CN101041854A - Polymorphism sites of primary liver cancer correlative coding small molecule RNA and usage thereof - Google Patents

Abstract

The invention discloses a nonencode small molecule ribonucleic acid hsa-mir-146a correlated with primary liver cancer and polymorphism site, which is characterized by the following: providing a method to analyze mononucleotide polymorphism in has-mir-146a fore-body, check polymorphism and relate to work efficiency of ripe body; possessing usage in aspect of pre-treat primary liver cancer.

Description

A polymorphic site of primary liver cancer-associated non-coding small molecule RNA and its application

technical field

The present invention relates to a non-coding small molecule RNA hsa-miR-146a related to primary liver cancer and a single nucleotide polymorphism site on its precursor, and also relates to a method for detecting the polymorphic site point, the method of analyzing its correlation with the processing efficiency of the small molecule RNA, and the use of the polymorphism on the hsa-miR-146a precursor in the prediction of primary liver cancer.

Background technique

Malignant tumors have become one of the main causes of human death, among which primary liver cancer ranks sixth in incidence and third in mortality among all tumors. Liver cancer ranks the second among various cancers within the city limits of our country (Zeng Yixin, Oncology (Second Edition). People's Medical Publishing House, Beijing, 2003, 5-6). The occurrence of malignant tumor is a multi-factor and multi-step process, in which the pathogenesis of genetic factors plays an important role. Single nucleotide polymorphism (SNP) refers to a single nucleotide sequence change at a specific position on the genome in a certain species. It has a high frequency in the human genome and is widely used as a genetic marker ( Collins, F.S., et al. A DNA polymorphism discovery resource for research on human genetic variation, GenomeRes, 1998, 8(12), 1229-31). More and more evidence shows that the SNPs in the protein coding sequence and its upstream and downstream regulatory sequences in protein coding genes not only play an important role in regulating gene expression and function, but also affect the progression of diseases including malignant tumors. occurrence, development and prognosis. At present, there have been many reports on the correlation between this type of SNP and cancer. Using SNP sites as markers for auxiliary diagnosis and treatment of diseases will be an important reference index for realizing personalized treatment of diseases.

MicroRNA (microRNA) is a class of small molecule non-coding ribonucleic acid widely present in various biological species, and its length is about 20-22 bases. It does not encode proteins or polypeptides, but promotes its degradation or inhibits its translation process by specifically binding to the 3'UTR (3' untranslated region) of the target gene's mRNA (messenger RNA) to achieve gene expression. regulation. Many evidences show that microRNA is involved in various biological processes of cells, and the abnormality of its own regulation directly affects the pathogenesis and progression of malignant tumors. Due to the close relationship between microRNA and tumorigenesis, the SNP sites in their genome sequences are likely to regulate their expression or processing, and thus are related to the pathogenesis of cancer.

After searching the domestic and foreign literature of the prior art, there has not been any research report on polymorphic sites and diseases in the microRNA molecular sequence, and there is no polymorphic site in the hsa-miR-146a molecule in the microRNA family disclosed by the present invention. Morphological loci have been associated with various diseases, including primary liver cancer.

Contents of the invention

The purpose of the present invention is to reveal the correlation between a single nucleotide polymorphism site on the non-coding small molecule RNA hsa-miR-146a precursor, the processing efficiency of the small molecule, and the susceptibility to primary liver cancer, as well as its Use in the prediction of primary liver cancer.

First, the present invention provides a method for detecting single nucleotide polymorphisms on the hsa-miR-146a molecular precursor.

Specific steps are as follows:

(a) Determine the single nucleotide polymorphism (number rs2910164) present in the non-coding small molecule RNA hsa-miR-146a precursor, that is, the 460th nucleotide in the sequence of SEQ ID NO: 1 (marked as N), the two polymorphisms are G or C respectively;

(b) using polymerase chain reaction to specifically amplify the genomic DNA fragment comprising the hsa-miR-146a precursor sequence;

(c) Restriction fragment length polymorphism (RFLP) method was used to genotype the polymorphic site in the amplified DNA fragment containing the hsa-miR-146a precursor.

