CN114703283A

CN114703283A - Application of MNS16A genotype as biomarker for predicting cancer chemotherapy sensitivity

Info

Publication number: CN114703283A
Application number: CN202210360980.0A
Authority: CN
Inventors: 汪洛
Original assignee: Hainan Yuanxuan Biotechnology Co ltd
Current assignee: Hainan Yuanxuan Biotechnology Co ltd
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-07-05

Abstract

The invention discloses an application of MNS16A genotype as a biomarker in predicting cancer chemotherapy sensitivity, and relates to the technical field of gene detection; the MNS16A genotype can be classified into functional genotypes of LL, SL and SS according to VNTR, when the genotype of an individual is SS, the individual is judged to be a chemotherapy-sensitive individual, and when the genotype of the individual is LL and SL, the individual is judged to be a chemotherapy-insensitive individual.

Description

Application of MNS16A genotype as biomarker for predicting cancer chemotherapy sensitivity

Technical Field

The invention relates to the technical field of gene detection, in particular to application of MNS16A genotype as a biomarker in predicting cancer chemotherapy sensitivity.

Background

Malignant tumor is one of the main diseases endangering human health in the new century, is also one of the global larger public health problems, and brings great disease burden to society. In the last 20 years, malignant tumors have become the first cause of death for residents in China. Therefore, tumor prevention and treatment is a major task in all humans. With social demands, various tumor treatment means (such as surgery, chemotherapy, radiotherapy and molecular targeted drug therapy) are rapidly developed, in recent years, the value of chemotherapy in the treatment process of malignant tumors is more and more emphasized, chemotherapy is not only used for palliative treatment of late patients and cases which have serious complications, are not suitable for surgical treatment, such as the advanced patients, but also used for auxiliary treatment of patients with high risk factors after pathological staging of surgery or complementary treatment with insufficient surgical resection range, and researches show that postoperative chemotherapy can effectively reduce death risk.

Chemotherapy sensitivity is the degree of sensitivity of cancer patients to chemotherapy drugs, and more clinical data show that different cancer patients have different responses to the treatment of the same drug, and even if the clinical diagnosis, the stage and the general condition are the same, different curative effects or adverse reactions can still be presented by using the same drug and treatment dose. Research shows that the gene biomarker can predict the sensitivity of tumor cells to chemotherapeutic drugs, can be used for clinically guiding the selection of cancer chemotherapeutic schemes, avoids over-treatment, and achieves the purposes of accurate treatment and individual medication, so that the combination of the gene biomarker and the chemotherapeutic sensitivity is necessary.

Telomeres are structures covering the distal ends of chromosomes and function to prevent chromosome degradation, end-to-end fusion, rearrangement, and chromosome loss. Telomerase is a holoenzyme that maintains telomeres, and its activity cannot be detected in most normal human cells, except in peripheral blood, cord blood and bone marrow leukocytes. About 90% of tumor cells have telomerase activity, while most normal cells are negative, indicating that telomerase can be a characteristic marker for tumor cells, and human telomerase consists of three parts: an RNA component (hTERC) which serves as an endogenous template for telomere repeat synthesis; 2. telomerase binding protein (TEP 1); 3. telomerase catalytic subunit (hTERT), also known as telomerase reverse transcriptase gene (human). The RNA component and the catalytic subunit of telomerase are essential components for telomerase activity. Research shows that telomerase catalytic subunit (hTERT) plays a determining role in telomerase activity, and the telomerase catalytic subunit is expressed at a high level in most tumor cells or tumor cell strains, but is not expressed or expressed at a low level in normal cells, so that the hTERT gene is considered to be one of the most common tumor markers. Current studies indicate that human telomerase expression levels are independent predictors of the prognosis of many cancers; recently, it was found that a polymorphic tandem repeat microsatellite (MNS16A) is present in the antisense transcription promoter region of hTERT gene, and that the VNTR polymorphism of MNS16A is related to hTERT mRNA expression, but it is not shown that MNS16A in hTERT gene is related to cancer prognosis.

Disclosure of Invention

The invention aims to provide application of MNS16A genotype as a biomarker in prediction of cancer chemotherapy sensitivity, and solve the technical problem that no appropriate gene biomarker is available in the prior art for prediction of cancer chemotherapy sensitivity.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of MNS16A genotype as a biomarker for predicting cancer chemotherapy sensitivity.

Further, the MNS16A genotype can be classified into functional genotypes of LL, SL and SS according to VNTR, when the genotype of an individual is SS, the individual is judged to be a chemotherapy-sensitive individual, and when the genotype of the individual is LL and SL, the individual is judged to be a chemotherapy-insensitive individual.

