Detection of nrde2 gene polymorphism associated with liver cancer and application thereof

Abstract

The application discloses application of single nucleotide polymorphism rs199890497 in human genome in detection of a liver cancer susceptibility gene. One technical solution protected by the application is application of a substance for detecting polymorphism or genotype (i.e. allele) of rs199890497 in human genome DNA in preparation of a product for detecting single nucleotide polymorphism related to liver cancer. The substance for detecting polymorphism or genotype (i.e. allele) of rs199890497 can be combined with other substances (such as a substance for detecting other single nucleotide polymorphism or genotype (i.e. allele) related to liver cancer) to prepare a product for screening liver cancer and personalized medication guidance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to methods, kits, and applications for detecting NRDE2 gene polymorphisms associated with liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. In my country, HCC ranks fourth in incidence and third in mortality among malignant tumors. Epidemiological and experimental studies have shown that HCC is a complex, multifactorial disease resulting from the combined effects of environmental and genetic factors. In my country, hepatitis B virus (HBV) infection is the most significant factor in the development of HCC, associated with approximately 80% of cases. Traditional case-control association studies based on candidate genes and the emerging genome-wide association studies (GWAS) have successively discovered several single nucleotide polymorphisms (SNPs) significantly associated with HCC, confirming the existence of genetic causes for HCC, namely, the presence of susceptibility genes. The identification of HCC susceptibility genes and the elucidation of their related molecular mechanisms will provide a theoretical basis for the prevention and clinical treatment of high-risk groups for HCC, and may ultimately lead to early prevention and individualized treatment of HCC, thereby improving the treatment outcomes.

[0003] Due to limitations in SNP microarray technology and the size of the study populations, previous genetic association studies of complex diseases have primarily focused on common genetic variants (minor allele frequencies greater than 5%), with limited research on low-frequency or rare genetic variants (minor allele frequencies less than 5%). Studies have shown that the identified common genetic variants are insufficient to explain the full heritability of complex diseases. However, a growing body of research in recent years has suggested that low-frequency or rare genetic variants may also be able to explain a portion of the heritability of complex diseases.

[0004] In recent years, with the development of high-throughput sequencing technology, it has become possible to systematically identify rare genetic variants associated with complex diseases. Based on genetic association studies of rare variants, researchers have successfully located susceptibility genes for various complex diseases. For example, researchers used whole-exome sequencing to conduct genetic association analysis of polyposis choroidal vascular disease and successfully discovered a rare missense mutation in the FGD6 gene that is significantly associated with the occurrence of this disease. Rare variants in the BRCA2 gene have been found to be significantly associated with the risk of pancreatic cancer. However, to date, there are no reported studies using high-throughput sequencing technology to identify rare genetic variants associated with liver cancer. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to screen individuals susceptible to liver cancer, how to predict a person's susceptibility to liver cancer, how to screen individuals carrying human liver cancer susceptibility genes, or how to assess the risk of developing liver cancer.

[0006] To solve the above-mentioned technical problems, the present invention first provides any one of the following applications (A1-A5) and the product (A6):

[0007] A1. Application of substances that detect polymorphisms or genotypes (i.e., alleles) of rs199890497 in the human genome in the preparation of products for screening or assisting in the screening of individuals susceptible to or not susceptible to liver cancer.

[0008] A2. Application of substances that detect polymorphisms or genotypes (i.e., alleles) of rs199890497 in the human genome in the preparation of products for detecting or assisting in the detection of liver cancer susceptibility.

[0009] A3. Application of substances that detect polymorphisms or genotypes (i.e., alleles) of rs199890497 in the human genome in the preparation of products for detecting or assisting in the detection of liver cancer risk.

[0010] A4. Application of substances that detect polymorphisms or genotypes (i.e., alleles) of rs199890497 in the human genome in the preparation of products for evaluating or assisting in the evaluation of liver cancer risk.

[0011] A5. Application of substances that detect polymorphisms or genotypes (i.e., alleles) of rs199890497 in the human genome in the preparation of products for screening or assisting screening of liver cancer susceptibility genotypes or liver cancer non-susceptibility genotypes.

