NL2033031B1 - Single nucleotide polymorphism (snp) molecular marker and kit for assessing tumor progression risk of patient with carcinoma of colon and rectum, and use thereof - Google Patents
Single nucleotide polymorphism (snp) molecular marker and kit for assessing tumor progression risk of patient with carcinoma of colon and rectum, and use thereof Download PDFInfo
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Abstract
The present disclosure provides a single nucleotide polymorphism (SNP) molecular marker and a kit for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, and use thereof, and relates to the technical field of biomedicine. In the present disclosure, the SNP molecular marker is provided for assessing the tumor progression risk of the patient with carcinoma of colon and rectum; experiments have confirmed that an rle766212 locus polymorphism. located, in a .KCNQlOIfl gene is significantly associated with tumor progression in the patient with carcinoma of 10 colon and rectum. Compared with patients with an rle766212 GG genotype, patients with an rle766212 GA genotype have a significantly lower risk in developing tumors of stages III and IV.
Description
SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MOLECULAR MARKER AND KIT FOR
ASSESSING TUMOR PROGRESSION RISK OF PATIENT WITH CARCINOMA OF
COLON AND RECTUM, AND USE THEREOF
The present disclosure relates to the technical field of bio- medicine, in particular to a single nucleotide polymorphism (SNP) molecular marker and a kit for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, and use thereof.
Carcinoma of colon and rectum is a common malignancy in the gastrointestinal tract. In the early stage of the carcinoma of co- lon and rectum, there are usually no obvious symptoms. With the development of the tumor, the following symptoms become noticea- ble: changes in bowel habits, blood in the stool, diarrhea, alter- nating diarrhea and constipation, and local abdominal pain. In the advanced stage, systemic symptoms such as anemia and weight loss are widely present. The carcinoma of colon and rectum has morbidi- ty and mortality only after gastric cancer, esophageal cancer, and primary liver cancer among malignant tumors of the digestive sys- tem. The cancer can spread to other tissues and organs through lymphatic, blood circulation, and direct spreading. The cancer can be diagnosed according to clinical manifestations, X-ray barium enema, or fiberoptic colonoscopy. A key to treatment lies in early detection, timely diagnosis, and radical surgery, and prognosis depends on early diagnosis and timely surgical treatment.
Single nucleotide polymorphism (SNP) refers to a DNA sequence polymorphism caused by single nucleotide variation at the genome level. The SNP, as the most common type of human heritable varia- tion, has a high density, stable inheritance, and easy typing, and has extremely high research values and prospects for use in the fields of disease diagnosis and drug development. However, there is still a lack of SNP molecular markers for assessing a tumor progression risk of a patient with carcinoma of colon and rectum.
In view of this, an objective of the present disclosure is to provide an SNP molecular marker for assessing a tumor progression risk of a patient with carcinoma of colon and rectum. By detecting the molecular marker, a genotype of an rs10766212 G>A locus of a test subject can be detected non-invasively and accurately, which is helpful to assess the tumor progression risk in patients.
To solve the above technical problems, the present disclosure provides the following technical solutions.
The present disclosure provides an SNP molecular marker for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, including an SNP located at an rs10766212 locus on a KCNQIOTI gene, where the locus is G or A.
Preferably, the KCNQIOT1 gene may have a nucleotide sequence shown in SEQ ID NO: 1.
Preferably, the SNP molecular marker may be located at posi- tion 47 of the nucleotide sequence shown in SEQ ID NO: 1, which is
G or A.
The present disclosure further provides a primer set of an
SNP molecular marker for detecting a tumor progression risk of a patient with carcinoma of colon and rectum, having nucleotide se- quences shown in SEQ ID NO: 2 and SEQ ID NO: 3.
The present disclosure further provides a kit for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, including the primer set.
Preferably, the kit may further include a sequencing primer with a nucleotide sequence shown in SEQ ID NO: 4 for the SNP mo- lecular marker.
Preferably, the kit may further include a cotton swab and a reaction solution.
Preferably, the reaction solution may include a DNA extrac- tion reagent and a PCR amplification reagent; the DNA extraction reagent may include Chelex-100 and a proteinase K; and the PCR am- plification reagent may include 2xTag PCR Master Mix.
The present disclosure further provides use of a reagent for detecting the SNP molecular marker in preparation of a kit for as-
sessing a tumor progression risk of a patient with carcinoma of colon and rectum.
