CN114517233B

CN114517233B - Primer probe combination for early warning and clinical diagnosis of colorectal cancer

Info

Publication number: CN114517233B
Application number: CN202111559960.8A
Authority: CN
Inventors: 胡晓彤; 郭兴中; 张陆敏; 费伟强; 李辉
Original assignee: Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
Current assignee: Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2023-10-31
Anticipated expiration: 2041-12-20
Also published as: CN114517233A

Abstract

A primer probe combination for early warning and clinical diagnosis of colorectal cancer belongs to the technical field of gene detection. The invention provides a primer probe combination for early warning and clinical diagnosis of colorectal cancer, which comprises a methylation primer of a USP44 gene and a probe, and further provides application of the primer probe combination. The invention searches a tumor marker with higher sensitivity and specificity, which can be used for early warning and clinical diagnosis of colorectal cancer, and develops a primer probe combination which can be used for early warning and clinical diagnosis of colorectal cancer.

Description

Primer probe combination for early warning and clinical diagnosis of colorectal cancer

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a primer probe combination for early warning and clinical diagnosis of colorectal cancer.

Background

Colorectal cancer is now the third largest cancer in the world and is one of the most common malignant tumors of the digestive system, with extremely high morbidity and mortality. Colorectal cancer occurs due to changes in expression of multiple genes, and factors closely related to the occurrence of cancer include environmental factors, acquired lifestyle and dietary factors, etc. Colorectal cancer is usually the cause of the middle and late stages of the disease in diagnosis, and the main causes are that the early stage has no obvious symptoms, the screening method is not convenient enough, and the like. These factors often lead to poor prognosis and reduced quality of life for the patient.

The tumor marker has the advantages of convenient detection, convenient screening, minimally invasive, low cost, high sensitivity, high specificity and the like. The damage of multiple genes is responsible for the development of colorectal cancer. The nature of the gene promoter region exhibiting elevated methylation levels is that the DNA molecule is abnormal, which is one of the important causes of inactivation of expression of many oncogenes. In normal tissue cells, the CpG islands of the gene regulatory region are in an unmethylated state, and when the cells are cancerous, the CpG island regions tend to be highly methylated, so that DNA methylation research is an important field of tumor molecular biology research. The methylation level of the gene is generally related to the occurrence of tumors, can be used for early diagnosis of the tumors, and becomes an important molecular biological marker. Abnormal methylation often occurs in the promoter region of early tumor genes, which provides a research idea for early diagnosis, prognosis and efficacy evaluation of most tumors.

Detection of abnormal methylation of promoters has a number of advantages over other types of tumor molecular markers. Abnormal methylation levels of gene promoters exist in almost all kinds of tumors. More importantly, DNA released by cancerous cells can also be found in the peripheral blood. Through previous experimental study, it can be found that abnormal methylation of promoter of tumor-associated gene existing in tumor tissue can be detected in peripheral blood plasma, serum, body fluid related to tumor-associated organs (such as saliva, sputum, urine, stool, etc.). However, these biological samples are relatively easy to obtain, and thus detection of the methylation status of the promoter regions of some tumor-associated genes can be the basis for early diagnosis of tumors. The abnormal methylation region of the promoter of the gene is the same in different types of tumors, which also provides convenience for detection. Abnormal methylation is a positive signal compared to markers such as allele deletions, and is readily distinguishable from negative background in normal tissue.

In 2000, a methylation specific quantitative PCR technique MethyLight based on TaqMan probes was established by Eads et al. The method has the advantages of high flux, high sensitivity, simple operation and the like, can accurately detect methylated alleles even under the condition of high proportion of unmethylated alleles, and can be used for rapid analysis of multiple samples and multiple gene loci. In addition, the method has the characteristics of repeatability, small required sample size and no need of electrophoresis separation, and can provide reliable technical support for the research of clinical samples. The sample is easy to obtain and the dosage is small.

Methylated DNA immunoprecipitation (MeDIP-chip) is one of the most widely used detection techniques at present, and is based on the principle of immunoprecipitation and chip site needle hybridization. The MeDIP-chip is specifically enriched for DNA fragments that are methylated on the genome by 5' -methylcytosine antibodies. And (3) carrying out image analysis by using NimbleScan, converting the image signal into a digital signal, and calculating the signal value ratio of a Cy3 channel (IP sample) to a Cy5 channel (Input sample) after correction. Researchers can use the media-chip technology to quickly and efficiently find methylation regions on the genome, and thus compare differences in DNA methylation modification patterns between different cells, tissues or disease samples. The MeDIP-chip can detect methylation status of a large number of sites at the same time, but is expensive and has high cost for clinical detection.