[0012] A6. Products containing substances that detect the polymorphism or genotype (i.e., allele) of rs199890497 in the human genome, which are any of products a)-d):

[0013] a) Products that detect single nucleotide polymorphisms or genotypes associated with liver cancer;

[0014] b) Products that identify or assist in the identification of single nucleotide polymorphisms or genotypes associated with liver cancer;

[0015] c) Products for screening or assisting in the screening of liver cancer patients;

[0016] d) Products for detecting or assisting in the detection of susceptibility to liver cancer.

[0017] In the aforementioned applications and products, the detection of the polymorphism or genotype of rs199890497 in human genomic DNA can specifically refer to the detection of the nucleotide types of rs199890497. The genotype of rs199890497 is TT or AT, where TT is a homozygous genotype with a T locus in rs199890497, and AT is a heterozygous genotype with both A and T loci in rs199890497. Individuals with the genotype AT of rs199890497 have a higher or higher risk of developing liver cancer than individuals with the polymorphism or genotype TT of rs199890497.

[0018] In the above application, the liver cancer susceptibility genotype is AT, and the liver cancer non-susceptibility genotype is TT; AT is a heterozygous genotype with A and T at rs199890497, and TT is a homozygous genotype with T at rs199890497.

[0019] The aforementioned applications and products are applicable to Chinese people, such as those from southern China. These Chinese people may be from Guangdong and / or Guangxi.

[0020] In the aforementioned applications and products, the substance for detecting the polymorphism or genotype (i.e., allele) of rs199890497 in human genomic DNA can be the reagents and / or instruments required to determine the polymorphism or genotype of rs199890497 by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chips. Among these, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0021] In the above applications and products, the substance for detecting the polymorphism or genotype of rs199890497 in human genomic DNA may contain PCR primers and / or single-base extension primers for amplifying human genomic DNA fragments including rs199890497.

[0022] The PCR primers do not have specific sequence requirements, as long as they can amplify the genomic DNA fragment including rs199890497, such as the single-stranded DNA shown in SEQ ID No. 1 and SEQ ID No. 2 in the sequence listing. The single-base extension primers can be designed based on the upstream or downstream (excluding the SNP site) of rs199890497 in the human genome. The nucleotide extended by the single-base extension primer corresponds to the nucleotide at the rs199890497 site in the human genome, that is, the 3' terminal nucleotide of the single-base extension primer corresponds to the adjacent nucleotide of rs199890497 in the human genome (i.e., the nucleotide before or after rs199890497).

[0023] In the above applications and products, the product may be a reagent, kit, or system. The system may include a combination of reagents or kits and instruments, such as a product consisting of PCR primers, single-base extension primers, and a mass spectrometer; a combination of PCR primers and a DNA sequencer; or a combination of PCR reagents, DNA sequencing reagents, and a DNA sequencer. The product may include the substance described above for detecting the polymorphism or genotype of rs199890497 in human genomic DNA.

[0024] In one embodiment of the present invention, PCR primers are used to amplify genomic DNA fragments including DNA rs199890497. Using the obtained PCR amplification product as a template, single-base extension primers are used to perform a single-base extension reaction. The sequence of the obtained extension product is detected to determine the polymorphism and genotype of rs199890497.

[0025] In one embodiment of the present invention, a device for screening liver cancer patients is provided, comprising:

[0026] A sequencing device, wherein the sequencing device is used to sequence the NRDE2 gene fragment including rs199890497 in the whole genome of the subject in order to obtain sequencing results;

[0027] A comparison device, which is connected to the sequencing device, and is used to determine the gene type of rs199890497 based on the sequencing results;

[0028] An analysis device, connected to the comparison device, is used to determine the risk of liver cancer based on the SNP type.

