SNP (Single Nucleotide Polymorphism) marker related to auxiliary diagnosis of hepatocellular carcinoma as well as primer and application of SNP marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) marker for screening genes related to hepatocellular carcinoma as well as primers and application of the SNP marker, the SNP marker is a combination of rs12150387, rs1278927 and rs7789137, the primers corresponding to the SNP marker are provided, the SNP marker can be used for auxiliary diagnosis of hepatocellular carcinoma patients at all stages, the disease state of the patients can be screened in time, and the diagnosis efficiency of the hepatocellular carcinoma patients is improved. And a convenient reference is provided for early diagnosis and precise treatment of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to an SNP marker related to the auxiliary diagnosis of hepatocellular carcinoma, its primers and applications. Background Art

[0002] Hepatocellular carcinoma is a malignant tumor with a high incidence and poor prognosis globally. Its pathogenesis is complex and involves multiple molecules and signaling pathways. In recent years, with the development of genomics and transcriptomics technologies, revealing the molecular characteristics of hepatocellular carcinoma by integrating multi-omics data has become a research hotspot. GWAS and eQTL analysis can identify genetic variations related to diseases and their effects on gene expression, while single-cell RNA sequencing and spatial transcriptomics technologies provide expression information at the cellular level and spatial localization. In addition, Bulk RNA-seq data provides a basis for the analysis of overall expression levels, and CRISPR technology helps to evaluate the dependence of genes in cells.

[0003] SNP, full name single nucleotide polymorphism, refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. The presence of SNP sites is closely related to the occurrence and development of certain diseases. By detecting SNP sites in a large population and statistically analyzing the disease incidence rate, it can be found that certain SNP sites have a significant association with the genetic risk of specific diseases. These SNP sites related to diseases are usually related to specific allele variations, which in turn affect the susceptibility and disease risk of individuals. Therefore, SNPs can provide a reliable scientific basis for the prediction, diagnosis, and treatment of diseases.

[0004] Currently, the preferred treatment method for early hepatocellular carcinoma is mainly surgical resection of the lesion. The key lies in early detection and early treatment. At present, the main indicators for early screening of HCC in clinical practice are the serum alpha-fetoprotein (AFP) level and imaging B-ultrasound detection, but both have certain limitations. Therefore, if SNPs related to hepatocellular carcinoma can be screened out as biomarkers and corresponding primers can be developed, it will surely be of great significance for the early diagnosis and precise treatment of hepatocellular carcinoma. Summary of the Invention

[0005] The present invention discloses an SNP marker related to the auxiliary diagnosis of hepatocellular carcinoma, its primers and applications, screens out SNPs of genes related to hepatocellular carcinoma, and provides corresponding primers, which can provide a basis for the early diagnosis and precise treatment of hepatocellular carcinoma.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, an SNP marker related to the auxiliary diagnosis of hepatocellular carcinoma is provided, and the marker is a combination of rs12150387, rs1278927, and rs7789137.

[0008] In the second aspect of the present invention, primers for detecting the above-mentioned SNP markers are provided, including:

[0009] The primer sequences for detecting the rs12150387 locus are shown in SEQ ID No: 1 to 3;

[0010] The primer sequences for detecting the rs1278927 locus are shown in SEQ ID No: 4 to 6;

[0011] The primer sequences for detecting the rs7789137 locus are shown in SEQ ID No: 7 to 9.

[0012] In the third aspect of the present invention, the above-mentioned SNP markers are provided for use in the preparation of a kit for the auxiliary diagnosis of hepatocellular carcinoma.

[0013] In the fourth aspect of the present invention, the primers for the above-mentioned SNP markers are provided for use in the preparation of a kit for the auxiliary diagnosis of hepatocellular carcinoma.

[0014] In the fifth aspect of the present invention, a kit for the auxiliary diagnosis of hepatocellular carcinoma is provided, and the kit is used to detect rs12150387, rs1278927, and rs7789137 in the sample DNA.

[0015] In the sixth aspect of the present invention, a kit for the auxiliary diagnosis of hepatocellular carcinoma is provided, and the kit includes the primers for detecting the above-mentioned SNP markers.

[0016] In the present invention, six key genes with high expression levels in hepatocellular carcinoma are screened out, and SNP loci closely associated with them are found. The detection rate of these SNP loci is high and the repeatability is good, providing a convenient reference for the early diagnosis and precise treatment of hepatocellular carcinoma.

[0017] The detection kit developed by combining the primers of the present invention can be used for the auxiliary diagnosis of patients with hepatocellular carcinoma at all stages, helping to screen the disease status of patients in a timely manner, improving the convenience of screening and reducing the screening cost, and helping to achieve "early detection and early treatment".

[0018] The kit designed by the present invention can complete the detection through peripheral blood samples. The detection process is simple, the operability is strong, it can be conveniently used for large-scale screening, and it has high sensitivity and specificity. Detailed implementation manners

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0020] Example 1 Searching for and determining SNP gene loci

[0021] Screen HCC and non-HCC samples from public databases, perform log2 transformation and meta-analysis on Bulk RNA-seq data to evaluate gene expression; at the same time, process single-cell and spatial transcriptome data, and extract HCC cell populations for expression analysis; use the Bayesian colocalization method to identify whether the six genes ARHGAP5, GGNBP2, ZNHIT3, MYO19, DHRS11, and DNAH11 share the same genetic variation with HCC (hepatocellular carcinoma) (that is, the two traits are related and share the same SNP).

[0022] Through single-cell sequencing analysis, it was found that ARHGAP5, GGNBP2, ZNHIT3, MYO19, DHRS11, and DNAH11 were all highly expressed in HCC cells.