The invention analyzes the methylation expression difference of related genes in normal colorectal mucosa and colorectal cancer tissues systematically, and combines clinical data to search a tumor marker with higher sensitivity and specificity, which can be used for early warning and clinical diagnosis of colorectal cancer.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to design a technical scheme of a primer probe combination for early warning and clinical diagnosis of colorectal cancer.

The invention is realized by the following technical scheme:

in one aspect, the invention provides a primer probe combination for early warning and clinical diagnosis of colorectal cancer, which comprises a methylation primer of a USP44 gene and a probe;

the methylation primer of the USP44 gene comprises nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;

the probe of the USP44 gene comprises a nucleotide sequence shown as SEQ ID NO. 3.

Further, the 5 '-end modification group of the probe of the USP44 gene is FAM, and the 3' -end modification group is MGB;

the invention also provides application of the primer probe combination in preparation of a kit for early warning and clinical diagnosis of colorectal cancer.

In another aspect, the invention provides a kit for early warning and clinical diagnosis of colorectal cancer, which comprises the primer probe combination.

In another aspect, the invention provides a method for using the above kit, comprising the steps of:

(1) Extracting genomic DNA of a sample;

(2) Performing bisulfite modification treatment on the DNA extracted in the step (1) to obtain converted DNA;

(3) Combining and mixing the methylated DNA obtained in the step (2) with a primer probe to prepare a PCR reaction system, performing PCR amplification detection, and obtaining a detection result according to the PCR amplification result.

The invention primarily screens the DMRs with obvious difference in the data of the Illumina850K chip. The sample size was then expanded by MethyTarget for further screening. The USP44 gene was screened for methyl light detection in combination with TCGA database data. The results showed that there was a statistical difference (p < 0.001) in whether methylation occurred in colorectal cancer tumor tissue and distal normal tissue for USP44-DMR-7280-8479 bp. Meanwhile, the immunohistochemical result shows that the expression degree of the USP44 gene in tissues is closely related to the differentiation degree of colorectal cancer and lymph node metastasis and can be used as a potential index of colorectal cancer prognosis.

Based on this, the present invention has developed a primer probe for showing USP44-DMR-7280-8479bp, which can be used for early warning and clinical diagnosis of colorectal cancer.

Drawings

FIG. 1 is a differential methylation site heat map;

FIG. 2 shows methylation and expression levels of USP44 in the TCGA database in colorectal cancer and normal populations;

FIG. 3 shows expression of USP44 RNA levels in part of colorectal cancer and distal normal tissue;

FIG. 4 shows the expression of USP44 in colorectal cancer tissue;

FIG. 5 is a graph showing USP44 protein versus patient survival;

fig. 6 shows the tumor tissue and distal normal tissue of a patient with colorectal cancer detected by methyl light.

Detailed Description

The invention is further illustrated by the following examples.

Experimental materials:

human blood sample

The human colorectal cancer blood samples used in this example were taken from colorectal cancer patients who were hospitalized at the university of Zhejiang medical institute affiliated with Shao Yifu hospital without receiving new adjuvant therapy during the period of 9 months in 2018 to 10 months in 2019, and the normal human blood samples were taken from the university of Zhejiang medical institute affiliated with Shao Yifu hospital biological sample library. Both were submitted to informed consent by the patient himself or herself or family members and blood samples were taken. The collected blood samples are all collected in an EDTA-containing vacuum blood collection tube, and are stored in a refrigerator at-80 ℃ after being filled into a 600 mu L freezing tube within two hours. The study selects 50 primary colorectal cancer blood samples in total; 50 normal human blood samples were tested for MethylTarget.

Human tissue sample

The human colorectal cancer samples and normal colorectal tissue samples used in this example were collected between 3 months in 2014 and 10 months in 2016 and excluded from colorectal cancer patients receiving neoadjuvant therapy. After the patient himself or herself or family member signs the informed consent, tumor tissue and distal normal tissue 5cm or more from the tumor are collected. All sample tissues collected were stored in liquid nitrogen for subsequent extraction of tissue DNA and RNA.