The present disclosure provides an SNP molecular marker for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, having a single nucleotide sequence with a poly- morphism of G or A at the rsl0766212 locus on the KCNQIOTI gene.
The experiments have confirmed that an rsl0766212 (G or A) locus polymorphism located in a KCNQIOTI gene is significantly associat- ed with tumor progression in the patient with carcinoma of colon and rectum. Compared with patients with an rs10766212 GG genotype, patients with an rs10766212 GA genotype have a significantly lower risk in developing tumors of stages III and IV. Identifying a gen- otype of an rs10766212 G>A locus using a reagent for detecting the
SNP molecular markers may help to assess the tumor progression risk of the patient with carcinoma of colon and rectum, which is of great value for implementing individualized therapy.
FIG. 1 shows an agarose gel electrophoresis diagram of a PCR amplification product containing an rs10766212 locus provided by the present disclosure; and
FIG. 2 shows a sequencing result of an rs10766212 G>A locus of a KCNQIOTI gene provided by the present disclosure, where ar- rows indicate peak positions of the rs10766212 locus.
The present disclosure provides an SNP molecular marker for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, including an SNP located at an rs10766212 locus on a KCNQ/IOTi gene, where the locus may be G or A.
In the present disclosure, the KCNQIOTI gene containing the
SNP molecular marker has a nucleotide sequence shown in SEQ ID NO: 1: TTAAAGTGTAATCTGGAGGTTACAATAAGCAGCTTAAGCAAAAACArTTCTGCTTCAGGTTAA-
TAATCTACTCAAATATTTTTGTCTGCCCTATTCTCTCCTTCTCAGTACTCTTA (>
NC 000011.10: 2611876-2611990 Homo sapiens chromosome 11, GRCh38. pl4 Primary Assembly). r is A or G. The SNP molecular marker is preferably located at position 47 of the nucleotide sequence shown in SEQ ID NO: 1, which may be G or A.
The present disclosure further provides a primer set of an
SNP molecular marker for detecting a tumor progression risk of a patient with carcinoma of colon and rectum, having nucleotide se- quences shown in SEQ ID NO: 2 and SEQ ID NO: 3.
In the present disclosure, the primer set has nucleotide se- quences specifically as follows:
F: CACTTTTGTTCCTCTCTTCCTCC (SEQ ID NO: 2);
R: CCCAGTTTCTAATAGGCCACAG (SEQ ID NO: 3). The primer set can specifically amplify a DNA fragment containing the rs10766212 lo- cus. There is no special limitation on a source of the primers, and synthetic methods well known to those skilled in the art can be used for artificial synthesis. Preferably, a company is en- trusted to conduct the artificial synthesis.
The present disclosure further provides a kit for assessing a tumor progression risk of a patient with carcinoma of colon and rectum, including the primer set.
In the present disclosure, the kit further includes prefera- bly a sequencing primer with a nucleotide sequence shown in pref- erably CACTTTTGTTCCTCTCTTCCCTCC (SEQ ID NO: 4) for the SNP molecu- lar marker. There is no special limitation on a source of the se- quencing primer, and synthetic methods well known to those skilled in the art can be used for artificial synthesis. The sequencing primer can rapidly and accurately type the rs10766212 locus of the subject by a sequencer. There is no special limitation on a source of the sequencer, and conventional commercially-available sequenc- ers well known to those skilled in the art can be used, preferably an ABI3730 sequencer. The sequencing result is preferably analyzed using chromas software.
In the present disclosure, the kit further includes prefera- bly a cotton swab and a reaction solution. The cotton swab is used for sampling a test sample of the kit; the test sample of the kit includes preferably oral mucosal cells. The reaction solution in- cludes a DNA extraction reagent and a PCR amplification reagent; the DNA extraction reagent includes preferably Chelex-100 and a proteinase K; and the PCR amplification reagent includes prefera- bly 2xTaq PCR Master Mix. The kit can be used to non-invasively detect a genotype of the rs10766212 locus of the test subject; preferably, the kit can non-invasively extract a genomic DNA, and accurately detect a genotype of the rs10766212 G>A locus of the test subject based on the sequencer. 5 In the present disclosure, a method for DNA extraction is preferably a Chelex-100 method, including preferably the following steps: adding the Chelex-100 to a sample, adding 6 pL of the pro- teinase K, and mixing, water bathing, cooling, and centrifuging to obtain a supernatant, namely the DNA. Preferably, the resulting
DNA is diluted to a working concentration of 5 ng/pL to 10 ng/uL.
