CN111575295A

CN111575295A - Human CNOT1 mutant gene and application thereof in liver cancer chemotherapy

Info

Publication number: CN111575295A
Application number: CN202010539085.6A
Authority: CN
Inventors: 陈艳; 冀丽娟; 李永柱
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-06-13
Filing date: 2020-06-13
Publication date: 2020-08-25

Abstract

The invention provides a g. [42415insATCA ] mutation form of a human CNOT1 gene, wherein the sequence of a fragment in which mutation is located is shown as SEQ ID NO: 1 is shown in the specification; hepatoma patients with g. [42415insATCA ] mutation in the CNOT1 gene were resistant to FOLFOX4 chemotherapy regimen. The CNOT1 mutant gene fragment in which g. [42415insATCA ] mutation occurs can be amplified and mutation detected using a kit containing 5'-CTTCTTGAAAAAGTAAGTTCTTA-3' and 5'-ATTCTAGTTTTGTCTACATCTC-3' primers and a 5 '-FAM-TTCAGAATCAGTTTA-MGB-NFQ-3' probe or a method for constructing digital PCR or real-time fluorescent quantitative PCR using the same. The g. [42415insATCA ] mutation form of the CNOT1 gene provided by the invention is not reported at present, and the mutation can be used as a gene marker of drug resistance of FOLFOX4 scheme chemotherapy of liver cancer, can be applied to prediction and evaluation of drug resistance, and has the advantages of accuracy, specificity, timeliness and effectiveness.

Description

Human CNOT1 mutant gene and application thereof in liver cancer chemotherapy

Technical Field

The invention belongs to the field of medical molecular biology, and particularly relates to a human CNOT1 mutant gene and application thereof in liver cancer FOLFOX4 scheme chemotherapy drug resistance evaluation.

Background

Liver cancer is the main cancer most frequently suffered and easily killed by Chinese people, according to the latest national cancer report (2019 edition) issued by the national cancer center, the incidence rate of liver cancer is ranked the fourth highest on malignant tumors, the death rate is the second highest on malignant tumors, and the current domestic new liver cancer cases and death cases account for more than half of the world. Early symptoms of liver cancer are not obvious, more than 60% of patients are diagnosed in middle and late stages, and the optimal chance of surgical treatment is lost. Chemotherapy is an important treatment mode for liver cancer in middle and late stages, however, liver cancer patients have different sensitivities to chemotherapeutic drugs, and the prognosis of the liver cancer patients resistant to chemotherapy is very poor; in addition, the cancer focus of the liver cancer has heterogeneity and is easy to generate new drug resistance mutation in the chemotherapy process, so secondary drug resistance often appears in the chemotherapy process, and the treatment scheme needs to be changed in time. At present, the response of liver cancer patients to chemotherapy drugs cannot be predicted and evaluated accurately in time clinically, so that most liver cancer chemotherapy patients continuously carry out experimental chemotherapy, the condition of the patients is delayed, and unnecessary drug toxic and side effects are borne. Therefore, it is important to effectively detect primary resistance before chemotherapy and to monitor whether secondary resistance occurs in time during chemotherapy. Currently, the clinical evaluation of the curative effect of liver cancer chemotherapy mainly depends on imaging means and serological indexes, however, certain lag often exists in the imaging means, the general specificity of serum markers (such as serum alpha-fetoprotein (AFP)) is not strong, and the two can not effectively and accurately reflect the molecular essence of cancer foci, so that the change trend of the cancer foci is difficult to analyze and predict, and the value of the therapy guidance is limited. Therefore, a need exists for finding specific and accurate molecular markers for liver cancer chemotherapy, predicting and evaluating the chemotherapy curative effect, and finding drug-resistant people and drug-resistant time nodes in time so as to effectively guide the selection and timely change of a chemotherapy scheme.

The FOLFOX4 chemotherapy scheme which is clinically used in the treatment of cancers at present and combines the advantages of common chemotherapeutics such as fluorouracil, oxaliplatin and the like is superior to the traditional chemotherapeutics (such as adriamycin) in terms of overall response rate, disease control rate, progression-free survival time and overall survival time, and has better tolerance and safety, so the FOLFOX4 scheme is recorded by the primary liver cancer diagnosis and treatment standard issued by the ministry of health of the original country since 2011 and is recommended to be used for treating the advanced primary liver cancer. The invention aims at primary liver cancer (hereinafter referred to as liver cancer) patients receiving FOLFOX4 chemotherapy treatment, blood samples before and during chemotherapy are collected, extracellular free DNA (cell-free DNA, namely cfDNA) of the patients is extracted, circulating tumor DNA (circulating tumor DNA, namely ctDNA) in the blood samples is analyzed by a secondary DNA sequencing technology to monitor the cancer focus mutation change information, and the results of the analysis of clinical efficacy evaluation information show that two specific mutations of a CNOT1 (CCR 4-NOT transcription complex mutation 1) gene and two specific mutations of NPAS2 (neural PAS domain protein 2) can be used as markers of liver cancer chemotherapy resistance of FOLFOX4 scheme, while the lack of effective chemotherapy efficacy and drug resistance specific evaluation molecular marker FOOX 4 scheme has a great difficulty in liver cancer chemotherapy, so that the primary liver cancer patients are difficult to realize precise chemotherapy.