Finally, 71 cases of primary colorectal cancer specimens and 10 cases of normal intestinal mucosa specimens are selected for immunohistochemical experiments. 18 colorectal cancer tissues and corresponding distal normal tissues were selected for RT-PCR detection, and 56 colorectal cancer tissues and corresponding distal normal tissues (8 of which were repeated with RT-PCR experiments) were selected for methyl light detection.

Example 1: extraction of whole blood sample DNA

(1) Whole blood samples were added to sterile centrifuge tubes and the volume was adjusted to 250. Mu.L with an Elutation Buffer.

(2) 25. Mu.L of proteinase K solution and 250. Mu.L of BL Buffer were added and centrifuged at 13000rpm for 15 seconds.

(3) Incubate for 10 min at 65 ℃. Vortex once briefly during incubation.

(4) 260. Mu.L of absolute ethanol was added. Centrifuge at 13000rpm for 20 seconds.

(5) The DNA separation column was inserted into a 2 ml collection tube.

(6) All samples were transferred to a DNA separation column using a pipette.

(7) Centrifuge at 13000rpm for 1 minute.

(8) The filtrate and collection tube were discarded.

(9) The DNA separation column was inserted into a new 2 ml collection tube.

(10) 500. Mu.L of HBC Buffer was added to the DNA separation column.

(11) Centrifuge at 13000rpm for 1 minute. .

(12) The filtrate was discarded and the DNA separation column was returned to the collection tube.

(13) 700 mu L DNA Wash Buffer was added.

(14) Centrifuge at 13000rpm for 1 minute.

(15) The filtrate was discarded and the DNA separation column was returned to the collection tube.

(16) Repeating the steps 13-15, and performing a second DNA Wash Buffer cleaning step.

(17) The empty DNA separation column was centrifuged at 13000rpm for 2 minutes to dry the columnar matrix.

(18) The DNA separation column was transferred to a 2 ml centrifuge tube without nuclease.

(19) Adding 100-200 μL of solution Buffer, heating to 65deg.C

(20) Standing at room temperature for 5 min.

(21) Centrifugal force at 13000rpm for 1 min

(22) Repeating the steps 20-22 for a second elution.

(23) The eluted DNA was stored at-20 ℃.

Example 2: extraction of tissue Total RNA and DNA

30mg of tissue was placed in an RNase free polishing tube, 700. Mu.L of the lysate and 4 ceramic beads were added, and the tissue cells were sufficiently disrupted by using a tissue homogenizer, 13000g, and centrifuged for 5 minutes.

2.1 extraction of RNA

The liquid was carefully transferred to a DNA adsorption column (sleeved on a 2 ml collection tube), centrifuged to not less than 13000g for 1 minute, and the liquid in the collection tube was recovered. The liquid in the collection tube was transferred to a fresh 1.5 ml EP tube, 1/2 times the volume of absolute ethanol was added and the shaking was reversed for 15s. All the liquid was aspirated into an RNA adsorption column fitted over a 2 ml collection tube and centrifuged 13000g for 1 min. The liquid in the collection tube was discarded and returned under the RNA adsorption column. 500 mu L RNA wash buffer1 was added to the RNA adsorption column and centrifuged 13000g for 1 minute. The liquid in the collection tube was discarded and returned under the RNA adsorption column. 500 mu L RNA wash buffer2 was added to the RNA adsorption column and centrifuged 13000g for 1 minute. The collection tube was discarded and 500. Mu. L RNA wash buffer2 was added repeatedly and centrifuged 13000g for 1 minute. After the liquid in the collecting pipe is discarded, the centrifugal force is more than or equal to 13000g for 1 min, so as to thoroughly spin-dry the inner membrane of the RNA adsorption column. The RNA adsorption column was transferred to a fresh 1.5 ml EP tube, 30-70. Mu.L of DEPC water preheated at 70℃was directly added to the column, incubated at room temperature for 5 minutes, and centrifuged at 13000g for 1 minute. The RNA adsorption column was removed and 1.5 ml of EP tube was stored in a refrigerator at-80 ℃.

