Non-invasive method for the early diagnosis of gastric cancer using as a biomarker the methylation levels in the DNA sequence of microRNA-335-5p promoter
11512354 · 2022-11-29
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Abstract
The invention is directed to a method for the early detection of gastric cancer, by detecting the increase in DNA methylation of the promoter region of the microRNA-335-5p in samples obtained non-invasively, preferably in plasma. Thus, it is a contribution for the early detection of gastric cancer, without invasive procedures, with rapid collection of the sample and of the delivery of the results, and of lower cost than the technologies that employ invasive diagnostic techniques to the human and animal body in general.
Claims
1. A method for detecting a concentration of methylated DNA of a promoter of microRNA-335-5p in plasma, comprising: obtaining a sample of the plasma from a live subject in a non-invasive manner; adding one or more primers to the sample, wherein the one or more primers amplify a CpG island of the promoter of microRNA-335-5p, said CpG island defined from base 1683 to base 1808 of SEQ ID No. 1; after adding the one or more primers, performing polymerase chain reaction (PCR) cycles on the sample; and after performing the PCR cycles, detecting positive amplification bands in the sample, wherein the primers include a forward primer having a sequence of SEQ ID No. 2 and a reverse primer having a sequence of SEQ ID No. 3.
2. The method of claim 1, wherein the PCR cycles each include 30 seconds at 95° C., 30 sec. at 52° C. and 40 sec. at 72° C.
3. The method of claim 2, wherein 35 of the PCR cycles are performed.
Description
DESCRIPTION OF THE FIGURES
(1)
(2) The evaluated region was verified to correspond to the promoter region of the MEST gene through the online program PROSCAN Version 1.7 (Biolnformatics and Molecular Analysis Section, NIH), (https://www-bimas.cit.nih.gov/molbio/proscan/). bp, base pairs
(3) The following table summarizes the prediction results.
(4) TABLE-US-00001 CpG Size of the CpG Start-end of the CpG Islands island (bp) island (bp) CpG Island 1 318 460-777 CpG Island 2 465 836-1300 CpG Island 3 757 1546-2302 CpG Island 4 584 2362-2945
(5)
DETAILED DESCRIPTION OF THE INVENTION
(6) The invention is directed to a non-invasive method of early diagnosis of gastric cancer, using as a biomarker the levels of methylation of the DNA sequence of the microRNA-335-5p promoter in plasma.
(7) Specifically, the invention presented here is a method for the early detection of gastric cancer, by detecting the increase in DNA methylation of the promoter region of microRNA-335-5p in samples obtained non-invasively, preferably in plasma. Thus, it is a contribution for the early detection of gastric cancer, without invasive procedures, with rapid collection of the sample and delivery of the results, and of lower cost than the technologies that employ invasive diagnostic techniques to the human body and animal body in general.
(8) MicroRNA-335-5p (miR-335) is a transcript located on chromosome 7q32.2, in the second intron of the MEST gene (Mesoderm Specific Transcript Homolog), which codes for 17 different mRNAs, which can be transcribed from multiple transcription initiation sites, controlled by chromosomal methylation of CpG islands. The mature sequence of miR-335 in humans corresponds to 16-UCAAGAGCAAUAACGAAAAAUGU-38 (http://www.mirbase.org, accession: MIMAT0000765, ID: hsa-miR-335).
(9) The promoter region of miR-335 is considered 5000 bp upstream of the start of transcription (ATG) and is shown in SEQ ID No. 1.
(10) The inventors have determined that microRNA-335-5p is a potential tumor suppressor gene in gastric cancer and that it is rendered inactive by DNA methylation in its promoter region. Based on this result and on the need for a reliable biomarker that is fast to quantify at low concentrations from non-invasively isolated samples and that is low cost to people, the method of the invention, which allows for early detection of the development of gastric cancer, was developed; this, since surprisingly the inventors have established that determining the degree of DNA methylation of the promoter region of microRNA-335-5p in plasma allows to correlate an increase in said degree of methylation within patients with gastric cancer in comparison to healthy population.
(11) Studies developed by the inventors show that patients with gastric cancer have high levels of methylation compared to the levels observed in healthy subjects from the general population.