Furthermore, the SS genotype is a homozygote with S alleles, the LL genotype is a homozygote with L alleles, the SL genotype is a heterozygote with S and L alleles, the S allele is a DNA fragment with 243bp or 272bp on the telomerase gene, and the L allele is a DNA fragment with 302bp or 333bp on the telomerase gene.

The invention also provides application of the MNS16A genotype as a biomarker in preparing a kit for predicting cancer chemotherapy sensitivity.

Further, the kit comprises PCR specific amplification primers for detecting the MNS16A genotype.

Further, the sequence of the specific amplification primer is as follows: the sequence of the front primer was 5'-AGGATTCTGATCTGAAGGGTG-3' and the sequence of the reverse primer was 5'-TCTGCCTGAGGAGACGTATG-3'.

Further, the kit also comprises PCR buffer solution, dNTP, magnesium chloride, Taq polymerase and deionized water.

Compared with the prior art, the invention has the advantages that:

the invention can effectively predict the sensitivity of cancer patients to chemotherapeutic drugs by detecting the MNS16A genotype on the hTERT gene, is used for clinically guiding the selection of cancer chemotherapeutic schemes, makes more reasonable treatment schemes, avoids ineffective chemotherapy, reduces medical cost, improves effective cure rate and achieves the aims of accurate treatment and individual medication.

Drawings

FIG. 1 shows the results of genotyping human telomerase (hTERT) MNS16A in 299 patients with glioblastoma in accordance with the present invention;

FIG. 2 is a graph of the overall survival analysis for different genotypes.

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure. In the following examples, reagents and equipment not specifically described are commercially available, and experimental procedures not specifically described are carried out according to manufacturer's specifications or conventional techniques in the art, unless otherwise defined, and all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

The technical scheme of the invention specifically comprises the following contents:

(1) collecting peripheral blood samples of patients by a standard operation process, and collecting complete demographic data and clinical information by a system;

(2) carrying out regular follow-up visit on a patient, and carrying out complete follow-up visit data registration;

(3) genotyping a patient to obtain genotype information of the patient;

(4) performing statistical analysis based on the genotype of the patient, and performing sensitivity analysis;

specifically, the experimental method related in the invention mainly comprises the following parts:

this example provides a biomarker useful for predicting cancer chemotherapy sensitivity, providing a novel treatment for cancer patients

(1) Selection of study samples:

the sample is a glioblastoma multiforme patient in a primary malignant glioma patient, has complete case data (including diagnosis and treatment schemes, medical history and the like), all patients receive chemotherapy, all patients are initial patients without tumor incidence history, and patients do not receive tumor-related radiotherapy before admission, and the final study population is 299 patients with GBM, the diagnosis age of the patients ranges from 20 to 65 years, and the average age of the patients is 49.9 years.

(2) Peripheral blood genome DNA extraction:

leukocyte pellets were obtained from peripheral blood samples by centrifugation, and genomic DNA was extracted from the leukocyte pellets using a Qiagen DNA blood mini kit (Qiagen, Valencia, CA) according to the instructions for use, and the DNA purity was evaluated by electrophoresis on a 1% agarose gel, and the concentration of DNA was determined by ultraviolet spectroscopy.

(3) Preparation method of kit

A kit for prediction of cancer chemotherapy sensitivity comprising: DNA fragment comprising a variable number of tandem repeat polymorphisms of the hTERT gene MNS16A, pre-primer sequence: 5'-AGGATTCTGATCTGAAGGGTG-3', reverse primer sequence: 5'-TCTGCCTGAGGAGACGTATG-3', and auxiliary components such as PCR buffer solution, dNTP, magnesium chloride, Taq polymerase and deionized water.

(3) MNS16A genotyping

Specifically amplifying DNA fragments containing variable number of tandem repeat polymorphisms of the hTERT gene MNS16A to obtain genotype information, wherein the MNS16A genotype can be classified into functional genotypes of LL, LS and SS according to VNTR.

(4) Statistical analysis

The two-sided χ 2 test was used to determine any statistically significant differences in the distribution of the MNS16A genotype in demographic variables and clinical characteristics (age, gender, surgery); estimating the total median survival rate of three different MNS16A genotypes by adopting a Kaplan-Meier method, and using a logarithmic rank test to test and apply to calculating a P value; establishing a Cox proportional risk model according to the identified relevant prognostic factors, calculating a risk ratio (HR) and a 95% confidence interval (95% confidence interval, 95% CI), analyzing the influence of the MNS16A genotype polymorphism on the chemotherapy sensitivity of cancer patients, dividing the diagnosis age of the patients into two groups according to the average age of the patients (50 years and more than or equal to 50 years), and dividing the surgical resection types of the patients into the following three groups: gross total resection, sub-total resection and biopsy, with the patient's survival time calculated from the date of first registration at the cancer center, with the last contact date or death date as the censorship node; statistical analysis was done using the SAS software program with a statistical significance level of P < 0.05, all tests were two-sided.

