CN102796820B - Hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method and use - Google Patents

Abstract

The invention provides hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method and use. The hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism and its construction method have the advantages that 1, through a detection method provided by the invention, polymorphism of a single nucleotide located in an exon 4 area of a human klotho gene can be detected fast and simply and it is diagnosed that if an individual has susceptibility of hepatocellular carcinoma so that the hepatocellular carcinoma-susceptible population screening and hepatocellular carcinoma prevention are promoted; 2, a T allele point of rs648202 is used as a drug design target point for drug screening so that an active molecule adjusting a klotho gene expression level is selected and development of a novel antitumor drug is promoted; 3, through primer sequences and HaeIII endonuclease provided by the invention, simple, efficient and specific detection of rs648202 polymorphism is realized; and 4, according to hepatocellular carcinoma-related klotho gene single-nucleotide polymorphism, a hepatocellular carcinoma genetic diagnosis kit can be constructed.

Description

A kind of single nucleotide polymorphism of klotho gene associated with primary hepatocellular carcinoma and its construction method and application

technical field

The invention provides a construction method and application of a klotho gene single nucleotide polymorphism related to primary hepatocellular carcinoma, belonging to the field of genetic engineering biotechnology.

Background technique

Primary hepatocellular carcinoma (liver cancer, HCC) is one of the most common malignant tumors in the world. In December 2007, the data released by the American Cancer Society (ACS) showed that there were 667,000 cases of liver cancer and 598,000 deaths per year. Among them, about 50% of new cases and deaths are in China, that is, liver cancer poses a greater threat to the health of our people and has become the second cancer killer in our country. , society brings serious economic and psychological burden.

Single nucleotide polymorphism (SNP) refers to the polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans, accounting for more than 90% of all known polymorphisms. SNPs, as stable genetic early mutations, are closely related to diseases. When the frequency of a SNP in patients is significantly higher than that in non-patients, it is suggested that the SNP is related to the disease. By analyzing the haplotype and linkage disequilibrium of the two, any unknown pathogenic gene in the genome can be located . The role of SNP in the localization of disease-causing genes mainly includes: one is to look for disease-causing SNP, the appearance of this SNP can lead to changes in gene expression and (or) protein expression product structure, thereby leading to the occurrence of certain diseases Or make an individual susceptible to a certain disease; the second is that SNP, as a genetic marker, is linked to a disease or phenotype.

Klotho is an aging-related gene discovered in 1997. It is located on human chromosome 13q12 and is about 50kb in length, consisting of 5 exons and 4 introns. Loss of Klotho gene expression in mice leads to various phenotypes similar to human aging, such as arteriosclerosis, osteoporosis, emphysema, shortened lifespan, skin atrophy, infertility, movement disorders, etc. In recent years, more and more studies have found that klotho gene can regulate IGF, Wnt, TGF-βl, FGF and other signaling pathways, participate in the process of oxidative stress, and is related to the body's immune deficiency, and plays a role in the occurrence and development of malignant tumors. Similar to the important role of tumor suppressor genes. Therefore, klotho can be used as a marker for tumorigenesis and grading and as a target for anti-tumor therapy.

Searching domestic and foreign literature and patents of the prior art has not found any relevant reports on the relationship between klotho gene polymorphisms and susceptibility to hepatocellular carcinoma.

Contents of the invention

The purpose of the present invention is to fill up the gap in the prior art, and provide a method for constructing the single nucleotide polymorphism of klotho gene related to primary hepatocellular carcinoma and its application.

The present invention provides a method for detecting single nucleotide polymorphism of klotho gene related to primary hepatocellular carcinoma, which adopts PCR-RFLP method, comprising the following steps:

(1) Using the whole genome DNA of the human being to be tested comprising the klotho gene as a template, PCR amplifies the DNA sequence comprising the rs648202 single nucleotide polymorphism in the exon 4 region of the klotho gene, i.e. the 2190th in SEQ ID No.1 bit to bit 2352 of the sequence shown,

Wherein, the PCR primer sequence is as follows:

The upstream primer is F: 5'-ATAGCCTTGCAGGCTGATTG-3'

The downstream primer is R: 5'-TTCTTTGGTTCAGCCAGTCC-3'

(2) After digesting the PCR amplification product with restriction endonuclease Hae III, the rs648202 single nucleotide polymorphism was typed by agarose gel electrophoresis, and the length of the DNA fragment after digestion of the CC genotype was still 163bp ; DNA fragments of three lengths: 163bp, 100bp, and 63bp will be produced after enzyme digestion for the CT genotype; DNA fragments of two lengths: 100bp and 63bp will be produced after enzyme digestion for the TT genotype.