The protein encoded by the CNOT1 gene is an important subunit component of a CCR4-NOT transcription complex, the main function of the CCR4-NOT complex is to inhibit the translation of messenger RNA by regulating the degradation of the messenger RNA as a de-adenylase, and the complex is also widely involved in transcription initiation, extension and protein modification, so that the protein has important effects on the growth and proliferation of cells, the CNOT1 protein is indispensable for the function of the CCR4-NOT complex, and the specific mutation of the CNOT1 gene can influence the function of the CCR4-NOT complex. The CCR4-NOT protein complex is highly conserved, and partial studies show that the function disorder of the complex is obviously related to the occurrence and development of tumors, but the association research of the complex in liver cancer is few, and only sporadic reports such as the discovery that the CNOT7 gene is related to the IFN-gamma tolerance of hepatocellular carcinoma in the Chonren research (Chonren. CNOT7 gene knockout research for reversing the IFN-gamma tolerance of hepatocellular carcinoma [ D ]. Shanxi: Shanxi university of medical science, 2017.). The invention discovers two specific mutations of CNOT1 gene for the first time, and applies the two specific mutations to the prediction and evaluation of liver cancer FOLFOX4 scheme chemotherapy resistance, which has significant meaning for the selection and timely change of a liver cancer patient chemotherapy scheme, especially the FOLFOX4 scheme, can effectively improve the benefit of primary liver cancer patient FOLFOX4 scheme chemotherapy, and avoid ineffective chemotherapy and unnecessary chemotherapy toxic and side effects; meanwhile, the method has important value for the research on the primary liver cancer FOLFOX4 scheme chemotherapy drug resistance mechanism and the research and development of effective chemotherapeutic drugs.

Disclosure of Invention

The invention provides two specific mutations of CNOT1 gene, a detection kit and a method thereof, wherein the two specific gene mutations can be used as molecular markers of primary liver cancer FOLFOX4 scheme chemotherapy resistance and applied to prediction and analysis of drug resistance.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the object (1) of the present invention is to provide a human CNOT1 mutant gene in which the g. [42415insATCA ] mutation occurs, which is formed by inserting 4 nucleotides of ATCA into the 42415 th site of the wild-type CNOT1 gene, and the sequence within the range of 58519951 and 58629826 of the sequence shown by NCBI accession number NC-000016.10 is the reference sequence of the CNOT1 wild-type gene, and the sequence of the fragment in which the g. [42415insATCA ] mutation occurs is shown as SEQ ID NO: 1, the rest sequences are referred to CNOT1 wild type gene reference sequence; hepatoma patients with g. [42415insATCA ] mutation in the CNOT1 gene were resistant to FOLFOX4 chemotherapy regimen. The CNOT1 gene with g [42415insATCA ] mutation can be applied to the development of a FOLFOX4 scheme chemotherapy drug resistance detection kit, a detection method and a therapeutic drug for liver cancer.

The object (2) of the present invention is to provide a human CNOT1 mutant gene in which a g. [42481_42482delAG ] mutation occurs, which is formed by deletion of 2 nucleotides of AG at the 42481-42482 site of the wild-type CNOT1 gene, and the sequence within the range of 58995511-58629826 of the sequence shown by NCBI accession number NC _000016.10 is the reference sequence of the CNOT1 wild-type gene, and the sequence of the fragment in which the g. [42481_42482delAG ] mutation occurs is as shown in SEQ ID NO: 2, the rest sequences are referred to CNOT1 wild type gene reference sequence; hepatoma patients with g [42481_42482delAG ] mutations in the CNOT1 gene were resistant to FOLFOX4 chemotherapy regimen. The CNOT1 gene with g [42481_42482delAG ] mutation can be applied to the development of a FOLFOX4 scheme chemotherapy drug resistance detection kit, a detection method and a therapeutic drug for liver cancer.

The invention aims to provide a detection primer and a probe for detecting g. [42415insATCA ] mutation of CNOT1 gene, wherein the sequence of the fragment of g. [42415insATCA ] mutation of CNOT1 gene is shown as SEQ ID NO: 1, the rest sequences are referred to CNOT1 wild type gene reference sequence; the detection primers are 5'-CTTCTTGAAAAAGTAAGTTCTTA-3' and 5'-ATTCTAGTTTTGTCTACATCTC-3', and the detection probe is 5 '-FAM-TTCAGAATCAGTTTA-MGB-NFQ-3'; the detection primers and the probes can be used for constructing a CNOT1 gene g. [42415insATCA ] mutation detection kit and a detection method, and are particularly used for constructing a CNOT1 gene g. [42415insATCA ] mutation detection method based on digital PCR or real-time fluorescence quantitative PCR.