(5) Analysis of results

The MNS16A genotype distribution did not statistically significantly differ in age, sex, or surgical treatment, however, the genotype distribution significantly varied with length of survival, with the SS genotype surviving longer than the SL or LL genotype patients; median survival for patients with SS genotype was 25.1 months (95% CI ═ 14.9-30.9 months), for patients with SL genotype was 14.7 months (95% CI ═ 13.9-18.8 months), and for patients with LL genotype was 14.6 months (95% CI ═ 12.6-17.4 months) (P ═ 039); the L allele has a dominant effect, with statistical significance as measured by the trend towards increased HRs in patients with increased L alleles (p.02); in summary, it can be concluded that the MNS16A genotype can be used as a biomarker for predicting cancer chemotherapy sensitivity, patients with SS genotype survive more frequently as chemotherapy-sensitive individuals, and patients with LL genotype and patients with SL genotype have shorter survival and higher risk of death as chemotherapy-insensitive individuals.

In some embodiments, the invention provides MNS16A genotype as a biomarker for predicting cancer chemotherapy sensitivity and application thereof, and develops a detection kit for predicting the chemotherapy sensitivity, so as to be used for accurately and individually judging the cancer chemotherapy curative effect and guiding the individual treatment to improve the prognosis of cancer patients.

In some embodiments, the invention provides the MNS16A genotype as a biomarker for predicting cancer chemotherapy sensitivity, the MNS16A genotype being classifiable as a functional genotype of LL, LS, and SS according to VNTR.

In some embodiments, the MNS16A genotype PCR-specific amplification primer pre-primer sequence is 5'-AGGATTCTGATCTGAAGGGTG-3' and the reverse primer sequence is 5 ' -TCTGCCTGAGGAGACGTATG-3.

In some embodiments, the invention provides use of the MNS16A genotype as a biomarker in the preparation of a kit for predicting cancer chemotherapy sensitivity, the kit comprising PCR-specific amplification primers for the MNS16A genotype, and auxiliary components such as PCR buffer, dNTP, magnesium chloride, DNA polymerase, deionized water, and the like.

Example 1

One embodiment of the kit for predicting cancer chemotherapy sensitivity of the invention comprises a PCR specific amplification primer of MNS16A genotype, and auxiliary components such as PCR buffer solution, dNTP, magnesium chloride, DNA polymerase, deionized water and the like.

Example 2

Data collection and arrangement of research samples

The collected samples are glioblastoma multiforme patients in primary malignant glioma patients, 299 patient samples meeting the standard are selected for subsequent research analysis by sorting clinical data, the diagnosis age of a research population ranges from 20 to 65 years, the average age is 49.9 years, all patients have complete case data (including diagnosis and treatment schemes, medical history and the like), the patients receive chemotherapy, the patients are all primarily diagnosed patients without tumor development history, and the patients do not receive tumor-related radiotherapy before admission.

Example 3

Patient DNA amplification and genotyping

(1) Extracting genome DNA:

taking a peripheral blood sample of the patient in example 2, obtaining a leukocyte precipitate from the peripheral blood sample by centrifugation, extracting genomic DNA from the leukocyte precipitate using a Qiagen DNA blood mini kit (Qiagen, Valencia, CA) according to the instructions, evaluating the DNA purity by electrophoresis in a 1% agarose gel, and determining the concentration of DNA by ultraviolet spectroscopy;

(2) MNS16A genotyping

Using a detection kit, firstly, specifically amplifying a DNA fragment containing the variable number of tandem repeat polymorphism of the MNS16A of the hTERT gene, wherein the primer sequences are as follows:

the sequence of the pre-primer is as follows: 5'-AGGATTCTGATCTGAAGGGTG-3', reverse primer sequence: 5'-TCTGCCTGAGGAGACGTATG-3', respectively;

the reaction system (10ul) contained: 1 XPCR buffer (50mmol/L KCl, 10mmol/L Tris-HCl, pH8.3), 40ng genomic DNA, 5pmol PCR primer, 1.5mmol/L magnesium chloride, 0.1mmol/L dNTP and 1U Taq polymerase. The amplification reaction is carried out on a PCR amplification instrument, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min, 35 cycles of 95 ℃ for 30 sec, 60 ℃ for 45 sec, 72 ℃ for 1 min, and 72 ℃ for 10 min;

after the PCR reaction is finished, carrying out DNA electrophoresis (0.25 mu g/mL ethidium bromide and 2% agarose gel) to detect a PCR product, wherein the result is shown in figure 1, the generated genotypes can be divided into different combinations of 243bp, 272bp, 302bp and 333bp, the DNA fragment of 243bp or 272bp is classified as an S allele, and the DNA fragment of 302bp or 333bp is classified as an L allele; the MNS16A genotype can be classified into the functional genotypes of LL, SL and SS according to VNTR, 10% of samples are randomly selected for repeated genotyping, and the result is completely consistent with the previous result.