2.2 extraction of DNA

The DNA adsorption column in the first step of extracting RNA is inserted into a new 2 ml collecting pipe, and the centrifugation is more than or equal to 13000g for 1 minute. The liquid in the collection tube was discarded and returned under the DNA adsorption column. To the DNA adsorption column, 700. Mu. L DNA wash buffer was added and centrifuged 13000g for 1 minute. After the liquid in the collecting pipe is discarded, the centrifugal force is more than or equal to 13000g for 1 min, so as to thoroughly spin-dry the inner membrane of the DNA adsorption column. The DNA adsorption column was transferred to a fresh 1.5 ml EP tube, 50-100. Mu.L of eluent was added, incubated at room temperature for 2 minutes, and centrifuged at 13000g for 1 minute. The DNA adsorption column was removed and 1.5 ml of EP tube was stored in a refrigerator at-80 ℃.

Example 3: bisulfite treatment of DNA

(1) Mu.g of DNA was placed in a 1.5 ml EP tube, 1 XTE buffer was added and the total volume was adjusted to 30. Mu.L

(2) 3.3. Mu.L of 3M NaOH (freshly prepared) was added and the concentration was adjusted to 0.3M and incubated at 37℃for 15 minutes.

(3) 0.055g of Hydroquinone (Hydroquinone) was weighed into 50 ml of enzyme-free water at a concentration of 10mM, 1.82g of sodium bisulphite was weighed, and 430. Mu.L of 3M NaOH and 2.8 ml of enzyme-free water were added to dissolve. 10mM hydroquinone was added and mixed well to give a total volume of 2.4 ml M bisulfite solution.

(4) To the EP tube containing DNA 333 mu L bisulfite solution was added, mixed well and incubated at 55℃for 4 hours.

(5) To the EP tube, 1 ml of QX1 buffer and 16. Mu.L of QIAEX buffer were added and mixed by spin for 1 hour.

(6) Centrifugation at 13000rpm for 1 min, discarding the supernatant, adding 1 ml PE buffer, washing the precipitate.

(7) Centrifuge at 13000rpm for 1 min, discard supernatant, add 500. Mu.L PE buffer, wash the pellet.

(8) Centrifugation at 13000rpm for 1 min, discarding the supernatant, oven baking at 37℃for 5 min, until the pellet is semi-dry.

(9) 50. Mu.L of TE buffer was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes.

(10) 5.56. Mu.L NaOH (3M) was added and the final concentration was adjusted to 0.3M and incubated at 37℃for 15 minutes.

(11) 27.78 μl of the prepared ammonium acetate (ph=7.0) was added to give a final concentration of 3M, and mixed well.

(12) Add 50 μl NaOAc (ph=5.2), correct pH < 7.0, mix well.

(13) 1 ml of QX1 buffer was added and the mixture was spun and mixed for 20 minutes.

(14) Repeating the steps (6) - (7), centrifuging at 13000rpm for 1 min, and discarding the supernatant.

(15) Add 30. Mu.L TE buffer (1X) to dissolve DNA, mix well, stand at room temperature for 5 minutes and centrifuge at 13000rpm for 3 minutes.

(16) The supernatant was transferred to a new 1.5 ml EP tube.

(17) Add 20. Mu.L TE buffer (1X), mix well, dissolve DNA well and incubate for 10 minutes at 37 ℃.

(18) Centrifugation at 13000rpm for 3 min, transferring the supernatant to the EP tube in step (14)

(19) The new EP tube was centrifuged again at 13000rpm for 3 minutes and the supernatant was transferred to another 1.5 ml EP tube and stored at-20℃for further use.

Example 4: immunohistochemistry

4.1 preparation of reagents

3％H ₂ O ₂ -methanol: according to methanol: 30% H ₂ O ₂ Equal to 9: 1. (e.g., 10 ml of 30% H in 90 ml of methanol) ₂ O ₂ 。

10% blocked serum: goat serum was mixed with PBS1:9 (e.g. 100. Mu.L goat serum plus

900μL PBS)。

PBS and repair solution: PBS and citrate powder were dissolved in 1L of 16Ω water for use.

4.2 Experimental procedure

(1) Dewaxing and hydration

Prior to dewaxing, the tissue slides should be clamped on a metal staining clamp and left at room temperature for 60 minutes or baked in a 60℃incubator for 20 minutes.