The object (4) of the present invention is to provide detection primers and probes for detecting g. [42481_42482delAG ] mutation in CNOT1 gene, wherein the sequence of the fragment in which g. [42481_42482delAG ] mutation in CNOT1 gene is as shown in SEQ ID NO: 2, the rest sequences are referred to CNOT1 wild type gene reference sequence; the detection primers are 5'-CTTCTTGAAAAAGTAAGTTCTTA-3' and 5'-ATTCTAGTTTTGTCTACATCTC-3', and the detection probe is 5 '-VIC-GTACAAGGTGAGTGA-MGB-NFQ-3'; the detection primers and the probes can be used for constructing a CNOT1 gene g. [42481_42482delAG ] mutation detection kit and a detection method, and are particularly used for constructing a CNOT1 gene g. [42481_42482delAG ] mutation detection method based on digital PCR or real-time fluorescence quantitative PCR.

Two mutation forms of g. [42415insATCA ] and g. [42481_42482delAG ] of the CNOT1 gene provided by the invention are not reported at present, and the two mutations can be used as gene markers of drug resistance of FOLFOX4 scheme chemotherapy of liver cancer, so that the CNOT1 gene can be applied to prediction and evaluation of drug resistance and has the advantages of accuracy, specificity, timeliness and effectiveness; in addition, primers and probes for specific detection of mutations are provided. The method has obvious significance for the selection and timely change of a chemotherapy scheme of a liver cancer patient, particularly a FOLFOX4 scheme, can effectively improve the benefit of the FOLFOX4 scheme chemotherapy of the liver cancer patient, and avoids ineffective chemotherapy and unnecessary toxic and side effects of chemotherapy; meanwhile, the method has important value for the research on the drug resistance mechanism of FOLFOX4 scheme chemotherapy of the liver cancer and the research and development of effective chemotherapeutic drugs.

Drawings

FIG. 1 is a diagram showing the specific mutation in exon 5 of the human CNOT1 gene. The middle white box represents exon 5, and the two black boxes represent introns; the left and right arrows point to the occurrence of the g. [42415insATCA ] mutation and g. [42481_42482delAG ] mutation, respectively.

Detailed Description

The resistance of the current clinical chemotherapy scheme mainly depends on the evaluation on imaging, and the chemotherapy effect is reflected by the change of the size, the size and the position of a cancer focus. In fact, cancer patients contain circulating tumor DNA (ctDNA) released from cancer foci in their blood, and more studies have confirmed that genetic mutation information of cancer foci, which is an intrinsic driving factor of the development and change of cancer foci, can be reflected by analyzing ctDNA. Therefore, by dynamic analysis and comparative analysis of blood sample ctDNA in the chemotherapy process of liver cancer FOLFOX4 chemotherapy regimen resistance and beneficial patient population, and combined with clinical curative effect evaluation, the FOLFOX4 chemotherapy regimen resistance gene marker can be researched.

The invention is further illustrated below with reference to specific examples.

Example 1 screening of liver cancer FOLFOX4 protocol chemotherapy drug resistance Gene markers

(1) Basic information of patients in group

15 cases of primary liver cancer patients diagnosed by clinical pathology in the period from 2017, 1 month to 2017, 12 months are selected, and are treated by adopting a chemotherapy scheme of FOLFOX4 (no intervention treatment, biological treatment and other treatment in the period), and the patient numbers, basic information and chemotherapy curative effect evaluation information are shown in table 1, wherein patients 1-5 are primary drug resistant groups (chemotherapy ineffective groups), patients 6-10 are secondary drug resistant groups (chemotherapy ineffective groups), and patients 11-15 are non-drug resistant groups (chemotherapy beneficial groups). The primary drug resistant group shows continuous progress without remission in the chemotherapy process, and usually has chemotherapy drug resistant mutation due to the existence of cancer focus before chemotherapy; the secondary drug resistance group means that the early stage of chemotherapy is remission, and the later stage of chemotherapy is changed into progress, and usually because a cancer focus has chemotherapy drug resistance mutation in the chemotherapy process; the non-drug resistant group means that only remission and no progress occur in the equivalent process of chemotherapy, and usually the reason is that a cancer focus does not exist before chemotherapy and the cancer focus does not have chemotherapy drug resistant mutation in the chemotherapy process.

The FOLFOX4 chemotherapy reference protocol was: adding 250ml of 5% glucose injectionOxaliplatin 85mg/m²2h d1 intravenous drip, 250ml of normal saline is added with 200mg/m calcium folinate²2h d1-2, Fluorouracil (5-FU) 400mg/m for intravenous drip²D1-2, 5-FU 600mg/m for intravenous injection²Continuous intravenous pumping 22h d1-2, 14d for 1 chemotherapy cycle. The patient is subjected to not less than 2 cycles of chemotherapy each time and continues until the patient either develops disease progression or an intolerable toxic response occurs. The patients are evaluated for curative effect and adverse reaction after every 1 or 2 chemotherapy cycles, and 10-20ml of blood samples are extracted at the same time (on the premise that the patients are fully informed and meet clinical diagnosis and treatment requirements).

The size of the tumor before and after chemotherapy is detected according to means such as CT or MRI and the like, the maximum diameter of the tumor body is determined, and the recent curative effect evaluation standard adopts the RECIST 1.1 standard of 2009 edition. Complete Remission (CR): all target lesions disappear; partial Remission (PR): the sum of the longest diameters of the target lesions is reduced by at least 30% compared with the baseline; progression (PD): the sum of the longest diameter of the target lesion is compared with the smallest sum of the longest diameter of the target lesion recorded after the treatment is started, the relative increase is 20 percent, and the absolute value of the sum is increased by at least 5mm or a new lesion appears; stable (SD): there was either a reduction in baseline lesion length but no PR or an increase but no PD. The treatment effect evaluation is based on the previous cancer focus or the cancer focus before treatment, and the patient is continuously followed during and after the treatment process.