SEQ ID NO: 1

1 CCCCTACAGA TTAGTTTTTTG TTTTGACAGG GTCTCTCTCT GTGGCCCAGA

51 CTGGAGTGCA GTGGTGCAAT CATAGCTCAC TGCAACCTCC AATTCCCAGG

101 CTCAAGCGAT CCTCCCACCA CAGGCCATCA TGCATGGCTC ATTTTTTATT

151 TTTAGTAGAG ACAAATTCTC CATGTTGCCC AGGCTAGTCC TGAACTCCTG

201 GGCTCAAGAG ATCCACCCAC ATCAGCCTTC CAGACTGCTG GCCTGGTCTC

251 CTCCAGATGT TTATAACTCA TGAGTGCCAG GACTAGACCT GGTACTAGGA

301 AGCAGCTGCA TTGGATTTAC CAGGCTTTTC ACTCTTGTAT TTTACAGGGC

351 TGGGACAGGC CTGGACTGCA AGGAGGGGTC TTTGCACCAT CTCTGAAAAG

401 CCGATGTGTA TCCTCAGCTT TGAGAACTGA ATTCCATGGG TTGTGTCAGT

451 GTCAGACCTN TGAAATTCAG TTCTTCAGCT GGGATATCTC TGTCATCGTG

501 GGCTTGAGGA CCTGGAGAGA GTAGATCCTG AAGAACTTTT TCAGTCTGCT

551 GAAGAGCTTG GAAGACTGGA GACAGAAGGC AGAGTCTCAG GCTCTGAAGG

601 TATAAGGAGT GTGAGTTCCT GTGAGAAACA CTCATTTGAT TGTGAAAAGA

651 CTTGAATTCT ATGCTAAGCA GGGTTCCAAG TAGCTAAATG AATGATCTCA

701 GCAAGTCTCT CTTGCTGCTG CTGCTACTCG TTTACATTTA TTGATTACTT

751 ACGATGATTC AGGTACTGTT GTAAGTGCTT TACATGCTGT TATACGAGAC

801 TCTTGGGAGA AATCACTTTA ATGAAGCTTG AGACACATGG CATTGCCATG

851 CAATGATTTT TCCCCCCTCT TCACGGGATC AGAGGGAACT AATAGAATG

The invention also provides nucleotide primers for specifically amplifying hsa-miR-146a molecular DNA fragments, the length of which is 20-22 bases.

Amplification primers are as follows:

Forward primer: 5'-GGAGGGGTCTTTGCACCATC-3' (SEQ ID NO: 2)

Reverse primer: 5'-TGCCTTCTGTCTCCAGTCTTCC-3' (SEQ ID NO: 3)

Secondly, the present invention provides a method for detecting the polymorphic site on hsa-miR-146a precursor (401-499 sequence of the sequence shown in SEQ ID NO: 1) to the mature body (sequence shown in SEQ ID NO: 1 No. 422-441 sequence) the technical method of processing efficiency influence, the specific steps are as follows:

(a) Constructing small molecule RNA expression vectors with the two polymorphic sites identified above (genotypes are G or C respectively), and using them to transfect model cells respectively;

(b) collecting the model cells transfected with the expression vector, and using RNA extraction technology to obtain ribonucleotides in the cells;

(c) Northern blot technology was used to detect the expression levels of the precursor and mature molecules of hsa-miR-146a in the obtained ribonucleotides.

The invention also provides nucleotide primers for specifically cloning hsa-miR-146a expression fragments in the expression vector, and oligonucleotide probes for specifically binding hsa-miR-146a precursors and mature bodies. Cloning primers are as follows:

Forward primer: 5'-ATGCTCGAGCCTGGTCTCTCTCAGATGTT-3' (SEQ ID NO: 4)

Reverse primer: 5'-ATGTCTAGATTCTCACAGGAACTCACACTCC-3' (SEQ ID NO: 5)

Probe: 5'-AACCCATGGAATTCAGTTCTCA-3' (SEQ ID NO: 6)

Finally, the present invention provides a prognostic kit for detecting primary liver cancer. include:

(1) A pair of primers for specifically amplifying the genomic fragment containing the hsa-miR-146a precursor sequence;

(2) Restriction enzymes required for detecting the genotype of the polymorphic site rs2910164 in the amplification product.

The beneficial effects of the present invention are: the kit for analyzing the sequence of the single nucleotide polymorphism site in the hsa-miR-146a precursor sequence provided by the present invention can be applied to the auxiliary diagnosis of primary liver cancer patients, Evaluate the susceptibility of individuals to primary liver cancer, which is beneficial to the prevention, early diagnosis and treatment of primary liver cancer; it can be used as a potential target of drugs to screen for pharmaceutical molecules that can regulate the expression of hsa-miR-146a, Facilitate the discovery of new anti-liver cancer drugs.