1) Placing the tissue chip in xylene (xylene I) for soaking for 10 minutes, and replacing the xylene (xylene II) and then soaking for 10 minutes;

2) Soaking in absolute ethanol for 3 minutes;

3) Soaking in 95% ethanol for 3 min;

4) Soaking in 85% ethanol for 3 min;

5) Soaking in 75% ethanol for 3 min;

6) Placing the tissue slide clamp in a stainless steel barrel, placing the tissue slide clamp under a tap, aligning the distal end of the chip clamp to the water outlet of the tap, and flushing with tap water for 5 minutes;

7) Tissue slides were clamped in a plastic box like a Western ice box, poured into PBS, and soaked for 1 min 3 times.

(2) Antigen retrieval exposes the antigenic determinant

The autoclave was placed on an induction cooker, and distilled water was added to the autoclave in an amount of about 1 liter. Placing a tissue slide clamp in a stainless steel barrel, adding 250 milliliters of citrate antigen repair liquid into the barrel, immersing the slide in the repair liquid for 5 minutes, then covering a stainless steel pressure cooker cover, locking the cover, starting an electromagnetic oven power supply, starting pressurizing, lifting a small valve, boiling for about 5 minutes after the small valve is lifted, or boiling for about 2.5 minutes after an intermediate air release valve is uniformly deflated, closing the electromagnetic oven power supply, lifting the intermediate air release valve and tilting to one side of the intermediate air release valve, discharging internal air towards the far end direction of the intermediate air release valve, opening the locked pressure cooker cover after the air is discharged (the small valve is sunken), taking out the stainless steel cooker with the slide clamp, placing the stainless steel cooker in a window, opening the window, and naturally cooling the slide.

(3) Blocking nonspecific proteins

1) The PBS was soaked for 3 minutes and repeated 3 times.

2)3％H ₂ O ₂ -methanol soaking for 30 minutes at room temperature.

3) Washing with tap water for 10 min, soaking in PBS for 3 min, repeating for 3 times, and removing excessive liquid (without touching tissue) by spin-drying or paper towel, and drawing a circle around tissue with a histochemical pen (10% of sealing serum is prepared during distilled water washing time).

4) 100 mu L of blocking nonspecific antigen (which should be added immediately after the tissue is dried and the water is absorbed) of 10% blocking serum is quickly dripped into the tissue in the circle, and the blocking solution is buckled for 30 minutes (a primary antibody is prepared) at room temperature without cleaning.

(4) Incubation with primary antibody

Sheep serum on sections was removed, residual serum around the tissue was wiped off with filter paper, diluted primary antibody (1:200) was directly added, primary antibody (about 80. Mu.L) was added dropwise, and the mixture was placed in a wet box overnight at 4 ℃. The next day, taking out from the refrigerator, was re-warmed at 37 ℃ for 45 minutes.

(5) Second antibody incubation

1) The primary antibody is buckled off, the slide is inserted into a plastic slide frame, then the whole slide is put into a plastic box, PBS is added for soaking, and the slide is put on a micro-oscillator for oscillating for 5 minutes at the lowest rotation speed and repeated for 3 times.

2) The water around the circle was sucked off with filter paper, secondary antibody (i.e. solution a in DAB kit, directly squeeze the solution a bottle and drop one drop) was added, room temperature for 30 minutes.

3) The solution was immersed in PBS for 5 minutes and repeated 3 times (procedure 14) while preparing DAB developer (solution C: solution b=1: 50).

(6) Color development

The PBS was removed and DAB was developed for about 1 minute (the extent of staining was observed under a microscope to control the staining time). The water was rinsed for 15 minutes.

(7) Counterstaining, dehydrating, transparency and sealing.

1) Counterstaining: hematoxylin is evenly dripped into the mixture for 2 drops, 40 seconds and is washed by tap water for 15 to 30 minutes.

2) Dehydrating: the slide was placed on an iron frame (not a plastic frame, since xylene was corrosive to plastic at the back), and then eluted with 75% ethanol- > 85% ethanol- > 95% ethanol- > absolute ethanol II- > absolute ethanol I sequentially for 3 minutes each stage.

3) And (3) transparency: xylene I for 3 min and xylene II for 3 min.

4) Sealing piece: the paper towel sucks and removes the dimethylbenzene, a drop of neutral resin is dripped on the glass slide, then the cover glass is covered, the cover glass is lightly squeezed by forceps, air bubbles are removed, (small air bubbles are not remained at the tissue part), and the cover glass is placed in a fume hood for drying after sealing.

