer. In addition, the present invention also has very high accuracy, and each sample is repeated 3 times, and the result is the same. Methylated bands were amplified in 5/7 metastatic prostate cancer specimens and all metastatic prostate cancer cell lines. The present invention only needs 2.5-25ng DNA (100mg tissue or less) to detect.

Those skilled in the art can expect that nested PCR can also be used to carry out the MSP method of the present invention, so as to increase the accuracy and specificity of the method of the present invention.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Improvements and equivalent changes made according to the disclosure of the present invention shall be included in the protection scope of the present invention.

sequence listing

<110> Applicant: Gao Xin

<120> Kit for early diagnosis of prostate cancer metastasis

<160>6

<210>1

<211>27

<212>DNA

<213> Artificial sequence

<400>1

tttttgtagt ttttgagagc gagttac 27

<210>2

<211>29

<212>DNA

<213> Artificial sequence

<400>2

ctctaccata acgcgaatcg aatacgacg 29

<210>3

<211>29

<212>DNA

<213> Artificial sequence

<400>3

gttttttgta gtttttgaga gtgagttat 29

<210>4

<211>34

<212>DNA

<213> Artificial sequence

<400>4

ctctcctcta ccataacaca aatcaaatac aaca 34

<210>5

<211>26

<212>DNA

<213> Artificial sequence

<400>2

tgattttgag atttagttag gtagtg 26

<210>6

<211>23

<212>DNA

<213> Artificial sequence

<400>2

tttcctcttt acatctacct cac 23

Claims (5)

1. two pairs of primers that are used for prostate cancer transfer early diagnosis are right; It is characterized in that; First primer is to having the sequence shown in SEQ ID NO:1, the SEQ ID NO:2, and second primer is to having the sequence shown in SEQ ID NO:3, the SEQ ID NO:4, and through its methylation-specific polymerase chain reaction; Detect the prostate cancer tissue midbrain methylation status that albumen test regulates the protein-4 gene promoter area that declines, have or not the transfer of prostate cancer with judgement.

2. one kind is used for the test kit that prostate cancer shifts early diagnosis; It is characterized in that; Said test kit comprises first primer of SEQ ID NO:1, SEQ ID NO:2 to right with second primer of SEQ ID NO:3, SEQ ID NO:4; And through its methylation-specific polymerase chain reaction, detect the prostate cancer tissue midbrain methylation status that albumen test regulates the protein-4 gene promoter area that declines, have or not the transfer of prostate cancer with judgement.

3. test kit as claimed in claim 2 is characterized in that, it is right that said test kit also comprises the outer primer of SEQ ID NO:5, SEQ ID NO:6.

4. like claim 2 or 3 described test kits, it is characterized in that said test kit also comprises the PCR reaction solution.

5. like claim 2 or 3 described test kits, it is characterized in that said test kit also comprises extracting genome DNA reagent.

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