阅读说明：本技术 一种抑癌基因甲基化联合检测试剂盒及其应用 (Cancer suppressor gene methylation joint detection kit and application thereof ) 是由 向廷秀 彭溦雁 唐俊 邱祝 于 2020-08-11 设计创作，主要内容包括：本发明公开了选自LRRC3B、PENK和RASSF1基因中的至少一个基因的甲基化检测试剂在制备用于诊断受试者是否患有结直肠癌或用于预测受试者是否具有患结直肠癌风险或用于判断结直肠癌预后的试剂盒中的应用,属于生物检测技术领域。本发明通过联合检测LRRC3B、PENK和RASSF1三个抑癌基因的启动子甲基化程度,能够实现对结直肠癌进行预防、早期诊断、临床治疗及预后评价。(The invention discloses application of a methylation detection reagent of at least one gene selected from LRRC3B, PENK and RASSF1 genes in preparing a kit for diagnosing whether a subject has colorectal cancer or predicting whether the subject has the risk of having the colorectal cancer or judging the prognosis of the colorectal cancer, and belongs to the technical field of biological detection. The invention can realize prevention, early diagnosis, clinical treatment and prognosis evaluation of colorectal cancer by jointly detecting the methylation degree of the promoters of three cancer suppressor genes of LRRC3B, PENK and RASSF 1.)

1. Use of a methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes in the manufacture of a kit for diagnosing whether a subject has colorectal cancer or for predicting whether a subject is at risk of having colorectal cancer or for determining a prognosis for colorectal cancer.

2. The use of claim 1, wherein the methylation detection reagent is used to detect the promoter methylation status of the gene.

3. The use of claim 2, wherein the methylation detection reagent comprises a specific primer for detecting the methylation status of the gene promoter.

4. The use of claim 3, wherein the specific primers for detecting the methylation state of the LRRC3B gene promoter comprise an upstream primer having a nucleotide sequence shown by SEQ ID number 1 and a downstream primer having a nucleotide sequence shown by SEQ ID number 2; the specific primers for detecting the unmethylated state of the LRRC3B gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID number 3 and a downstream primer with a nucleotide sequence shown in SEQ ID number 4.

5. The use as claimed in claim 3, wherein the specific primers for detecting the methylation status of the PENK gene promoter comprise an upstream primer having a nucleotide sequence shown by SEQ ID number 5 and a downstream primer having a nucleotide sequence shown by SEQ ID number 6; the specific primers for detecting the non-methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 7 and a downstream primer with a nucleotide sequence shown by SEQ ID number 8.

6. The use according to claim 3, wherein the specific primers for detecting the methylation state of the RASSF1 gene promoter comprise an upstream primer having the nucleotide sequence shown by SEQ ID number 9 and a downstream primer having the nucleotide sequence shown by SEQ ID number 10; the specific primers for detecting the unmethylated state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID number 11 and a downstream primer with a nucleotide sequence shown in SEQ ID number 12.

7. The use of any one of claims 1-6, wherein the methylation detection reagent further comprises a reagent for extracting DNA from a sample.

8. The use of any one of claims 1 to 5, wherein the sample is whole blood or plasma and the DNA is plasma-free DNA.

9. Use of a promoter methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes in the preparation of a kit suitable for use in a method comprising:

s1, extracting DNA of the sample of the subject,

s2, detecting the promoter methylation state of the gene by using the detection reagent,

s3, diagnosing whether the subject has colorectal cancer or predicting whether the subject is at risk of having colorectal cancer or judging the prognosis of colorectal cancer according to the methylation state detected by the S2.

10. A kit for diagnosing whether a subject has colorectal cancer or predicting whether a subject has a risk of having colorectal cancer or for judging a prognosis of colorectal cancer, comprising a promoter methylation state detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes, wherein a specific primer for detecting the methylation state of the promoter of LRRC3B gene comprises an upstream primer having a nucleotide sequence represented by SEQ ID number 1 and a downstream primer having a nucleotide sequence represented by SEQ ID No. 2; the specific primers for detecting the methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 5 and a downstream primer with a nucleotide sequence shown by SEQ ID number 6; the specific primers for detecting the methylation state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID number 9 and a downstream primer with a nucleotide sequence shown by SEQ ID number 10.