Example 5: reverse transcription and polymerase chain reaction of RNA

5.1 reverse transcription

1-2. Mu.g of total RNA of the extracted cells or tissues was subjected to a reverse transcription reaction using a reagent from Promega corporation, the system being 25. Mu.L.

According to the system proportion, 25 mu L of reagent is added into an eight-joint tube to be uniformly mixed, the mixture is put into a PCR instrument to be reacted for 1 hour at 42 ℃, the temperature is changed to 72 ℃, the reaction time is 10 minutes, and the reverse transcription product cDNA is put at-20 ℃ to be preserved for standby.

5.2 polymerase chain reaction

GAPDH is used as an internal reference, and the primer sequence is as follows

GAPDH-F：TCCTGTGGCATCCACGAAACT；

GAPDH-R：GAAGCATTTGCGGTGGACGAT。

mu.L of the cDNA obtained by the above reverse transcription was taken and subjected to PCR reaction using a reagent from Promega corporation, the system being 12. Mu.L.

Adding 12 mu L of reagent into an eight-joint tube according to the system, mixing uniformly, and adopting a gradient cooling method (touchdown PCR) in a PCR instrument, wherein the program is set as follows:

95 ℃ for 5 minutes;

95 ℃ for 30 seconds;

60-50 ℃,30 seconds (10 cycles, 1 ℃ per cycle reduction);

72 ℃ for 30 seconds

95 ℃ for 30 seconds

50 ℃ for 30 seconds

72 ℃,30 seconds (30 cycles);

72 ℃ for 10 minutes

Then, the temperature was maintained at 4℃and 4-8. Mu.L of the product was subjected to agarose gel electrophoresis to analyze the band results.

Example 6: methyllight quantitative PCR

Since the TaqMan probe requires cleavage of the probe by 5 '. Fwdarw.3' exonuclease activity of the DNA polymerase, the reporter fluorophore is separated from the quencher fluorophore. Therefore, it is possible to use a general DNA polymerase, not to select Pfu DNA polymerase without 5 '. Fwdarw.3' exonuclease activity for qPCR system of Methyllight, and select COL2A gene as reference gene, reference gene probe: 5'-VIC 3' -BHQ1.

COL2A primer and probe sequence:

COL2A1-mF TCTAACAATTATAAACTCCAACCACCAA

COL2A1-mR GGGAAGATGGGATAGAAGGGAATAT

COL2A1-probe CCTTCATTCTAACCCAATACCTATCCCACCTCTAAA

the reaction system is as follows:

the reaction conditions were as follows:

experimental results

1 Illumina850K methylation chip detection

Colorectal cancer and normal whole blood samples were collected 3 times each, total DNA was extracted, and DNA was treated with bisulfite and then detected using Illumina850K methylation chip to obtain sequencing results and thermodynamic diagrams (fig. 1).

Except for differential methylation regions where primers cannot be designed or do not react well (Differentially Methylated Region, DMR), we initially screened 45 DMR with statistical significance (p < 0.05) (Table 1).

TABLE 1 differential methylation regions

2 MethylTarget methylation sequencing

2.1 the 45 DMR initially screened were designed for the corresponding MethyTarget primer (table 2).

TABLE 2 MethylTarget primer and Gene-related information

3 TCGA database analysis

We analyzed the relevant data for the above candidate genes one by one in the TCGA database and found that there were significant differences in methylation levels and expression of the USP44 gene in colorectal cancer and normal populations (FIG. 2). The methylation level of the USP44 promoter region was higher in the colon cancer population than in the normal population (fig. 2. A), and also in the rectal cancer population than in the normal population (fig. 2. B). USP44 expression levels were lower in the colon cancer population than in the normal population (fig. 2. C). USP44 expression levels were also lower in the rectal cancer population than in the normal population (figure 2.D). USP44 gene is found in other cancer species such as: methylation levels and expression in lung cancer, prostate cancer, breast cancer, etc. are also significantly different. In combination with published literature, it was found that no one has conducted intensive studies on colorectal cancer with the USP44 gene, so we selected USP44 as the target gene for the first study.