Description of drawings

Figure 1: The effect of polymorphic sites on the processing efficiency of hsa-miR-146a precursor to mature body

Detailed ways

Example 1: Collection of blood samples and extraction of genomic DNA

A total of 671 sporadic primary liver cancer specimens were collected from the Cancer Hospital Affiliated to Sun Yat-sen University, Guangdong Province, with an average age of 46.8±12.2 years (standard deviation), of which 65 were female. Also from Guangdong Province, there were 655 unrelated healthy controls, with an average age of 45.8±13.7 years (standard deviation), including 64 female samples.

After the genomic DNA was extracted from the blood samples collected above by the conventional phenol-chloroform method, the samples were uniformly diluted to 10 ng/ul.

Example 2: Typing of SNPs

The present invention adopts the single nucleotide polymorphism technology (PCR-SSCP) based on the polymerase chain reaction, and the polymorphic site rs2910164 (its allele point pair is G) existing in the molecular precursor of hsa-miR-146a /C) Genotyping was performed in case and control groups.

The PCR reaction was carried out on the MJ PTC-200 instrument.

Amplification primers are as follows:

Forward primer: 5'-GGAGGGGTCTTTGCACCATC-3' (SEQ ID NO: 2)

Reverse primer: 5'-TGCCTTCTGTCTCCAGTCTTCC-3' (SEQ ID NO: 3)

PCR reaction reagents and procedures:

Carry out an amplification reaction with a total volume of 20ul for each sample, and the system contains 20ng of the prepared genomic DNA dilution, 2ul of 10×Taq Buffer, 0.8ul of 5mM dNTP, 1.6ul of 25mM MgCl ₂ , and 0.25 units of Taq DNA polymerase and 0.5ul each of the above primers (diluted to 10uM), and finally add ddH ₂ O to make the total volume 20ul. After the reaction was pre-denatured at 94°C for 2 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and finally a fill-in at 72°C for 2 minutes were performed.

Use the restriction fragment length polymorphism (RFLP) method to amplify the hsa-miR-146a precursor containing hsa-miR-146a with a length of 210bp (located at sequence 372-581 of the sequence shown in SEQ ID NO: 1). The polymorphic loci in the DNA fragments were genotyped. The specific method is: take 3 ul of the amplified PCR reaction product, add 1 ul of digestion buffer, 1 unit of restriction endonuclease MamI, add ddH ₂ O to a total volume of 10 ul, and digest at 37°C overnight. Afterwards, run electrophoresis on a 12% denaturing polyacrylamide gel, and perform genotype analysis after developing with silver staining: after enzyme digestion, if the genotype of rs2910164 in the test sample is homozygous G, 82nt, 77nt and 51nt bands; if the genotype is homozygous C, bands of 82nt, 51nt, 45nt and 32nt appear; if the genotype is heterozygous GC, bands of 82nt, 77nt, 51nt, 45nt and 32nt appear .

Example 3: Detection of the impact of the polymorphic site on the processing efficiency of hsa-miR-146a precursor to mature body

The present invention is based on cell model detection and uses in vitro detection of hsa-miR-16a molecular processing efficiency, and the technical path is as follows:

(1) Based on the commercialized pCDNA3.0 vector backbone of Invitrogen Company, using samples of the two homozygous genotypes as templates, the genomic DNA sequence containing the hsa-miR-146a precursor was cloned into the backbone to construct become two expression vectors of the G or C polymorphism of the microRNA;

Cloning primers are as follows:

Forward primer:

5'-ATGCTCGAGCCTGGTCTCTCTCAGATGTT-3' (SEQ ID NO: 4)

Reverse primer:

5'-ATGTCTAGATTCTCACAGGAACTCACACTCC-3' (SEQ ID NO: 5)

(2) The two polymorphic microRNA expression vectors constructed in step 1 and the empty vector pCDNA3.0 were introduced into 293T cells (obtained from the American Type Culture Collection) by conventional calcium phosphate transfection method, and transfected Then continue to culture the cells for 24 hours;

(3) Collect the total RNA of the 293T cells transfected in step 2 and the untransfected cells, electrophoresis on 15% denatured polyacrylamide, transfer membrane and isotope probe hybridization according to the conventional Northern blot method, The expression levels of hsa-miR-146a molecular precursor and mature body in different RNA samples were detected by phosphorscreening. The specific hybridization probe used is: 5'-AACCCATGGA ATTCAGTTCTCA-3' (SEQ ID NO: 6);

(4) Compare the expression level ratio of mature and precursor in cell samples transfected with two polymorphic expression vectors, that is, the processing efficiency of hsa-miR-146a molecular precursor to mature.