[0029] To uncover rare genetic variants associated with liver cancer, the inventors conducted a genome-wide association study based on high-throughput sequencing technology. Because high-throughput sequencing can effectively and systematically detect rare genetic variants, this study is expected to systematically identify rare genetic variants associated with liver cancer. In the discovery phase, the inventors included high-throughput sequencing data from 348 liver cancer cases (case group) and 1749 control individuals (control group) from southern China. In the validation phase, the inventors used the Sequenom mass spectrometry genotyping method to detect SNPs in three independent populations: Validation Population 1 (672 cases and 768 controls), Validation Population 2 (794 cases and 742 controls), and Validation Population 3 (923 cases and 886 controls). Ultimately, rs199890497 on the NRDE2 gene was successfully identified as a novel susceptibility site for liver cancer. Its molecular and physiological mechanisms were explored, and molecular markers of single nucleotide polymorphisms and genotypes related to the NRDE2 gene were developed, providing excellent allelic variants and rapid selection methods for the preparation of liver cancer screening or assisted screening products. Attached Figure Description

[0030] Figure 1 The quantile map is drawn from the genome-wide association results of the excavated population according to Embodiment 1 of the present invention.

[0031] Figure 2 This is a Manhattan plot drawn from the genome-wide association results of the excavated population according to Embodiment 1 of the present invention. Fisher's exact test was performed on each rare genetic variant based on the case-control population from the excavation phase. The horizontal axis represents genomic location, arranged in ascending order of chromosome number. The vertical axis represents the log-transformed p-value.

[0032] Figure 3 This is a map of the chromosomal location region of rs199890497, plotted based on the association results of the discovery and validation populations according to Embodiment 1 of the present invention. The regions are 250 kilobases (KB) upstream and downstream. The genomic location is based on the human reference genome (HG19 version). P-values ​​for rs199890497 in the discovery population are shown as circles. The pooled P-values ​​for rs199890497 in both the discovery and validation populations are shown as diamonds. Linkage disequilibrium (LD) values ​​(r²) of other SNPs with rs199890497 are indicated by varying shades of color.

[0033] Figure 4This study, based on Example 2 of the present invention, observed the growth, invasion, and migration of HepG2 cells with stable overexpression of wild-type and mutant NRDE2. (a) CCK-8 assay demonstrated that stable knockdown of NRDE2 promoted the growth of HepG2 cell lines; (b) cell colony formation assay demonstrated that stable knockdown of NRDE2 promoted the colony formation ability of HepG2 cell lines; (c) stable knockdown of NRDE2 promoted the migration and invasion ability of HepG2 cell lines; (d) CCK-8 assay demonstrated that stable overexpression of wild-type (but not mutant NRDE2) inhibited the growth of HepG2 cell lines; (e) cell colony formation assay demonstrated that stable overexpression of wild-type NRDE2 (but not mutant NRDE2) inhibited the colony formation ability of HepG2 cell lines; (f) stable overexpression of wild-type NRDE2 (but not mutant NRDE2) inhibited the migration and invasion ability of HepG2 cell lines. ***, P<0.001.

[0034] Figure 5 This invention relates to Example 2 of the study, which describes the observation of cell growth, invasion, and migration in Huh7 cells with stable overexpression of wild-type and mutant NRDE2. (a) CCK-8 assays demonstrated that stable knockdown of NRDE2 promoted the growth of the Huh7 cell line; (b) cell colony formation assays demonstrated that stable knockdown of NRDE2 promoted the colony formation ability of the Huh7 cell line; (c) stable knockdown of NRDE2 promoted the migration and invasion ability of the Huh7 cell line; (d) CCK-8 assays demonstrated that stable overexpression of wild-type (but not mutant) NRDE2 inhibited the growth of the Huh7 cell line; (e) cell colony formation assays demonstrated that stable overexpression of wild-type NRDE2 (but not mutant NRDE2) inhibited the colony formation ability of the Huh7 cell line; (f) stable overexpression of wild-type NRDE2 (but not mutant NRDE2) inhibited the migration and invasion ability of the Huh7 cell line. ***, P<0.001. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Example 1: Genome-wide association study of liver cancer based on high-throughput sequencing data

[0038] This genome-wide association study is divided into two phases: the discovery phase and the validation phase.