(12)
(13) Thus, the invention contributes to the state of the art, the detection of methylated DNA from the promoter region of microRNA-335-5p as a biomarker of gastric cancer, in non-invasively obtained samples, preferably from plasma, where an increase in levels of said methylated DNA, is indicative of a developing gastric cancer. While low circulating levels of methylated DNA from said promoter region of microRNA-335-5p, it is indicative of an absence of disease or very early stages of this cancer.
(14) Thus, the invention overcomes previously unsolved limitations in the current state of art, such as providing methods of gastric cancer diagnostics methods characterized by being non-invasive, highly accurate, fast in the delivery of results and of low purchase and operational cost.
(15) The method of detecting the methylated DNA sequence comprising the promoter of microRNA-335-5p expression can be detected by any means available in the art, such as Polymerase Chain Reaction for Methylated Sequences (MSP), quantitative MSP, expression microarrays, bisulfite sequencing, or pyrosequencing.
(16) In one embodiment, the invention resolves the previously stated limitations by employing PCR or MSP-specific methylation primers that specifically allow detection of low concentration levels of the methylated DNA sequence of the miR-335 promoter.
(17) If a PCR technique for methylated sequences is chosen for the embodiment of the invention, the primers can be designed for any region of the promoter containing a methylation site, preferably one of the four CpG islands defined in
(18) All possible embodiments for the detection of the methylated sequence of this promoter are comprised within the scope of the present invention.
(19) As indicated, in the prior art, the expression of miR-335 has mainly been detected by the detection of a 24-nucleotide long RNA, with all the difficulties that RNA detection brings, mainly due to its tendency to degradation in samples. On the contrary, the method of the invention focuses on the DNA of the promoter region of said RNA. As DNA is more resistant to degradation than RNA, our invention also provides greater stability over time and in the storage of its components, as well as of the sample to be analyzed, which, since it is obtained non-invasively (preferably plasma), is easily accessible, and this further reducing costs and increasing public accessibility to both diagnosis and early curative treatments.
(20) Next, a preferred embodiment of the invention is described, without limiting the technical variants that an expert in the field can incorporate or modify, and which are within the scope of the inventive concept that we claim in this application.
EXAMPLES
(21) The plasma was obtained using conventional techniques, which (0.5-1 ml) were used to extract DNA from 41 patients with gastric cancer and from 30 healthy donors, using the “QIAamp DNA Mini Kit” according to the indications of suppliers (QIAGEN, USA). The extracted DNA was dissolved in 20 μL TE buffer and bisulfite conversion was performed using the EZ DNA Methylation-Gold™ Kit reagents (Zymo Research Corporation, Irvine, Calif., USA).
(22) The specific PCR for methylated DNA (MSP) of the miR-335 promoter was performed according to the procedure of Zhengrong Li, et al. (2014), using the primers that amplify the second CpG islet between bases 1683 and 1808 of said promoter:
Primer1(forward):SEQ ID No.2;
Primer2(reverse):SEQ ID No.3.
(23) The conditions of the MSP were: denaturation 3 min. at 95° C., followed by 35 cycles of 30 sec. at 95° C., 30 sec. at 52° C. and 40 sec. at 72° C. The experiments were performed three times independently. The reported results represent the best combination of 3 pairs of primers evaluated for the miR-335 promoter region. The other evaulated sequences had sensitivity and specificity problems for determination in plasma.
(24) The results showed positive amplification bands in 23 of 41 (56.1%) plasma samples from patients with gastric cancer, but only in 9 of 30 (27.8%) plasma samples from healthy donors, thus, being the observed difference significant, (p=0.029, Pearson's correlation).
(25)
(26) This figure shows the “M” amplification, which corresponds to the result of the PCR product with specific primers for DNA methylation of the sequence comprising the promoter of the expression of microRNA335; and MyoD, an internal and constitutive marker in plasma was used as a DNA conversion control. CP corresponds to the positive control of methylation (methylated gastric cancer cell line); CN corresponds to the negative control of methylation (peripheral blood lymphocytes).
(27) As can be seen, a strong degree of methylation of this region is seen in 4 of the 6 patients with gastric cancer, while in healthy volunteers a low degree of methylation is seen only in 2 cases (lanes 4 and 6).
(28) In this way, it is found that an increase in the methylation of the miR-335 promoter region in plasma can be correlated with the presence of gastric cancer. Where, the method of the invention can be used by itself or in combination with other biological markers for the early detection of gastric cancer in plasma samples from an individual.