The results are shown in Figure 1. When the allelic position is G, the processing efficiency from the precursor to the mature body of the molecule is significantly improved compared with that when the allelic position is C.

Example 4: Correlation analysis between single nucleotide polymorphism sites in hsa-miR-146a molecular precursor and primary liver cancer

Statistical method: GDA (http://hydrodictyon.eeb.uconn.edu/people/plewis/software.php) was used to perform chi-square analysis with 10,000 simulations, and the Hardy-Weinberg equilibrium of healthy control group specimens was calculated. Using SPSS professional statistical software, the chi-square test was used to conduct statistical analysis on the genotype distribution frequency of polymorphic sites in the patient group and control group samples, and the confidence interval was set at 95% (ie 95% CI). The significant difference level was set at p<0.05, and the Odds Ratio (OR) value was calculated at the same time.

statistical results:

The genotype and allele distribution frequency of the rs2910164 polymorphic site in the specimens of the primary liver cancer patient group and the healthy control group are shown in the following table: group Total number of specimens Number of Genotypes (Frequency%) Number of alleles (frequency%) CC GC GG C G patient 671 226(33.7) 339 (50.5) 106(15.8) 791 (58.9) 551 (41.1) control 665 259 (38.9) 327(49.2) 79 (11.9) 845(63.5) 485(36.5) P-value 0.041 0.015 OR (95%CI) 1.21 (1.04<OR<1.42)

According to the distribution frequency in the above table, the genotype distribution of the samples in the control group was found to be in Hardy-Weinberg equilibrium by calculation using GDA software.

It can be seen from the above table that the G allele of the single nucleotide polymorphism site rs2910164 located on the hsa-miR-146a precursor has a significant distribution frequency in the primary liver cancer patient population (P=0.015) Greater than its distribution frequency in the healthy control group, it is a landmark locus of primary liver cancer susceptibility. The polymorphic site associated with the susceptibility to primary liver cancer is located at the stem part of the stem-loop structure of the hsa-miR-146a precursor molecule, and the polymorphism changes the complementary pairing of bases on the stem (if the If the site is a G polymorphism, it can form an RNA-specific G:U pairing with the U base on the other arm; if the site is a C polymorphism, the pairing will be destroyed), thereby changing the stem complementary integrity. This is likely to affect the secondary structure of the RNA, thereby affecting the processing efficiency of its precursor to the mature body. As shown in Example 3, the difference in this allelic site has been confirmed to affect the production of its mature body, which also from A functional perspective supports the above statistical conclusions.

Embodiment 5: detection kit

A kit for detecting the susceptibility of primary liver cancer is prepared, which contains the following oligonucleotide primer pairs, which are used to specifically amplify the genome fragment containing the hsa-miR-146a precursor sequence from the genome sample of the test subject.

Forward primer: 5'-GGAGGGGTCTTTGCACCATC-3' (SEQ ID NO: 2)

Reverse primer: 5'-TGCCTTCTGTCTCCAGTCTTCC-3' (SEQ ID NO: 3)

The kit also includes restriction endonuclease MamI, which is used to digest the PCR product amplified by the above primer pair. The digested product is subjected to 15% denatured polyacrylamide gel electrophoresis and developed, so that the G-C allele type of the rs2910164 polymorphic site in the specimen can be detected conveniently.

Examples of the utility of the present invention:

(1) The method established by the present invention can be used to analyze the G-C allele type of the single nucleotide polymorphism site rs2910164 on the human hsa-miR-146a molecular precursor sequence, to be applied to primary liver cancer Auxiliary diagnosis and judgment of whether an individual has primary liver cancer susceptibility, which is beneficial to the prevention and treatment of primary liver cancer;

(2) The method for studying the processing efficiency of this type of small molecule non-coding ribonucleic acid introduced in the present invention can be used to screen for effective drugs targeting the above-mentioned rs2910164 polymorphic site, and to search for drugs that can regulate the processing and expression of hsa-miR-146a molecules. active molecules to promote the discovery of new anti-liver cancer drugs;