The present invention also provides an isolated nucleic acid, which has the sequence shown from No. 2190 to No. 2352 in SEQ ID No. 1, and the base at No. 2255 in SEQ ID No. 1 is T.

The present invention also provides a specific primer for detecting the single nucleotide polymorphism of klotho gene related to primary hepatocellular carcinoma, the upstream primer is F: 5'-ATAGCCTTGCAGGCTGATTG-3', and the downstream primer is R: 5 '-TTCTTTGGTTCAGCCAGTCC-3'.

The present invention also provides a diagnostic kit for the susceptibility of primary hepatocellular carcinoma, which comprises:

(1) Primers for specifically amplifying the single nucleotide polymorphism rs648202 of the klotho gene, wherein the upstream primer is F: 5'-ATAGCCTTGCAGGCTGATTG-3', and the downstream primer is R: 5'-TTCTTTGGTTCAGCCAGTCC-3';

(2) Reagents needed to detect whether there is a change in the amplified product compared with the normal klotho gene.

Utilizing the nucleic acid sequence of the polymorphic site associated with primary hepatocellular carcinoma provided by the present invention, a kit for genetic screening of primary hepatocellular carcinoma susceptible populations can be constructed and used as a target for drug design. Points to find out the active molecules that regulate the expression of klotho gene, thereby promoting the discovery of new anti-tumor drugs.

The present invention has following technical effect:

(1) The method established by the present invention can easily and quickly detect the genotype of the single nucleotide polymorphism site rs648202 located in the exon 4 region of the klotho gene in the human chromosome 13q12 region, and judge by detecting the presence or absence of the T allele Whether the individual is susceptible to primary hepatocellular carcinoma, which is conducive to the screening of the susceptible population of primary hepatocellular carcinoma and the prevention of primary hepatocellular carcinoma;

(2) Use the T allele of rs648202 as the drug design target for drug screening, screen out active molecules that can regulate the expression level of klotho gene, and promote the discovery of new anti-tumor drugs;

(3) The rs648202 polymorphism can be detected easily, efficiently and specifically by using the primer sequences and Hae III endonuclease provided by the present invention;

(4) using the single nucleotide polymorphism of klotho gene related to primary hepatocellular carcinoma provided by the present invention to construct a kit for genetic diagnosis of primary hepatocellular carcinoma;

(5) Since klotho participates in pathological processes related to cell activities such as malignant proliferation, apoptosis, invasion and metastasis, the present invention provides experience and application basis for in-depth exploration of the relationship between klotho and other diseases in the future.

Description of drawings

Figure 1 is the agarose gel electrophoresis image after PCR amplification of the exon 4 region of the klotho gene using specific primers and Hae III digestion. Among them, M: DNA molecular weight standard; l: CT genotype; 2: CC genotype; 3: TT genotype; 4: blank control.

Detailed ways

The present invention will be further described below in conjunction with specific examples. It should be noted that the following examples are only used to illustrate the present invention, but not to limit the scope of the present invention.

Example 1

(1) Collection of blood samples and DNA extraction

A total of 212 patients with sporadic primary hepatocellular carcinoma were collected from the Second Affiliated Hospital of Nanjing Medical University. All patients were confirmed by histopathology, including 150 males and 62 females. The average age of the patients was 62 years old; the control group 288 cases, the average age was 61 years old. There was no significant difference in age and sex composition between the case group and the control group (P>0.05). The above results indicated that the two groups were balanced and comparable in terms of age and gender. All research subjects signed the informed consent.

White blood cells were isolated from the blood samples collected above using conventional methods, and the genomic DNA of white blood cells was extracted by the classic phenol -chloroform method, and the samples were uniformly diluted to 0.1 μg/μl as DNA templates, and the remaining DNA was placed at -20 Store at ℃.

(2) Design and synthesize nucleotide primers, the primer sequences are as follows,

The upstream primer is F: 5'-ATAGCCTTGCAGGCTGATTG-3',

The downstream primer is R: 5'-TTCTTTGGTTCAGCCAGTCC-3'.

Perform polymerase chain reaction (PCR)

Carry out PCR reaction system: the total amount is 20 μl

Set the reaction conditions in the PCR instrument:

After the final amplification, a 163bp DNA sequence containing the rs648202 single nucleic acid polymorphism in the exon 4 region of the klotho gene was obtained, that is, the sequence shown in the 2190th to 2352nd positions in SEQ ID No.1.

(3) Perform single nucleotide polymorphism typing on the amplified DNA sequence

For the DNA fragment products obtained in step (2), restriction endonucleases are used to carry out restriction fragment length polymorphism typing.