Technical Field

The invention relates to the technical field of biological detection, in particular to a cancer suppressor gene methylation joint detection kit and application thereof.

Background

The incidence of colorectal cancer (CRC) is in the 3 rd place in China, and the 4 th place of cancer cause of death. Colorectal cancer is a malignant tumor which can be prevented and early diagnosed, and the 'three early' (early detection, early diagnosis and early treatment) is the most effective method for improving the treatment effect of the colorectal cancer. And (3) displaying clinical data: the survival rate of early cancer patients after operation for 5 years is up to more than 90 percent. However, due to the lack of specific clinical presentation in early stages of colorectal cancer and the lack of highly sensitive and specific early diagnostic techniques, most patients have been diagnosed with advanced cancer, losing the best treatment opportunity, resulting in a low 5-year survival rate (less than 20%) after surgery.

Early diagnosis of tumors often depends on two key factors: the high specificity and sensitivity of the detection index and the non-invasive and easy-to-operate detection method. Fecal Occult Blood Testing (FOBT) is the most extensive method for screening colorectal cancer at present, and provides important diagnostic value for discovery of colorectal cancer. However, FOBT is affected by diet and drugs, has poor specificity, and cannot distinguish polyps from cancers. Such as barium enema, Double Contrast Barium Entera (DCBE) and the like, and has the advantages of simple and easy imaging examination, little pain, and incapability of evaluating the infiltration depth and the external invasion range of the colorectal cancer. Electronic Colonoscopy (CS) is the gold standard for diagnosis of colorectal cancer, can be used for excision and biopsy of diseased parts, but belongs to invasive operation and is not suitable for early screening. The tumor markers such as CEA and CA199 used in the current colorectal cancer classical screening have poor specificity and sensitivity.

Therefore, the method is of great significance in finding and developing economic, simple and high-sensitivity specific biomarkers for early screening and diagnosis of colorectal cancer.

Disclosure of Invention

In order to overcome the drawbacks of the prior art, the inventors found in previous studies that LRRC3B, PENK and RASSF1 were highly expressed in most normal tissues, but were abnormally low expressed in colorectal cancer patients and appeared negatively associated with tumor staging. Further studies showed that the methylation level of the promoters of the three tumor suppressor genes of serum free LRRC3B, PENK and RASSF1 can predict the onset of colorectal cancer. Thus, the present invention has been completed.

The present invention provides in a first aspect the use of a methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK and RASSF1 genes in the manufacture of a kit for diagnosing whether a subject has colorectal cancer or for predicting whether a subject is at risk for having colorectal cancer or for determining the prognosis of colorectal cancer.

In some embodiments of the invention, the methylation detection reagent is used to detect the promoter methylation status of the gene.

In some embodiments of the invention, the methylation detection reagent comprises a specific primer for detecting the methylation status of the gene promoter.

In some preferred embodiments of the present invention, the specific primers for detecting the methylation state of the promoter of LRRC3B gene include an upstream primer having the nucleotide sequence shown in SEQ ID No.1 and a downstream primer having the nucleotide sequence shown in SEQ ID No. 2; the specific primers for detecting the unmethylated state of the LRRC3B gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 4.

In some preferred embodiments of the present invention, the specific primers for detecting the methylation state of the PENK gene promoter include an upstream primer having a nucleotide sequence shown in SEQ ID No.5 and a downstream primer having a nucleotide sequence shown in SEQ ID No. 6; the specific primers for detecting the non-methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 8.

In some preferred embodiments of the invention, the specific primers for detecting the methylation state of the promoter of the RASSF1 gene include an upstream primer having the nucleotide sequence shown in SEQ ID NO.9 and a downstream primer having the nucleotide sequence shown in SEQ ID NO. 10; the specific primers for detecting the unmethylated state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.11 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 12.

In some embodiments of the invention, the methylation detection reagent further comprises a reagent for extracting sample DNA.

In the present invention, the sample is whole blood or plasma, and the DNA is plasma-free DNA.