[0039] Ethical Statement

[0040] Each participant signed an informed consent form, and this study was approved by the Medical Ethics Committee of the Academy of Military Medical Sciences.

[0041] Research subjects

[0042] All study subjects were Chinese adults.

[0043] Sample inclusion criteria

[0044] Healthy individuals (control group, hereinafter referred to as control): ① No tumors; ② No hyperlipidemia or hypertension; ③ No systemic inflammatory response; ④ No cysts, suspicious nodules, etc.

[0045] The inclusion criteria for liver cancer patients (liver cancer cases, hereinafter referred to as cases) are a clinical diagnosis of primary hepatocellular carcinoma, no radiotherapy or chemotherapy, and pathological confirmation.

[0046] The specific diagnostic criteria are as follows:

[0047] Positive pathological diagnosis from tissue biopsy, serum alpha-fetoprotein (AFP) >400 ng / mL, and one positive imaging diagnosis; or negative tissue biopsy, serum AFP ≤400 ng / mL, but at least two positive imaging diagnoses, and a history of liver cancer epidemiology.

[0048] None of the liver cancer cases and control individuals mentioned above were related. All patients signed informed consent forms.

[0049] The discovery phase included 348 liver cancer cases and 1749 control individuals. All individuals were from southern China. The validation phase included three independent case-control populations: Shanghai (validation population 1, 672 cases and 768 controls), Guangdong / Guangxi (validation population 2, 794 cases and 742 controls), and Shaanxi (validation population 3, 923 cases and 886 controls). Gender and age information for the case-control populations is shown in Table 1.

[0050] Table 1. Summary of population information used in genome-wide association studies.

[0051]

[0052]

[0053] 1. Establishment of a method for detecting the rs199890497 polymorphic site in the NRDE2 gene

[0054] Using high-throughput sequencing data from 348 hepatocellular carcinoma cases and 1749 control individuals during the discovery phase, we employed GATK (The Genome Analysis Toolkit) software to detect genetic variations. After rigorous quality control of these variations, a total of 219,479 rare genetic variations (minor allele frequencies less than 1%) were detected.

[0055] We employed a single-locus-based analysis strategy to conduct a point-by-point association study on the aforementioned 219,479 rare genetic variations. The inflation coefficient of the association results was 1.039, which is less than 1.05. Furthermore, the quantile plots showed no significant shift, as... Figure 1 As shown in the figure, we found a total of 14 SNPs that were significantly associated with the risk of liver cancer (P<1.0×10-5). Figure 2 The rs199890497 site on the NRDE2 gene has drawn our attention. This site is a missense variant on the NRDE2 gene that will lead to changes in the amino acid composition of the NRDE2 protein (p.N377I).

[0056] rs199890497 is a SNP site on human genomic DNA. This variant is a transition (T / A, or A / T on its complementary strand). rs199890497 is located at 14:90303001 (GRCh38), HGVS:NC_000014.9:90303000:T:A, within the NRDE2 gene (NCBI Reference Sequence:NC_000014.9:g.90303001T>A), and is SEQ ID No. 4 in the sequence listing. The genotype of rs199890497 is TT, TA, or AA. TT is a homozygous genotype with T at the rs199890497 site, AA is a homozygous genotype with A at the rs199890497 site, and TA is a heterozygous genotype with both T and A at the rs199890497 site.

[0057] 1.1 Extraction of genomic DNA

[0058] Genomic DNA is extracted from the sample to be tested and used as a template for genotyping.

[0059] 1.2 Primer Design and Synthesis

[0060] PCR amplification primers and single-base extension primers for rs199890497 were designed using Sequenom's Genotyping Tools and MassARRAY Assay Design software, and synthesized by a biotechnology company. The sequences of the PCR amplification-specific primer pairs for detecting the rs199890497 locus genotype of the NRDE2 gene are as follows: forward primer: 5'-ACGTTGGATGTTGGCCAGTTTCAGATCCAC-3' (SEQ ID No. 1 in the sequence listing); reverse primer: 5'-ACGTTGGATGAAAAGCGAAAGAGGTCCCTG-3' (SEQ ID No. 2 in the sequence listing); extension primer: 5'-TGCTCTCAATGGCCCGCTC-3' (SEQ ID No. 3 in the sequence listing).