(3) Utilize the sequence and genotyping method of the small molecule non-coding ribonucleic acid related to primary liver cancer and its polymorphic site provided by the present invention to construct a detection reagent for molecular genetic diagnosis of primary liver cancer box;

(4) Since the hsa-miR-146a molecule is likely to be involved in other pathological processes related to the dysregulation of cell activities such as proliferation and apoptosis, including other types of tumors, etc. Therefore, the present invention provides experience and application basis for in-depth research on the relationship between hsa-miR-146a molecule and other diseases in the future;

(5) There are similar single nucleotide polymorphism sites in the precursor sequences of other small molecule non-coding ribonucleic acids of the same type, and these sites may also regulate the processing and expression of the corresponding small molecules, Thereby affecting its biological function and even affecting the occurrence of various diseases. At present, there are no reports at home and abroad that the polymorphic sites that exist on this type of small molecule non-coding ribonucleic acid are related to diseases. Therefore, the present invention also announces the most valuable clues, experience and application technology for future research in this field. .

Sequence Listing

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Claims (9)

1, a kind of method that detects the single nucleotide polymorphism of hsa-mir-146a molecule, it is characterized in that: it may further comprise the steps:

(1) determines the mononucleotide polymorphic site rs2910164 that on the has-mir-146a molecular precursor, exists, i.e. the Nucleotide of the 460th of SEQ ID NO:1;

(2) adopt the polymerase chain reaction, specific amplification comprises the genomic DNA fragment of has-mir-146a precursor sequence;

(3) pleomorphism site in the dna fragmentation that includes the has-mir-146a precursor that amplification is obtained with restriction fragment length polymorphism (RFLP) method carries out gene type.

2, the method for the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 1, it is characterized in that: the sequence of the primer that adopt step (2) polymerase chain reaction is respectively SEQ ID NO:2, sequence shown in the SEQ ID NO:3.

3, the method for the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 1, it is characterized in that: the concrete grammar of step (3) is: with restriction enzyme MamI the PCR product of step step (2) is carried out enzyme and cut, the length of the dna fragmentation after cutting according to enzyme is then determined the polymorphic site genotype.

4, a kind of single nucleotide polymorphism that detects the hsa-mir-146a molecule may further comprise the steps the method for its precursor to ripe body working (machining) efficiency influence:

(1) structure has the microRNA expression vector that two kinds of pleomorphism site genotype of mirror are respectively G or C respectively, and distinguishes transfection pattern cell with it;

(2) the pattern cell behind the collection transfection expression carrier utilizes the ribonucleotide in the RNA extractive technique acquisition cell;

(3) adopt Northern blot technology, detect the precursor of has-mir-146a in the ribonucleotide that obtains and the expression level of ripe body molecule.

5, the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 4 is to the method for its precursor to ripe body working (machining) efficiency influence, it is characterized in that: step (1) is with SEQ ID NO:4, sequence shown in the SEQID NO:5 is that primer is right, two kinds of genotypic gene DNA fragments that mononucleotide polymorphic site rs2910164 are respectively G or C and comprise the has-mir-146a precursor sequence are cloned into respectively on the pCDNA3.0 carrier, are configured to the G of this microRNA or two expression vectors of C multiformity.

6, the single nucleotide polymorphism of detection hsa-mir-146a molecule according to claim 4 is to the method for its precursor to ripe body working (machining) efficiency influence, it is characterized in that: step (3) is specially: utilize the method for conventional Northern Blot to change film and isotope probe hybridization, hybridization probe is a sequence shown in the SEQ ID NO:6, and developing by phosphorus screen then detects the expression level of hsa-mir-146a molecular precursor and ripe body in the different RNA sample.

7, a kind of diagnostic kit of primary hepatocarcinoma is characterized in that, it comprises:

(1) it is right that specific amplification includes the primer of genomic fragment of hsa-mir-146a precursor sequence;

(2) detect the required restriction enzyme of polymorphic site rs2910164 genotype in the amplified production.

By the diagnostic kit of the described primary hepatocarcinoma of claim 7, it is characterized in that 8, specific amplification includes the primer of genomic fragment of hsa-mir-146a precursor sequence to having SEQ ID NO:2, sequence shown in the SEQID NO:3.

9, by the diagnostic kit of the described primary hepatocarcinoma of claim 7, it is characterized in that described restriction enzyme is restriction enzyme MamI.