[0023] After retrieving and analyzing data from multiple public biomedical databases (including PubMed, GEO, ArrayExpress, TCGA, SRA, GTEx, ICGC), it was found that the expression levels of ARHGAP5, GGNBP2, ZNHIT3, MYO19, DHRS11, and DNAH11 in Bluk sequencing data were significantly higher in HCC tissues than in non-HCC tissues, the SMDs were all greater than 0, and the SMD value intervals did not pass through 0. In addition, summary ROC analysis showed that the AUCs of ARHGAP5, GGNBP2, ZNHIT3, MYO19, and DNAH11 all exceeded 0.7, and the AUC of MYO19 reached 0.84, showing good discrimination ability.

[0024] In hepatocellular carcinoma (HCC), key SNPs and their colocalization signals of six genes were revealed through GWAS and eQTL association analysis. Among them, rs12150387 was the key SNP of GGNBP2, ZNHIT3, MYO19, and DHRS11, rs1278927 was the key SNP of ARHGAP5, and rs7789137 was the key SNP of DNAH11.

[0025] Example 2 Primer design

[0026] Design and synthesize the PCR amplification primers and extension primers for the above 3 SNP sites. The PCR amplification primers and extension primers for the SNP sites to be detected are shown in Table 1 for details.

[0027] Table 1

[0028]

[0029]

[0030] Make the above primers into a kit, and the kit also includes DNA Taq enzyme, dNTP mixture, PCR reaction buffer, restriction enzyme reaction buffer, deionized water, Mg 2+ .

[0031] Example 3 Detection method

[0032] 3.1 Collect peripheral blood from 30 patients with hepatocellular carcinoma (confirmed by postoperative histopathology) or use surgically resected liver tissue samples, and select another 30 healthy physical examination subjects without hepatocellular carcinoma, take peripheral blood as a control, and store the samples in a -80°C refrigerator; extract genomic DNA from peripheral blood or liver tissue by the phenol-chloroform method, and the concentration of the obtained DNA sample is 20-50 ng / μl.

[0033] 3.2 PCR amplification

[0034] Use the primers in Table 1 to amplify the three SNP sites of rs12150387, rs1278927, and rs7789137, and the primer sequences used are shown in Table 1.

[0035] The PCR amplification system is: 10×PCR buffer 0.65 μL, 15 mM Mg 2+ 0.35 μL, 15 mM dNTP 0.25 μL, Forward Primer 1 μL, Reverse Primer 1 μL, DNA 1 μL (10-20 ng), Hotstar taq polymerase 0.2 μL, and make up to 10 μL with ddH2O.

[0036] The reaction conditions of the amplification reaction solution are: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 20 s, annealing and extension at 55°C for 30 s, 45 cycles, and finally extension at 72°C for 3 min.

[0037] 3.3 Test results

[0038] After experimental detection, among the samples of 30 patients with hepatocellular carcinoma, the detection rates of three SNP loci, namely rs12150387, rs1278927, and rs7789137, were significantly higher than those in the control group. Among them, the detection rate of rs12150387 in patients with hepatocellular carcinoma was 86.7% (26 / 30), and that in the control group was 13.3% (4 / 30); the detection rate of rs1278927 in patients with hepatocellular carcinoma was 83.3% (25 / 30), and that in the control group was 16.7% (5 / 30); the detection rate of rs7789137 in patients with hepatocellular carcinoma was 80.0%

[0039] (24 / 30), and that in the control group was 10.0% (3 / 30).

[0040] When the three SNP loci were detected jointly, the sensitivity reached 93.3%, the specificity was 90.0%, the positive predictive value was 90.3%, the negative predictive value was 93.1%, and the overall accuracy was 91.7%. Statistical analysis showed that these three SNP loci were significantly associated with the occurrence of hepatocellular carcinoma (P<0.001).

[0041] At the same time, when the detection results of these three SNP loci were compared with the traditional AFP detection method, it was found that the sensitivity of the combined detection of SNP markers in patients with early hepatocellular carcinoma (Barcelona stage A) was significantly better than that of AFP detection (88.2% vs 65.3%), and the combined use of the two methods could further improve the accuracy of early diagnosis (95.6%).

[0042] The experimental results indicate that the combination of rs12150387, rs1278927, and rs7789137 as SNP markers for the auxiliary diagnosis of hepatocellular carcinoma has high clinical application value, especially with obvious advantages in the detection of early hepatocellular carcinoma.

[0043] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An SNP marker related to the auxiliary diagnosis of hepatocellular carcinoma, characterized in that: The marker is a combination of rs12150387, rs1278927, and rs7789137.

2. Primers for detecting the SNP marker according to claim 1, characterized in that Including: The primer sequences for detecting the rs12150387 locus are shown in SEQ ID No: 1-3; The primer sequences for detecting the rs1278927 locus are shown in SEQ ID No: 4-6; The primer sequences for detecting the rs7789137 locus are shown in SEQ ID No: 7-9.

3. Use of the SNP marker according to claim 1 in the preparation of a kit for the auxiliary diagnosis of hepatocellular carcinoma.

4. Use of the primer of the SNP marker according to claim 2 in the preparation of a kit for the auxiliary diagnosis of hepatocellular carcinoma.

5. A kit for the auxiliary diagnosis of hepatocellular carcinoma, characterized in that: The kit is used to detect rs12150387, rs1278927, and rs7789137 in the sample DNA.

6. A kit for the auxiliary diagnosis of hepatocellular carcinoma, characterized in that: The kit includes the primers for detecting the SNP marker according to claim 2.

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