[0061] 2. Sequenom classification

[0062] SNP genotyping in the Sequenom MassARRAY system employs matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). First, multiplex PCR amplification is performed on the DNA template. SNP-specific extension primers are then added to the amplification products for single-base extension. This reaction terminates at the SNP site. Therefore, the resulting extension products have varying molecular weights due to differences in SNP allelic polymorphism. Finally, the molecular weight of the extension products is detected using MALDI-TOF MS, and the differences in molecular weight are interpreted using analytical software to determine the genotype of the SNP site. The experimental steps for SNP genotyping in the Sequenom MassARRAY system mainly include PCR amplification, alkaline phosphatase (SAP) reaction, single-base extension, desalting purification reaction, and mass spectrometry analysis.

[0063] 2.1 PCR amplification: PCR amplification was performed in 384-well plates, with a total volume of 5 μL for each reaction system. The PCR reaction system was prepared according to Table 2.

[0064] Table 2 Components of each PCR reaction system

[0065] 10×PCR buffer 0.5 <![CDATA[MgCl2(25mM)]]> 0.4 dNTP mix (25mM) 0.1 HotStar Taq enzyme (5U / μL) 0.1 Ultrapure water 1.9 DNA shown in SEQ ID No. 1 0.5 DNA shown in SEQ ID No. 2 0.5 Total volume 4

[0066] Take out the prepared DNA sample and adjust the sample volume to 1 μL. Each 5 μL PCR reaction system contains 20-50 ng of template DNA, 0.5 U of Hotstar Tag, 0.5 pmol of each amplification primer, and 0.1 μL of 25 mM dNTPs.

[0067] 2.2 Treatment of PCR products with alkaline phosphatase: After the PCR reaction is completed, the PCR products are treated with SAP (shrimpalkaline phosphatase) to remove free dNTPs in the system. The SAP reaction system is prepared according to Table 3.

[0068] Table 3 SAP Reaction System

[0069] Ultrapure water 1.53 10×SAP buffer 0.17 SAP enzyme (1.7 U / ul) 0.3 Total volume 2

[0070] 2.3 Single base extension: After alkaline phosphatase treatment, a single base extension reaction was carried out. The total volume of the reaction system was 9 μL. The single base extension reaction system was prepared according to Table 4.

[0071] Table 4. Monobase Extension Reaction System

[0072] water 1.53 10× Single Base Extension Reaction Buffer 0.17 Single base extension reaction enzyme (1.7 U / ul) 0.3 Total volume 2

[0073] 2.4 Resin purification: Spread the Clean Resin resin evenly onto a 6 mg resin plate; add 16 μL of water to the corresponding well of the extension product; pour the dried resin into the extension product plate, seal the plate, and rotate vertically at low speed for 30 minutes to ensure that the resin and reactants are in full contact; centrifuge to allow the resin to settle to the bottom of the well.

[0074] 2.5 Chip Spotting: Start the MassARRAY Nanodispenser RS1000 spotter (SEQUENOM) and transfer the resin-purified extension product onto a 384-well SpectroCHIP (Sequenom) chip (SEQUENOM).

[0075] 2.6 Mass spectrometry detection: The spotted SpectroCHIP chip was analyzed using MALDI-TOF, and the detection results were classified and output using TYPER 4.0 software (sequenom).

[0076] 2.7 Statistical Analysis

[0077] Fisher's exact test was used for association studies of candidate SNP loci. The inflation coefficient measures the difference between the actual and expected p-values. Quantile plots and inflation coefficients were calculated using R software.