After the DNA is extracted, PCR amplification is preferably con- ducted; a PCR amplification system is preferably 20 pL, and pref- erably includes the following components: 1 pL of each of 10 pM primers F and R, 10 pL of the 2xTag PCR Master Mix, 1 pL of a DNA template (the sample to be tested), and 7 pL of ddH;0. A PCR ampli- fication program is preferably: at 95°C for 3 min; at 95°C for 30 sec, at 58°C for 30 sec, at 72°C for 30 sec, conducting 32 cycles; at 72°C for 10 min; and storage at 4°C.
The present disclosure further provides use of a reagent for detecting the SNP molecular marker in preparation of a kit for as- sessing a tumor progression risk of a patient with carcinoma of colon and rectum. Compared with patients with the rs10766212 GG genotype, patients with the rs10766212 GA genotype have a signifi- cantly lower risk in developing tumors of stages III and IV.
In order to make the objectives, technical solutions and ad- vantages of the present disclosure clearer, the present disclosure will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present disclosure.
Experimental methods in the following examples are conven- tional methods, unless otherwise specified.
The materials, reagents, and the like used in the following examples are all commercially available, unless otherwise speci- fied.
Example 1 1. The oral mucosal cells from 576 patients with carcinoma of colon and rectum in Shanghai Xuhui District Central Hospital were collected using cotton swabs. 2. A cell genome was extracted by a Chelex-100 method. 3. With a genomic DNA as a template, a DNA fragment contain- ing an rsl0766212 locus was amplified using a primer set shown in
SEQ ID NO: 2 and SEQ ID NO: 3; a PCR amplification system had a total volume of 20 pL, including: 1 pL of each of 10 pM primers F and R, 10 pL of 2xTag PCR Master Mix, 1 pL of a DNA template (a sample to be tested), and 7 pL of ddH;0.
A PCR amplification program was: at 95°C for 3 min; at 95°C for 30 sec, at 58°C for 30 sec, at 72°C for 30 sec, conducting 32 cycles; at 72°C for 10 min; and storage at 4°C. 4. After the amplification in step 3 was completed, a se- quencing reaction was conducted on the amplified DNA fragment us- ing a sequencing primer shown in SEQ ID NO: 4; an obtained se- quencing reaction product was purified and then sequenced on an
ABI3730 sequencer; specific operation steps were conducted accord- ing to instructions of a Sanger sequencing kit. 5. The data obtained by sequencing was processed using chro- mas software, and a genotype of the patient's rsl0766212 locus was analyzed and determined. 6. Logistic regression was conducted to analyze a relation- ship between an rs10766212 locus polymorphism of a KCNQIOTI gene and a tumor progression risk in patients with carcinoma of colon and rectum. The results were shown in Table 1.
Table 1 Relationship between rs10766212 G>A locus polymor- phism of KCNQ10T1 gene and tumor progression risk in patients with carcinoma of colon and rectum “Genotype HI/IV(n=246) I/i(n=330) OR(95%C) Pp
GG 205 (83.3%) 197 (59.7%) 1
GA 40 {16.3%) 125 {37.9%} 0.30 {0.20-0.46) <0.001
AA 1 (0.4%) 8 (2.4%) 0.12 {0.01-1.00) 0.05
The results showed that the SNP locus rs10766212 G>A located on the KCNQIOTI gene was significantly associated with the tumor progression in the patients with carcinoma of colon and rectum.
Compared with patients with an rs10766212 GG genotype, patients with an rs10766212 GA genotype have a significantly lower risk in developing tumors of stages III and IV. Identifying the genotype of the rs10766212 G>A locus may help to assess the tumor progres- sion risk of the patient with carcinoma of colon and rectum, which is of great value for implementing individualized therapy.