Table 115 basic information of primary liver cancer chemotherapy patients and FOLFOX4 protocol chemotherapy curative effect evaluation information

Note: data representing no efficacy assessment without chemotherapy.

(2) Capture sequencing of ctDNA

Before chemotherapy and on the day of evaluation of curative effect of each chemotherapy, 10ml-20ml of venous anticoagulation blood of a patient is collected at the same time, and plasma and mononuclear cells are separated within 2 hours, wherein a plasma sample contains ctDNA which can reflect the information of cancer focus gene mutation, and the mononuclear cells contain normal genome DNA which can be used for eliminating individual gene polymorphism. DNA samples were sent to wara genes on dry ice for whole exome capture and second generation DNA sequencing. Whole Exome Sequencing (WES) refers to a genome analysis method of high-throughput Sequencing after capturing and enriching exome region DNA of a Whole genome using a probe capture technology. Given the high cost of second-generation DNA sequencing, each patient sent only one blood sample (corresponding to the bold-labeled node in table 1) for whole exome sequencing, with the refractory drug sent the blood sample for the last clinical assessment of progress, i.e., PD, and the non-refractory drug sent the blood sample collected at the end of the first chemotherapy.

Blood sample ctDNA and genome DNA are respectively extracted by using a QIAamp Circulating Nucleic Acid Kit and a QIAamp DNAafter Mini Kit of Germany Qiagen company, and are captured by using a SureSelect Human All Exon V6+ UTR whole exome liquid phase capture chip (75 Mb in the capture Region of Agilent company in the United states), wherein the chip is a liquid phase targeting sequence capture system based on oligonucleotide synthesis technology, provides higher sequencing efficiency, and completely covers up-to-date gene databases including RefSeq, CCDS, GENCODE, miRBase, TCGA and UCSC, the exome Region and a non-translated Region (Untranstrained Region) therein, ensures high capture uniformity, and can effectively reduce sequencing cost.

The Hiseq4000 platform of Illumnia company in USA is adopted for sequencing, the average sequencing depth requirement of a normal genome DNA sample from a monocyte is more than 100 times, and the average sequencing depth requirement of a plasma ctDNA sample is more than 1000 times. Adopting Hiseq4000 matched analysis software to carry out preliminary analysis on a sequencing result, mainly filtering out adapters, low-quality bases and undetected bases from original off-line data, then adopting sequence comparison software BWA to compare an original sequencing sequence of each sample with a human reference genome sequence (hg 19), adopting a Samtools software package to position and compare the compared reads sequence in a genome, adopting variation detection software Varscan2 to carry out variation detection on the sample, and adopting a variation annotation tool Annovar to carry out annotation; and taking the sequencing result of the normal genome DNA sample of the blood cells as a reference, and then filtering the polymorphic sites of the population by using a thousand human genome database, an SNP database, a COSMIC (version 64) database and the like to finally obtain the mutation site information (including SNP, InDel, CNV and the like) of the ctDNA sample of the blood plasma.

(3) Comprehensive analysis of results

The second generation DNA sequencing results of 15 blood samples showed that the mean sequencing depth of ctDNA was 1214X. In total, 46 mutations were detected in all samples with a content of more than 1%, belonging to 17 different genes including TP53, CYP1a1, axi, ALDH2, CNOT1, NPAS2, etc. The search for drug-resistance related gene markers focuses on high-frequency gene mutation and gene mutation with difference in drug-resistant groups and non-drug-resistant groups. Further analysis combined with clinical information revealed that 2 high-frequency mutant genes, namely the CNOT1 gene and the NPAS2 gene, were detected only in the drug-resistant group (including the primary drug-resistant group and the secondary drug-resistant group) and not in the non-drug-resistant group, and the results are shown in Table 2 (the mutations were confirmed by Sanger sequencing). The samples of 6 patients No. 1, No. 2, No. 5, No. 6, No. 8 and No. 10 contained specific mutation of CNOT1 gene, and 4 patients No. 1, No. 2, No. 6 and No. 8 contained g. [42415insATCA ] mutation of CNOT1 gene, while 2 patients No. 5 and No. 10 contained g. [42481_42482delAG ] mutation of CNOT1 gene, and both mutations were located in the No. 5 exon (see FIG. 1). The sequence within the range of 58519951-58629826 in the sequence shown by NCBI accession number NC-000016.10 is the reference sequence (109876 bp in length) of the CNOT1 wild-type gene, and the start site of the CNOT1 gene is 1 (i.e., the 58519951-position of the sequence shown by NC-000016.10), then the g. [42415insATCA ] mutation of the CNOT1 gene indicates that 4 nucleotides ATCA are inserted into the 42415-position thereof, and the g. [42481_42482delAG ] mutation of the CNOT1 gene indicates that two nucleotides, namely AG, 42481-42482-position thereof, are deleted. Further, the g. [42415 instca ] and g. [42481_42482delAG ] mutations of the CNOT1 gene were verified by Sanger sequencing, wherein the g. [42415 instca ] mutation of the CNOT1 gene is in the sequence as set forth in SEQ ID NO: 1 (ATCA insertion mutation is at positions 415-418 of the sequence of SEQ ID NO: 1, the rest of the sequence is referred to as CNOT1 wild-type gene reference sequence), and the sequence of the g. [42481_42482delAG ] mutation is shown as SEQ ID NO: 2 (the AG deletion mutation is originally located at positions 481-482 of the sequence of SEQ ID NO: 2 before deletion, and the rest of the sequence is referred to as the CNOT1 wild-type gene reference sequence).