[0078] 2.8 Results of SNP Association Analysis for Sequenom Classification

[0079] Genomic DNA was extracted from peripheral blood samples of cases and controls, and PCR amplification, single-base extension reaction, and genotyping were performed. The genotype frequency distribution of the rs199890497 polymorphism site of the NRDE2 gene in the two groups is shown in Table 5.

[0080] Among 4104 healthy individuals, only 3 had the AT genotype, while the rest had the TT genotype, with a frequency of 0.07%. Among 2718 liver cancer patients, 30 had the AT genotype, while the rest had the TT genotype, with a frequency of 1.10%. An increased AT genotype significantly increases the probability of developing liver cancer (OR = 15.25; 95% CI = 4.74–78.16; P = 1.19 × 10⁻⁶). -9 Fisher's exact test revealed that rs199890497 was significantly associated with liver cancer in all three validation populations (P < 0.05, Table 5). Pooling the results from all populations, the p-value for rs199890497 reached 1.19 × 10⁻⁶. -9 (Table 5). The results above indicate that the NRDE2 gene polymorphism rs199890497 is significantly associated with liver cancer. Individuals carrying the AT genotype have a significantly higher risk of developing liver cancer than those carrying the TT genotype. Detection of the rs199890497 polymorphism or genotype can be used to screen individuals susceptible to liver cancer, predict susceptibility to liver cancer, screen individuals carrying liver cancer susceptibility genes, and assess the risk of developing liver cancer.

[0081] Table 5 Association analysis of rs199890497 among independent populations

[0082]

[0083]

[0084] a TT / AT / AA number of cases. OR, Odds Ratio. CI, Confidence Interval. Inf, infinite.

[0085] Example 2: Effects of NRDE2 gene polymorphism rs199890497 on the growth, migration, and invasion of liver cancer cells.

[0086] 1. CCK-8 cell proliferation experiment

[0087] Cells in the logarithmic growth phase were collected, and after cell counting, the cell density was adjusted. 2,000 cells were seeded into 96-well plates at a rate of 3 replicates per group. After a certain period of cell culture, serum-free cell culture medium (Dulbecco's modified Eagle's Medium, DMEM) containing 10% CCK-8 reagent was added. The absorbance (optical density, OD450) was measured at 450 nm using a microplate reader. Data were collected, and statistical analysis was performed based on the recorded data to plot cell growth curves.

[0088] 2. Plate cell colony formation assay

[0089] Cells in the logarithmic growth phase were digested and resuspended, and seeded into 6-well plates at a rate of 1,000 cells per well, with 3 replicates per group. After incubation for 8-14 days, the cells were removed, fixed with methanol for 15 minutes, stained with 0.5% crystal violet for 20 minutes, washed and dried, and the cell clones in the 6-well plates were photographed, counted, and statistically analyzed.

[0090] 3. Cell migration and invasion experiments

[0091] Cells in the logarithmic growth phase of both the experimental and control groups were seeded into 6-well plates and incubated for 36 hours. Then, the plates were replaced with serum-free DMEM (purchased from SciGen Biotech) and starved for 18 hours. Cells were then digested, resuspended in serum-free DMEM, and counted. Cells were then seeded in 7 × 10⁶ cells / wells in the upper chamber of a Transwell plate. 4 Cells were cultured in 500 μL of DMEM medium containing 20% ​​FBS in the lower chamber of a Transwell for 24-36 h. The chamber was then removed, washed once with phosphate-buffered saline (PBS), fixed with methanol for 15 min, stained with 0.5% crystal violet for 20 min, and excess stain was washed away. Cells that had not migrated were carefully wiped from the inside of the chamber and air-dried. Cells that had migrated through the chamber were photographed and counted under a microscope. For invasion assays, Transwell chambers containing Matrigel were used, and the remaining procedures were the same as for migration assays.

[0092] 4. Identification of the biological functions of NRDE2 and its rs199890497 (p.N377I) in hepatocellular carcinoma cells.