The second aspect of the present invention provides use of a promoter methylation detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes in the preparation of a kit suitable for use in a method comprising:

s1, extracting DNA of the sample of the subject,

s2, detecting the promoter methylation state of the gene by using the detection reagent,

s3, diagnosing whether the subject has colorectal cancer or predicting whether the subject is at risk of having colorectal cancer or judging the prognosis of colorectal cancer according to the methylation state detected by the S2.

In some embodiments of the invention, the subject is diagnosed with, or predicted to have a risk of, or is judged to have a poor prognosis for, colorectal cancer when the subject has at least one of LRRC3B, PENK, and RASSF1 genes with a level of methylation that is higher than normal.

The third aspect of the present invention provides a kit for diagnosing whether a subject has colorectal cancer or predicting whether a subject has a risk of having colorectal cancer or for judging a prognosis of colorectal cancer, comprising a promoter methylation state detection reagent for at least one gene selected from the group consisting of LRRC3B, PENK, and RASSF1 genes, wherein a specific primer for detecting the methylation state of the promoter of LRRC3B gene comprises an upstream primer having a nucleotide sequence shown in SEQ ID No.1 and a downstream primer having a nucleotide sequence shown in SEQ ID No. 2; the specific primers for detecting the unmethylated state of the LRRC3B gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 4. The specific primers for detecting the methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 6; the specific primers for detecting the non-methylation state of the PENK gene promoter comprise an upstream primer with a nucleotide sequence shown by SEQ ID NO.7 and a downstream primer with a nucleotide sequence shown by SEQ ID NO. 8. The specific primers for detecting the methylation state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.9 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 10; the specific primers for detecting the unmethylated state of the RASSF1 gene promoter comprise an upstream primer with a nucleotide sequence shown in SEQ ID NO.11 and a downstream primer with a nucleotide sequence shown in SEQ ID NO. 12.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the combined detection of LRRC3B, PENK and RASSF1 can be used as biomarkers for early screening, diagnosis, curative effect evaluation and prognosis judgment of colorectal cancer, and a combined detection kit based on the biomarkers can be used for early screening, diagnosis, curative effect monitoring and prognosis evaluation of colorectal cancer.

The detection method is convenient and quick, has low cost and is suitable for popularization and application.

The invention adopts plasma free DNA for detection, compared with the classical tumor marker, ctDNA has higher accuracy and specificity, is a novel tumor marker with no wound and wide clinical application prospect, and can qualitatively, quantitatively and track the disappearance, diffusion and recurrence of the tumor.

In addition, DNA methylation is a relatively frequent early event in tumorigenesis, and has the advantage over RNA, protein and gene mutations as early diagnosis and screening of tumors:

1) the DNA sample is extremely stable, is not similar to RNA and protein, and is greatly influenced by the environment;

2) the sensitivity of Methylation Specific PCR (MSP) and the method have simple operation, do not need expensive instruments and equipment, and are more feasible than quantitative detection methods (the inevitable existence of sequences derived from normal cells in clinical samples can seriously interfere the detection of genetic biomarkers which are characteristic of tumor cells, thereby causing the obstacle of early diagnosis of tumors by the quantitative detection method; the effect of the methylation of the screened cancer suppressor gene in the occurrence and development of the breast cancer is discussed, and the experimental method is simple and feasible;

3) methylation of the cancer suppressor gene promoter occurs in CpG islands mostly, the position is more limited compared with gene mutation, and probes and primers are easy to design;

4) a plurality of pairs of primers can be simultaneously designed for joint detection;

5) is suitable for various body fluid detection.

Drawings

FIG. 1 shows that Methtarget detects the methylation status of LRRC3B, PENK, and RASSF1 promoters.

FIG. 2 shows the expression levels and promoter relative methylation levels of LRRC3B, PENK and RASSF1 in a TCGA dataset obtained from the MethHC DNA methylation database (http:// meth. mb. nctu. edu. tw/php/index. php).

FIG. 3 shows a representative graph of the methylation status of LRRC3B, PENK and RASSF1 promoters in colon cancer patient tissues that are clinically and pathologically positive for MSP detection.

FIG. 4 shows a representative graph of the methylation status of the plasma free DNA LRRC3B, PENK and RASSF1 promoters as detected by the plasma free DNA methylation capture technique.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.