In addition to the CNOT1 gene mutation, 4 patients 3, 4, 7 and 9 contained the specific mutation of NPAS2 gene, of which 2 patients 3 and 7 contained the g. [ 105009-105012 delGCTT ] mutation of NPAS2 gene, and 2 patients 4 and 9 contained the g. [105054insGCCTA ] mutation of NPAS2 gene (the sequence in the range of 4989-181679 in the sequence with NCBI accession NG _023259.1 was the reference sequence of NPAS2 wild-type gene, and the length was 176691 bp).

TABLE 2 liver cancer FOLFOX4 chemotherapy regimen drug-resistant group specific high-frequency gene mutation information

Patient numbering	Mutant genes	Mutation site	Content of mutation
				1	CNOT1	g.[42415insATCA]	12.3%
2	CNOT1	g.[42415insATCA]	15.5%
				3	NPAS2	g.[105009_105012delGCTT]	20.4%
4	NPAS2	g.[105054insGCCTA]	13.5%
				5	CNOT1	g.[42481_42482delAG]	8.1%
6	CNOT1	g.[42415insATCA]	4.7%
				7	NPAS2	g.[105009_105012delGCTT]	9.4%
8	CNOT1	g.[42415insATCA]	11.9%
				9	NPAS2	g.[105054insGCCTA]	6.3%
10	CNOT1	g.[42481_42482delAG]	12.8%

From the above analysis, the specific mutations of CNOT1 and NPAS2 genes are specific to patients with liver cancer treated by FOLFOX4 chemotherapy-resistant drug combination, but are not detected in patients with non-drug-resistant drug combination. In addition, the CNOT1 and NPAS2 gene specific mutations exist in patients with primary drug resistance group and are detected in drug resistance node samples of patients with secondary drug resistance group after chemotherapy, and the patients are likely to have drug resistance caused by the CNOT1 and NPAS2 gene specific mutations in the process of chemotherapy. Therefore, preliminary results show that specific mutations of the CNOT1 and NPAS2 genes are related to drug resistance of liver cancer FOLFOX4 scheme chemotherapy, and the effectiveness and specificity of the specific mutations serving as drug resistance gene markers need to be further verified.

Example 2 validation of markers for liver cancer FOLFOX4 protocol chemotherapy resistance genes

If the specific mutations of the CNOT1 and NPAS2 genes are related to primary liver cancer FOLFOX4 chemotherapy resistance, the blood samples collected in the chemotherapy process of the liver cancer patients in example 1 can be used for analyzing the dynamic change information thereof, and clinical efficacy evaluation information is combined to deeply verify and reveal the specific association between the two genes. Blood ctDNA samples of 15 primary liver cancer patients before and during chemotherapy as shown in Table 1 were extracted by the method described in example 1. Specific mutations of CNOT1 and NPAS2 genes, specifically g. [42415insATCA ] mutation and g. [42481_42482delAG ] mutation of CNOT1 gene, and g. [233_238delCAGTCC ] mutation and g. [105096insAATCA ] mutation of NPAS2 gene, were subjected to quantitative analysis by Digital PCR (Quantstrudio 3D Digital PCRsystem of Life Tech company, USA, and 20K chip kit V2 kit) according to the instruction method by designing special primers and probes (PrimerV 5.0 software). All samples were tested for copy number of all 4 specific mutations of the CNOT1 and NPAS2 genes. Since the g. [42415insATCA ] mutation and g. [42481_42482delAG ] mutation of the CNOT1 gene are located in exon 5 of the gene at short intervals (less than 70bp, see FIG. 1), the same amplification primers were used, and the amplification primer probes, systems and conditions thereof are shown in Table 3, wherein the primer concentration is 0.4. mu. mol/l and the probe concentration is 0.2. mu. mol/l. Primers and probes are also designed, the NPAS2 gene mutation is detected by adopting a consistent method, the analysis of the final result shows that the gene mutation is also related to the drug resistance of FOLFOX4 scheme chemotherapy of liver cancer, and the related result is not shown in the situation of treatment of another scheme.

TABLE 3 amplification information of two specific mutations of the CNOT1 Gene

Note: FAM and VIC are fluorescence labeling groups, are modified on the 5' end base of the probe and are used for fluorescence detection of amplified fragments; NFQ is a non-fluorescence quenching group of ABI patent, and can absorb fluorescence emitted by FAM or VIC when used in a probe state; the MGB is a DNA minor groove binder, is beneficial to the combination of the probe and the amplification template, and improves the Tm value of the probe and the amplification template.