[0093] To explore the biological functions of NRDE2 and its associated rs199890497 (p.N377I) in hepatocellular carcinoma cells, we first stably knocked down NRDE2 in HepG2 and Huh7 cell lines. We designed and synthesized multiple shRNAs targeting NRDE2, transfected them into Huh-7 and HepG2 hepatocellular carcinoma cell lines, and evaluated the knockdown effect using qRT-PCR, thus obtaining two shRNAs that effectively knocked down NRDE2. The sequences are as follows: shRNA1: 5'-GCAAGCAGGUUGAACGCUA-3', shRNA2: 5'-GUUUAGUACCUUUUCGAUA-3'. These shRNAs were then constructed into a lentiviral vector (pLV-Luc) carrying a luciferase tag. After viral packaging, the virus was used to infect hepatocellular carcinoma cell lines. After selection with puromycin, the cells were identified by qRT-PCR and Western blotting (WB), thus obtaining hepatocellular carcinoma cell lines with stable NRDE2 knockdown.

[0094] Secondly, stable cell lines overexpressing wild-type and mutant NRDE2 were established: the CDS sequence of NRDE2 was amplified using qRT-PCR and constructed into a flag-tagged lentiviral vector (pLV-Flag). Simultaneously, the p.N377I point mutation was constructed using the QuickChange site-directed mutagenesis kit. After viral packaging, the cells were infected with hepatocellular carcinoma cell lines such as Huh-7 and HepG2. After selection with puromycin, the expression of wild-type and mutant NRDE2 was identified using RT-PCR and Western blotting, thus obtaining stable hepatocellular carcinoma cell lines with high expression of both wild-type and mutant NRDE2.

[0095] The results showed that knocking down NRDE2 significantly increased the growth, colony formation, migration, and invasion abilities of HepG2 and Huh7 cells. Figure 4 Chinese AC and Figure 5 (ac). Furthermore, we stably overexpressed wild-type NRDE2 and mutant NRDE2 (p.N377I) in HepG2 and Huh7 cell lines. In both HepG2 and Huh7 cell lines, overexpression of wild-type NRDE2 significantly reduced the growth, colony formation, migration, and invasion abilities of HepG2 cells. Figure 4 df and Figure 5 (df); however, after overexpression of mutant NRDE2 (p.N377I), the original tumor suppressor function of wild-type NRDE2 was lost. Figure 4 df and Figure 5(df). The above results suggest that NRDE2 may play a tumor suppressor role in liver cancer cells, and its rare variant p.N377I has a loss-of-function effect.

[0096] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of detecting polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for screening or assisting in the screening of susceptible or non-susceptible individuals for liver cancer.

2. Application of detecting polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for detecting or assisting in the screening of liver cancer susceptibility.

3. The application of detecting polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for detecting or assisting in screening the risk of liver cancer.

4. The application of detecting polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for evaluating or assisting in the evaluation of liver cancer risk screening.

5. Application of detecting polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for detecting single nucleotide polymorphisms associated with liver cancer.

6. Application of substances that detect polymorphisms or genotypes of rs199890497 in the human genome in the preparation of products for screening or assisting screening of liver cancer susceptibility genotypes or liver cancer non-susceptibility genotypes.

7. The application according to any one of claims 1-6, characterized in that: The substance used to detect the polymorphism or genotype of rs199890497 in the human genome contains PCR primers and / or single-base extension primers for amplifying human genomic DNA fragments including rs199890497.

8. The application according to claim 7, characterized in that: The PCR primers and / or single-base extension primers: The PCR primers consist of single-stranded DNA with nucleotide sequences of SEQ ID No. 1 and SEQ ID No. 2 in the sequence listing, respectively; the single-base extension primers consist of single-stranded DNA with nucleotide sequences of SEQ ID No. 3 in the sequence listing.

9. Equipment used for screening liver cancer patients, including: Sequencing device, the sequencing device being used to analyze the whole genome of a subject, including rs199890497. NRDE2 Gene fragments are sequenced to obtain sequencing results; A comparison device, which is connected to the sequencing device, and is used to determine the genotype of rs199890497 based on the sequencing results; An analysis device, connected to the comparison device, is used to determine the risk of liver cancer based on the genotype.

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