Example 4

Statistical analysis of the correlations was performed in conjunction with the genotyping obtained in example 3 and the data relating to glioblastoma multiforme patients in example 2:

as can be seen from table 1, the distribution of MNS16A genotypes was not statistically significantly different in age, gender, or surgical treatment. However, the distribution of this genotype varies significantly with the length of survival, and the SS genotype survives longer than patients with the SL or LL genotype. It can also be seen from the Kaplan-Meier survival curves of the hTERT MNS16A genotype of FIG. 2 that the SS genotype survives longer than the SL or LL genotype patients; as shown in table 2, median survival was significantly different for patients with the different MNS16A genotypes, 25.1 months (95% CI ═ 14.9-30.9 months) for patients with the SS genotype, 14.7 months (95% CI ═ 13.9-18.8 months) for patients with the SL genotype, and 14.6 months (95% CI ═ 12.6-17.4 months) (P ═ 039) for patients with the LL genotype; as can be seen from the one-and multi-factor Cox proportional risk analysis of table 3, the mortality risk was increased for both SL and LL genotypes compared to SS genotype (HR adjusted 1.78; 95% CI 1.11-2.85), and further analysis revealed that L allele (SL genotype, HR adjusted 1.69; 95% CI 1.03-2.76; LL genotype, HR adjusted 1.87; 95% CI 1.15-3.03) had dominant effect, with statistically significant HRs increase trend test for patients with increased L allele (p.02); multivariate Cox proportional risk analysis also confirms that patients with SL or LL genotypes are at higher risk of death than patients with SS genotypes, and that the effect of this genetic variation on the telomerase gene is independent of risk factors such as age of diagnosis and surgical treatment; in summary, it can be concluded that the MNS16A genotype can be used as a biomarker for predicting cancer chemotherapy sensitivity, patients with SS genotype survive more frequently as chemotherapy-sensitive individuals, and patients with LL genotype and patients with SL genotype have shorter survival and higher risk of death as chemotherapy-insensitive individuals. Therefore, the method can predict the chemotherapy effect when the treatment scheme is customized, so that a more reasonable treatment scheme is provided, the treatment effective rate is greatly improved, the follow-up comprehensive treatment is guided, the ineffective chemotherapy is avoided, the unnecessary toxic and side effects are reduced, the economic waste is avoided, and the burden of a patient is relieved.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

TABLE 1 MNS16A genotype distribution of selected variables

TABLE 2 median survival for the entire patient group, ranked by selected variables

TABLE 3 Single factor and multivariate Cox proportional Risk analysis of the entire patient group

Claims

Use of the MNS16A genotype as a biomarker for predicting cancer chemotherapy sensitivity.
2. Use according to claim 1, characterized in that: the MNS16A genotype can be classified into functional genotypes of LL, SL and SS according to VNTR, when the individual genotype is SS, the individual is judged to be a chemotherapy-sensitive individual, and when the individual genotype is LL and SL, the individual is judged to be a chemotherapy-insensitive individual.
3. Use according to claim 2, characterized in that: the SS genotype is a homozygote with S alleles, the LL genotype is a homozygote with L alleles, the SL genotype is a heterozygote with S and L alleles, the S allele is a DNA fragment with 243bp or 272bp on a telomerase gene, and the L allele is a DNA fragment with 302bp or 333bp on the telomerase gene.
Use of the MNS16A genotype as a biomarker for the preparation of a kit for predicting cancer chemotherapy sensitivity.
5. Use according to claim 4, characterized in that: the kit comprises PCR specific amplification primers for detecting the MNS16A genotype.
6. Use according to claim 5, characterized in that: the sequence of the specific amplification primer is as follows: the sequence of the front primer was 5'-AGGATTCTGATCTGAAGGGTG-3' and the sequence of the reverse primer was 5'-TCTGCCTGAGGAGACGTATG-3'.
7. Use according to claim 5, characterized in that: the kit also comprises PCR buffer solution, dNTP, magnesium chloride, Taq polymerase and deionized water.