4 RT-PCR detection of expression of USP44 in colorectal cancer tissue

Collecting 18 colorectal cancer patients who do not receive neoadjuvant treatment, namely tumor tissues and far-end normal tissues which are more than 5cm away from the tumor, extracting total RNA, performing reverse transcription into cDNA, and designing USP44 primers for PCR reaction, wherein the sequences of the primers are as follows:

USP44-F：AACGATTCAGGTGGTCAGGA

USP44-R：GTGTACCCAGAACCCTCCTT

the expression level of USP44 in these 18 colorectal cancers was then examined by RT-PCR experiments. The results show that: the expression of USP44 was significantly higher in distal normal tissue in 72.22% (13/18) cases than in tumor tissue (fig. 3), which essentially verifies the results of the TCGA database.

Correlation of expression of 5 USP44 with clinical pathological characteristics of colorectal cancer patients

We examined 71 primary colorectal cancer tissue specimens and 10 normal intestinal mucosa specimens by immunohistochemistry. According to HERACLES scoring criteria and the study by Wei et al, immunohistochemical scoring is performed herein according to the staining of the cytoplasm and cell membrane, with no staining of 0, low intensity staining of 1, medium intensity staining of 2, and high intensity staining of 3. Representative immunohistochemical staining results are shown in figure 4.

It was found that USP44 was highly expressed in the cytoplasm and nucleus of normal intestinal mucosal cells (fig. 4. A), whereas expression was reduced or absent in most colorectal cancer tissues (fig. 4. B.c.d).

SPSS 22.0 analysis of USP44 protein expression in tumor tissue correlated with patient clinical pathology, and the results showed that USP44 low expression was significantly correlated with lower differentiation of colorectal cancer cells (p=0.023) (table 4). USP44 low expression was significantly associated with more lymph node metastasis (p=0.047) (table 3).

Kaplan-Meie survival curves showed that USP44 expression levels were significantly correlated with overall survival in colorectal cancer patients (p=0.004), and that overall survival in patients with high USP44 expression was significantly higher than in patients with low USP44 expression (fig. 5).

TABLE 3 clinical pathological association of USP44 protein expression and colorectal cancer patients

*Statistically significant(p<0.005)

6 Methyllight detection

6.1 design of TaqMan probes and primers for USP44 promoter region

The USP44 genome sequence is from NCBI database, beacon Designer version 8.14.14 software detection, and the gene has 3492-3691bp,7076-7275bp,7280-8479bp,12132-12331bp,12497-12696bp,12753-12952bp,13113-13312bp and 15362-15561bp total 8 CpG islands. Primers and TaqMan probes were designed based on the individual CpG island positions (Table 4).

Table 4 USP44 gene methyl light detection primer and probe

After testing the effectiveness of the primers, we selected the USP44 methylation region DMR 7280-8479bp (hereinafter referred to as USP44-DMR-7280-8479 bp) for quantitative PCR. The 2 CpG sites marked in red are the sites with obvious difference in the CpG island, so that the probe is mainly aimed at the 2 CpG sites. The specific positions of the probes are underlined and thickened parts, the specific positions of the primers are the following grey areas, the 5 '-end modification group of the probes is FAM, and the 3' -end modification group of the probes is MGB.

6.2 quantitative PCR

DNA was extracted from tumor tissues and distal end normals of 56 colorectal cancer patients, and quantitative PCR was performed after modification treatment with bisulfite. The primer probe combination is as follows: 7280-8479-MF, 7280-8479-MR and 7280-8479-MP.

We define a CT value greater than 35 as unmethylated and less than 35 as methylated. Methylation was detected in 43 cases (76.8%) and not 13 cases (23.2%) in tumor tissue. Methylation was detected in 3 (5.4%) and not in 53 (94.6%) of the distal normal tissues. The Pearson chi-square test found that the two sets of data had statistically significant differences, p <0.001 (fig. 6).

Sequence listing

<110> Zhejiang university medical college attachment Shao Yifu Hospital

<120> a primer probe combination for early warning and clinical diagnosis of colorectal cancer

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Claims

1. Use of a primer probe combination comprising a methylation primer of a USP44 gene and a probe, wherein the methylation primer of the USP44 gene comprises a nucleotide sequence as shown in SEQ ID No.1 and SEQ ID No.2, and the probe of the USP44 gene comprises a nucleotide sequence as shown in SEQ ID No.3, for preparing a kit for predicting the total survival rate of colorectal cancer.

2. The use according to claim 1, characterized in that the 5 'end modification group of the probe of the USP44 gene is FAM and the 3' end modification group is MGB.