The dPCR detection results of the CNOT1 gene mutation show (see table 4) that only 6 patients, No. 1, No. 2, No. 5, No. 6, No. 8 and No. 10, contained the specific mutation of the CNOT1 gene, wherein all the 4 patient samples, No. 1, No. 2, No. 6 and No. 8, detected the g. [42415insATCA ] mutation of the CNOT1 gene, and all the 2 patient samples, No. 5 and No. 10, detected the g. [42481 — 42dell ag ] mutation of the CNOT cn 1 gene, and all the samples of the rest patients did not detect the two specific mutations of the CNOT1 gene, which is consistent with the results of the second-generation DNA sequencing in example 1, and can be mutually confirmed.

Two specific mutations of the CNOT1 gene were detected only in the drug-resistant group patients, and both the primary drug-resistant patients (patients No. 1, 2, and 5) and the secondary drug-resistant patients (patients No. 6, 8, and 10) contained the two specific mutations of the CNOT1 gene, which were not detected in the non-drug-resistant group patient samples. Further analysis revealed that the CNOT1 gene mutation was detected in the primary drug resistant group before chemotherapy, specifically 17.3 copies/. mu.l in patient No. 1, 87.0 copies/. mu.l in patient No. 2, and 56.7 copies/. mu.l in patient No. 5 (Table 4). In addition, the content of the specific mutation of the CNOT1 gene detected is increased gradually along with the disease progress in the chemotherapy process of the patients with primary drug resistance group, which is consistent with the trend of clinical disease change, so that the CNOT1 gene can be used as a drug resistance gene marker for the selection and change of the FOLFOX4 chemotherapy scheme, and is more timely and accurate compared with the conventional curative effect evaluation mode.

Taking patient No. 1 as an example, the curative effect of FOLFOX4 regimen chemotherapy cannot be predicted before routine clinical chemotherapy, and the g. [42415INSATCA ] mutation concentration of CNOT1 gene before chemotherapy is detected to be 17.3 copies/mul, so that the patient can be judged to be drug-resistant, namely, FOLFOX4 regimen chemotherapy is ineffective for the patient. Further, patient 1 evaluated as SD with the first chemotherapy showed no significant increase in cancer foci, and it was still difficult to clinically determine the efficacy of the FOLFOX4 regimen and predict the subsequent trend of the cancer foci, while the concentration of the mutant fragment rapidly increased to 67.6copies/μ l, and the clinical efficacy evaluated as SD was not effective with the FOLFOX4 regimen. If PD had been found to progress at the end of the second chemotherapy, the mutant concentration had continued to increase to 106.2 copies/. mu.l. The results of the analyses were similar for patients 2 and 5.

For patients with secondary drug resistance (patients No. 6, No. 8 and No. 10), no specific mutation of the CNOT1 gene is detected before chemotherapy, but during chemotherapy, and the content of the specific mutation of the CNOT1 gene is gradually increased along with the disease progress during the chemotherapy of the patients with secondary drug resistance, which is consistent with the trend of clinical disease change, so that the CNOT1 gene can be used as a drug resistance gene marker for selecting and changing FOLFOX4 chemotherapy schemes, and is more timely and accurate compared with the conventional curative effect evaluation mode. In the case of patient No. 10, no specific mutation of the CNOT1 gene was detected in the blood samples before and after chemotherapy, and the evaluation of clinical efficacy was PR, i.e., partial remission. Although the 3 rd clinical efficacy was evaluated as PR, the g. [42415 instca ] mutant fragment of CNOT1 gene was detected to be 5.4copies/μ l, at which time it could be predicted that a drug resistance mutation had occurred, i.e., the FOLFOX4 regimen had failed to work for the patient; however, at this time (third chemotherapy, evaluated as PR) conventional clinical trials have not been able to ascertain and predict the subsequent efficacy and chemotherapy continues to be performed using the original regimen. If the SD after the 4 th chemotherapy is changed to the PD of the 5 th and 6 th chemotherapy, the detected concentration of the mutant fragment in the process is continuously increased to 345.2 copies/mu l. The results of the analyses were similar for patients No. 6 and No. 8.

TABLE 4 dPCR detection results of CNOT1 Gene mutation

Note: the concentration units are copies/. mu.l, i.e.copy number/. mu.l.

From the above, g. [42415insATCA ] and g. [42481_42482delAG ] mutations of the CNOT1 gene can be used as drug resistance gene markers to be applied to selection and change of FOLFOX4 chemotherapy schemes of primary liver cancer patients, and have the advantages of timeliness, accuracy, specificity and effectiveness compared with a conventional chemotherapy effect evaluation mode.