The specific method is: add 5U of restriction endonuclease HaeIII and 1.0 μl of corresponding 10× enzyme digestion reaction buffer to 5 μl of PCR amplification product, add water to 10 μl, mix and digest at 37°C for 6 hours. Take 10 μl of the digested product and add 1.0 μl of loading buffer , mix thoroughly, add to the sample hole of 3% agarose gel, use DNA Marker as a reference, and the electrophoresis working solution is 1×TBE, so that the sample moves from the negative electrode to the positive electrode After about 40 minutes at a constant voltage of 80V, put the coagulation chamber plate on the quartz glass platform of the ultraviolet transilluminator for detection, observe whether there are fluorescent bands in each swimming lane, analyze the genotype of the digested product with a gel image analyzer, take pictures and Record the digestion results. As shown in Figure 1, the length of the DNA fragment after digestion of the CC genotype is still 163bp; DNA fragments of three lengths of 163bp, 100bp, and 63bp are produced after digestion of the CT genotype; DNA fragments of 100bp and 63bp are produced after digestion of the TT genotype DNA fragments of various lengths.

(4) Correlation analysis between rs648202 single nucleotide polymorphism in Klotho coding region and genetic susceptibility to primary hepatocellular carcinoma

Statistical method: use SPSS11.0 software to establish the database. The allele and genotype frequency distribution of the klotho gene of the cases and controls, as well as variables such as demographic variation, smoking and drinking, were tested by the ^χ2 test to determine whether there was a difference in genotype or allele in the selected population. distribution bias to determine its representativeness of the population as a whole. Univariate logistic regression analysis was used to obtain odds ratios (ORs) and 95% confidence intervals (CIs), all OR values adjusted for sex and age. SAS9.0 software was used for statistical analysis, P<0.05 was considered statistically significant difference, and all tests were two-sided tests.

The statistical results are as follows:

① Genetic balance test of genotype frequency at rs648202 locus of Klotho gene

The Hardy-Weniberg genetic balance test was performed on the genotype frequency of Klotho gene rs648202 site, χ ² =2.272, P=0.132, there was no statistically significant difference between the observed value and the expected value of the gene frequency at this site, indicating that the genotype frequency was in line with The Hardy-Weniberg ratio, the sample is representative of the group.

②The genotype and allelic distribution frequency of the rs648202 polymorphic site between the primary hepatocellular carcinoma patient group and the healthy control group are shown in Table 1 below:

Table 1 The genotype and allele frequency of Klotho gene rs648202 polymorphism and its relationship with liver cancer

The CC, CT and TT genotype frequencies were 57.1%, 37.7% and 5.2% in the cases, and 68.7%, 27.1% and 4.2% in the controls, respectively, and the distribution of the genotypes was significantly different (P=0.026 ). Comparing GA and AA with GG, the two are still statistically significant (P=0.007). Logistic regression analysis showed that, compared with CC wild type individuals, the risk of liver cancer in individuals carrying the mutated CT genotype increased significantly, with an increase of 59.6%, and the risk of individuals carrying the CT+TT mutation genotype increased by 60.4% .

These results suggest that rs648202 in exon 4 region of klotho gene is a marker locus for susceptibility to primary hepatocellular carcinoma. When the locus carries the T allele, that is, when the genotype of the locus is T/T or C/T, the risk of developing liver cancer is significantly higher than that of individuals with the genotype C/C, indicating that C/T or Individuals with T/T genotype are susceptible to primary HCC, while individuals with C/C genotype are less susceptible to HCC.

Organization Applicant

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<110> OrganizationName : The Second Affiliated Hospital of Nanjing Medical University

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Individual Applicant

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<110> LastName : Huang

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Application Project

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<120> Title : A single nucleotide polymorphism of klotho gene associated with primary hepatocellular carcinoma, its construction method and its

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the

Claims (1)

1. the purposes of a species-specific primer on the diagnostic kit for preparing the primary hepatocellular carcinoma (hcc) susceptibility, it is characterized in that, described Auele Specific Primer, upstream primer is F:5'-ATAGCCTTGCAGGCTGATTG-3', downstream primer is R:5'-TTCTTTGGTTCAGCCAGTCC-3', described diagnostic kit, for detection of the genotype in klotho gene rs648202 site, detects and adopts the PCR-RFLP method, comprises the following steps:

(1) the people's complete genome DNA to be measured that comprises the klotho gene of take is template, carry out with above-mentioned Auele Specific Primer the DNA sequence dna that comprises the rs648202 mononucleotide polymorphic that pcr amplification obtains klotho gene extron 4th district, i.e. sequence shown in the 2190th to the 2352nd in SEQ ID No.1;

(2) pcr amplification product is carried out after enzyme is cut carrying out the rs648202 single nucleotide polymorphism typing by agarose gel electrophoresis through restriction enzyme Hae III.

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