Example 2
A genotype of an rsl0766212 G>A locus of a KCNQIOTI gene of a test subject was detected based on a kit provided by the present disclosure: 1) DNA extraction
The oral mucosal cells were collected from subjects with car- cinoma of colon and rectum in Shanghai Xuhui District Central Hos- pital with a cotton swab, a surface of the cotton swab was peeled, put into a centrifuge tube, and added with 300 pL of a 5% chelex- 100 solution to immerse the specimen; 6 pL of a proteinase K was added, mixed well, treated in a water bath at 56°C for 30 min, mixed well, boiled in the water bath for 10 min, placed on ice for 10 min, and centrifuged at 14,000 r/min for 2 min; a supernatant
DNA was collected, a concentration of the DNA was determined using
Nano-100, and the sample was diluted to a working concentration of 5 ng/pL to 10 ng/pL. 2) PCR reaction (20 ulL): 1 pL (10 uM) of PCR amplification primers, 10 pL of 2xTaqg PCR
Master Mix, 1 pL of a DNA template, and 7 pL of ddH-0; program included: at 95°C for 3min + (at 95°C for 30 sec + at 58°C for 30 sec + at 72°C for 30 sec) x 32 cycles + at 72°C for 10 min + 4°C ©, The amplification results were shown in FIG. 1.
According to FIG. 1, the designed primers and PCR reaction conditions could specifically amplify the fragment containing the rsl0766212 locus. 3) Sanger sequencing
The PCR product was purified, sequenced, and purified accord- ing to operation steps in a Sanger sequencing kit (purchased from
Applied Biosystems, Cat. No. A38073). The products were sequenced using an ABI3730 sequencer, and sequencing results were analyzed by chromas software. The results were shown in FIG. 2, where sam- ples 1 to 3 each were patients with carcinoma of colon and rectum of stage II.
The results of sequencing analysis showed that sample 1 was
AA type, sample 2 was type GA, and sample 3 was type GG, suggest- ing that the tumor progression risk in patient 2 was 0.3 times that of patient 3.
The above are only the examples of the present disclosure and therefore do not limit the patent scope of the present disclosure.
Any equivalent structure or equivalent process transformation used according to the contents of the specification in the present dis- closure, no matter whether it is directly or indirectly used in any other related technical field, should be included within the scope of patent protection of the present disclosure.
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Sl <INSDQualifier valuersynthetic construct“/INSDQualifien value»
GZ </INSDOualifier> a2 </IN3DFeature gualsd
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Be]
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Citations (3)
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|---|---|---|---|---|
| WO2010019690A1 (en) * | 2008-08-12 | 2010-02-18 | The Ohio State University Research Foundation | Polymorphisms associated with developing colorectal cancer, methods of detection and uses thereof |
| US8658358B1 (en) * | 2009-04-21 | 2014-02-25 | Transgenomic, Inc. | Mutations associated with long QT syndrome |
| US9157121B2 (en) * | 2009-04-06 | 2015-10-13 | Vanda Pharmaceuticals, Inc. | Method of treatment based on polymorphisms of the KCNQ1 gene |
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Patent Citations (3)
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|---|---|---|---|---|
| WO2010019690A1 (en) * | 2008-08-12 | 2010-02-18 | The Ohio State University Research Foundation | Polymorphisms associated with developing colorectal cancer, methods of detection and uses thereof |
| US9157121B2 (en) * | 2009-04-06 | 2015-10-13 | Vanda Pharmaceuticals, Inc. | Method of treatment based on polymorphisms of the KCNQ1 gene |
| US8658358B1 (en) * | 2009-04-21 | 2014-02-25 | Transgenomic, Inc. | Mutations associated with long QT syndrome |
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| NAKANO SEIJI ET AL: "Expression profile of LIT1/KCNQ1OT1 and epigenetic status at the KvDMR1 in colorectal cancers", CANCER SCIENCE, vol. 97, no. 11, 1 November 2006 (2006-11-01), JP, pages 1147 - 1154, XP093022342, ISSN: 1347-9032, DOI: 10.1111/j.1349-7006.2006.00305.x * |
| THAN B L N ET AL: "The role of KCNQ1 in mouse and human gastrointestinal cancers", ONCOGENE, NATURE PUBLISHING GROUP UK, LONDON, vol. 33, no. 29, 26 August 2013 (2013-08-26), pages 3861 - 3868, XP037750247, ISSN: 0950-9232, [retrieved on 20130826], DOI: 10.1038/ONC.2013.350 * |
| XU MI-DIE ET AL: "Long non-coding RNAs in colorectal cancer: implications for pathogenesis and clinical application", MODERN PATHOLOGY, vol. 27, no. 10, 1 October 2014 (2014-10-01), GB, pages 1310 - 1320, XP055919624, ISSN: 0893-3952, Retrieved from the Internet <URL:https://www.nature.com/articles/modpathol201433.pdf> DOI: 10.1038/modpathol.2014.33 * |
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