The CCR4-NOT complex is a multi-subunit protein complex that is conserved in eukaryotes, is widely involved in transcription initiation, elongation, and protein modification, and thus has important roles in cell growth and proliferation. The NOT1 protein is one of the components of the CCR4-NOT complex, and the research shows that the CNOT1 of the human is composed of three regions of an N end (NOT 1-N), a middle (NOT 1-m) and a C end (NOT 1-C), wherein the C end (NOT 1-C) is a homologous conserved region of NOT 1. In addition, CNOT1 may also serve as a scaffold protein, providing binding sites for the catalytic module and other subunits of the complex, and CNOT1 as a scaffold complex, and also have many different cellular functions. The CNOT1 protein is indispensable for the function of a CCR4-NOT complex, and specific mutation of the CNOT1 gene can influence the function of the CCR4-NOT complex, so that the growth and the proliferation of cells are influenced. The g. [42415insATCA ] and g. [42481_42482delAG ] mutations of the CNOT1 gene both occur in the exon 5 of the gene, specifically in the junction region of the exon and the intron (on the exon side, see fig. 1), relate to the change of AG structure, and are compared with the corresponding amplified fragments of mRNA and DNA, Sanger sequencing results show that the occurrence of the two mutations both cause the variable shearing of the CNOT1 gene, and influence the protein translation and functional exertion of the gene.

Example 3 application value of liver cancer FOLFOX4 protocol chemotherapy drug resistance gene marker

The sample size is further enlarged to verify the accuracy and specificity of the CNOT1 gene specific mutation for evaluating the drug resistance of FOLFOX4 scheme chemotherapy of the liver cancer and the application value of the drug resistance. 50 cases of clinically and pathologically confirmed primary liver cancer patients from 2018 to 2019 are treated by FOLFOX 4in a chemotherapy scheme (in which no intervention treatment, biological treatment and the like are performed). 10-20ml of venous anticoagulation was collected before and at the time of evaluation of the effect of chemotherapy, and the g. [42415 instca ] and g. [42481_42482delAG ] mutation concentrations of CNOT1 gene in blood samples were determined in accordance with the method in example 2, and the results are shown in table 5. Judging the drug resistance through two specific mutations of CNOT1 gene, if the mutation is detected before chemotherapy and the blood sample concentration is not less than 5 copies/mul, judging the drug resistance as primary drug resistance; if the mutation is detected at a certain time in the chemotherapy process and the blood sample concentration is not lower than 5 copies/mu l, the secondary drug resistance is judged. For the determination of drug resistance by conventional clinical methods, see example 1.

The data in table 5 show that 11 patients before chemotherapy had two specific mutations in the CNOT1 gene detected and blood sample concentrations both exceeded 5copies/μ l (7 of g. [42415 instca ] mutations and 4 of g. [42481_42482delAG ] mutations), and these 11 patients were considered primary drug resistance by clinical efficacy assessment, whereas the results based on gene detection determinations were on average 2.3 months earlier than conventional clinical efficacy assessments, with a clear difference. Two specific mutations of the CNOT1 gene were not detected in the early stage of 15 patients in the chemotherapy process until two specific mutations of the CNOT1 gene were detected suddenly at one time (wherein 10 g. [42415INSATCA ] mutations and 5 g. [42481_42482delAG ] mutations) in the 15 patients were identified as secondary drug resistance according to the clinical efficacy evaluation, but the results determined based on the gene detection were 4.7 months ahead of the conventional clinical efficacy evaluation (the mutations were detected at the evaluation node before the clinical progress), and the two were very different. Therefore, the detection of g. [42415insATCA ] mutation and g. [42481_42482delAG ] mutation of the CNOT1 gene indicates that the FOLFOX4 scheme chemotherapy of liver cancer can appear or already appear drug resistance, and the mutation can be used as a gene marker of the drug resistance.

TABLE 5 analysis of specific mutation in CNOT1 Gene and its concordance with clinical efficacy assessment

Mutation detection node	Number of mutation detection cases	Clinical efficacy assessment, number of cases and consistency	Mean time earlier for gene detection than for clinical efficacy assessment
				Before chemotherapy	11 examples of	Primary drug resistance, 11 cases, 100%	2.3 months
In the course of chemotherapy	15 examples of	Secondary drug resistance, 15 cases, 100%	4.7 months

In conclusion, the invention firstly discloses two new specific mutations of the CNOT1 gene, namely g. [42415insATCA ] mutation and g. [42481_42482delAG ] mutation, and the detection of the two mutations indicates that the FOLFOX4 scheme chemotherapy of the liver cancer can appear or has appeared to be resistant to drugs, so that the method can be applied to the prediction and evaluation of the resistance of the FOLFOX4 scheme chemotherapy of the liver cancer, and has the advantages of accuracy, specificity and timeliness and effectiveness. The method has obvious significance for the selection and timely change of a chemotherapy scheme of a liver cancer patient, particularly a FOLFOX4 scheme, can effectively improve the benefit of the FOLFOX4 scheme chemotherapy of the liver cancer patient, and avoids ineffective chemotherapy and unnecessary toxic and side effects of chemotherapy; meanwhile, the method has important value for the research on the drug resistance mechanism of FOLFOX4 scheme chemotherapy of the liver cancer and the research and development of effective chemotherapeutic drugs.

Sequence listing

<110> exhibition

<120> human CNOT1 mutant gene and application thereof in liver cancer chemotherapy

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>914

<212>DNA

<213> Intelligent (Homo sapiens)

<400>1

tctcgacgct gtcctatgcc attgataatc cattgcacta tcagaaggtt ggtattacta 60

ttatctttaa gagtgtgatc tctccttagg ctttctaaaa agtccctaag ccttgatctc 120

ataatgtgat gtcttaatat aaaaatttca ggttgttatt ttggggtgtc ttttatagtt 180

tgtaacaaaa tcaaggcagt tcatttaata ataaatagaa tcttactaaa accagatcat 240

gtgaaggctt aagtttgtca tttcacagtt ttaggacata gtgtaatgtt tctagtaaca 300

ctacagaatt aagcaactcc tatgtagtgc catccttggg caaacagata ggttgttaaa 360

aacttcttga aaaagtaagt tcttatttta aactaaaatt tggtttgttt cagaatcagt 420

ttaaagcctg caccccactt atttgcccag ctgagtaaag tgctcaaatt aagcaaagta 480

caagaggtga gtgatttatg aaaagagatg tagacaaaac tagaattaaa aacagccatt 540

tgactctttg gttaatctaa tccttcttgt ttcaggtaat ttttggcctt gccctgttga 600

attcttccag ctcagatctt agaggtttcg gtgagttact ttttccaaaa atatcacgaa 660

gtgagacttt aaaatacgtg ggtttttaat catgaggctc tgtgatttag acttagagag 720

taagataatc tgaaattaat ttctctttat ctgtggtaaa aaacatgtaa tataaatgtt 780

accctcataa ccactttaag tgtacagttc catagggata attgtattca ggttgtgtaa 840

catctctctg gaactttatt ttgccaaaca gaactatgcc catcaacaac tctctatttt 900

cccctcccca ctgc 914

<210>2

<211>908

<212>DNA

<213> Intelligent (Homo sapiens)

<400>2

tctcgacgct gtcctatgcc attgataatc cattgcacta tcagaaggtt ggtattacta 60

ttatctttaa gagtgtgatc tctccttagg ctttctaaaa agtccctaag ccttgatctc 120

ataatgtgat gtcttaatat aaaaatttca ggttgttatt ttggggtgtc ttttatagtt 180

tgtaacaaaa tcaaggcagt tcatttaata ataaatagaa tcttactaaa accagatcat 240

gtgaaggctt aagtttgtca tttcacagtt ttaggacata gtgtaatgtt tctagtaaca 300

ctacagaatt aagcaactcc tatgtagtgc catccttggg caaacagata ggttgttaaa 360

aacttcttga aaaagtaagt tcttatttta aactaaaatt tggtttgttt cagagtttaa 420

agcctgcacc ccacttattt gcccagctga gtaaagtgct caaattaagc aaagtacaag 480

gtgagtgatt tatgaaaaga gatgtagaca aaactagaat taaaaacagc catttgactc 540

tttggttaat ctaatccttc ttgtttcagg taatttttgg ccttgccctg ttgaattctt 600

ccagctcaga tcttagaggt ttcggtgagt tactttttcc aaaaatatca cgaagtgaga 660

ctttaaaata cgtgggtttt taatcatgag gctctgtgat ttagacttag agagtaagat 720

aatctgaaat taatttctct ttatctgtgg taaaaaacat gtaatataaa tgttaccctc 780

ataaccactt taagtgtaca gttccatagg gataattgta ttcaggttgt gtaacatctc 840

tctggaactt tattttgcca aacagaacta tgcccatcaa caactctcta ttttcccctc 900

cccactgc 908

Claims

1. A human CNOT1 mutant gene is characterized in that the CNOT1 mutant gene is formed by inserting 4 nucleotides of ATCA into the 42415 th site of a wild-type CNOT1 gene, namely, a g. [42415insATCA ] mutation occurs, a sequence within the range of 58519951 and 58629826 of a sequence shown by an NCBI accession number NC-000016.10 is a reference sequence of the CNOT1 wild-type gene, and the sequence of a fragment in which the g. [42415insATCA ] mutation occurs is as shown in SEQ ID NO: 1, the rest sequences are referred to CNOT1 wild type gene reference sequence; hepatoma patients with g. [42415insATCA ] mutation in the CNOT1 gene were resistant to FOLFOX4 chemotherapy regimen.

2. The mRNA and encoded protein corresponding to the mutant gene of human CNOT1 according to claim 1.

3. The use of the human CNOT1 mutant gene according to claim 1, characterized in that the CNOT1 mutant gene is used in liver cancer FOLFOX4 scheme chemotherapy resistance detection kit, detection method and therapeutic drug development.

4. The kit for detecting a mutant gene in human CNOT1 as claimed in claim 1, wherein the kit comprises 5'-CTTCTTGAAAAAGTAAGTTCTTA-3' and 5'-ATTCTAGTTTTGTCTACATCTC-3' primers and 5 '-FAM-TTCAGAATCAGTTTA-MGB-NFQ-3' probe.

5. The method for detecting human CNOT1 mutant gene as claimed in claim 1, wherein the CNOT1 mutant gene fragment with g. [42415insATCA ] mutation can be amplified and mutation detected by using 5'-CTTCTTGAAAAAGTAAGTTCTTA-3' and 5'-ATTCTAGTTTTGTCTACATCTC-3' primers and 5 '-FAM-TTCAGAATCAGTTTA-MGB-NFQ-3' probe to construct a digital PCR or real-time fluorescent quantitative PCR-based method.