CN114807375A - Melanoma biomarker of urine sample and application thereof - Google Patents

Melanoma biomarker of urine sample and application thereof Download PDF

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CN114807375A
CN114807375A CN202210626270.8A CN202210626270A CN114807375A CN 114807375 A CN114807375 A CN 114807375A CN 202210626270 A CN202210626270 A CN 202210626270A CN 114807375 A CN114807375 A CN 114807375A
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melanoma
biomarker
mirna
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许明炎
张晓妮
林郁松
李慧
周书雄
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Haplox Biotechnology Shenzhen Co ltd
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Abstract

The application discloses a melanoma biomarker of a urine sample and application thereof. The melanoma biomarker of the urine sample is miRNA of the sequence shown in Seq ID No. 1. The melanoma biomarker of the urine sample is expressed at a high level in the urine of a melanoma patient, and the expression level in the urine of a normal healthy person is significantly lower; therefore, melanoma can be judged by detecting the expression level of the melanoma biomarker of the application in a urine sample. The biomarker provides a new scheme and way for detecting the melanoma, and is particularly suitable for early screening and large-scale screening of the melanoma.

Description

Melanoma biomarker of urine sample and application thereof
Technical Field
The application relates to the technical field of melanoma biomarkers, in particular to a melanoma biomarker of a urine sample and application thereof.
Background
Melanoma originates in melanocytes in the skin, is the most common type of skin cancer, and has a high degree of malignancy and a poor prognosis. Usually, melanoma is found to have multiple metastases, leading to a poor prognosis.
MicroRNA (miRNA) is a single-stranded non-coding RNA molecule with the length of about 20nt and conserved in evolution, and is combined with target messenger RNA (mRNA) through base complementary pairing, and the target mRNA is guided to degrade or the translation of the target mRNA is inhibited, so that the generation of target protein is prevented.
The misexpression of miRNA in cells can cause the occurrence of various diseases including melanoma, and the latest research shows that some miRNA are abnormally expressed in the blood of melanoma patients, but no consensus is reached about which miRNA is related to the occurrence and development of melanoma. Therefore, it is necessary to find miRNA related to the occurrence and development of melanoma, so as to provide an effective means for clinical diagnosis and treatment of melanoma.
Disclosure of Invention
The application aims to provide a novel melanoma biomarker suitable for a urine sample and application thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
one aspect of the application discloses a melanoma biomarker of a urine sample, wherein the melanoma biomarker is miRNA of a sequence shown in Seq ID No. 1;
Seq ID No.1：5’-UAGCUUAUCAGACUGAUGUUGA-3’。
it should be noted that, in the present application, through comparative analysis of mirnas extracted from melanoma and urine of healthy people, a new melanoma biomarker, namely, miRNA with a sequence shown in Seq ID No.1, is found, and the melanoma biomarker is expressed at a high level in urine of melanoma patients, while the expression level in urine of normal healthy people is significantly lower. The melanoma biomarker provides a new scheme and way for detecting melanoma through urine. Also, urine is a liquid metabolic waste product produced by the kidneys and excreted through the urinary tract; as a novel biological sample, the biological sample has the characteristics of non-invasive collection, easy acquisition and convenient mass adoption. The melanoma biomarker has obvious expression difference in urine, and can be used for early screening and large-scale screening of melanoma; compare in the method that carries out the melanoma detection through the blood sample, carry out urine sample detection screening melanoma based on the melanoma biomarker of this application, it is more convenient to sample.
In another aspect of the present application, a cloning vector is disclosed, which comprises the melanoma biomarker or its expression gene of the present application.
It will be appreciated that in order to facilitate the use of the melanoma biomarkers of the present application, the melanoma biomarkers of the present application or their expressed genes may be cloned into a suitable vector.
In yet another aspect of the present application, a cell or vesicle comprising a melanoma biomarker or expression gene thereof of the present application, or comprising a cloning vector of the present application is disclosed.
It will be appreciated that the melanoma biomarkers or genes expressed therein, or cloning vectors containing the melanoma biomarkers or genes expressed therein, may be introduced into cells or vesicles for ease of storage.
In a further aspect, the present application discloses the use of the melanoma biomarker of the present application, the cloning vector of the present application, or the cell or vesicle of the present application in the preparation of a reagent for detecting melanoma.
It should be noted that the applications of the present application mainly include, for example, designing primers and/or probes for specific detection based on the melanoma biomarkers of the present application, or using the cloning vector of the present application as a positive control, and the like, and are not particularly limited herein.
In one implementation manner of the present application, the reagent for detecting melanoma mentioned in the application of the present application is mainly a reagent for detecting melanoma by using a urine sample.
In another aspect, the application discloses an application of miRNA or a cloning vector, cell or vesicle containing the miRNA as a biomarker for detecting melanoma by a urine sample, wherein the miRNA is a sequence shown in Seq ID No. 1.
Yet another aspect of the present application discloses a kit for detecting melanoma, which includes reagents capable of specifically detecting the melanoma biomarkers of the present application.
In one implementation manner of the present application, the reagent capable of specifically detecting the melanoma biomarker of the present application, which is referred to by the kit of the present application, is a specific primer and/or probe designed for miRNA having a sequence shown in Seq ID No. 1.
It is understood that designing specific primers and/or probes for miRNA having the sequence shown in Seq ID No.1 is only one way to detect the melanoma biomarkers of the present application, and does not exclude other ways to detect the miRNA, and reagents capable of specifically detecting the melanoma biomarkers of the present application may be incorporated into the kit of the present application.
In one implementation of the present application, the kit of the present application further comprises a cloning vector of the present application and/or a cell or vesicle of the present application.
It is understood that the cloning vector of the present application and/or the cell or vesicle of the present application may be assembled into a kit as a positive control.
In one implementation manner of the present application, the kit of the present application further comprises a reagent for extracting urine miRNA.
It can be understood that the kit of the present application detects melanoma mainly by detecting the expression of the melanoma biomarker of the present application in a urine sample; therefore, for convenience of use, the required reagents for extracting the urine miRNA may be assembled into a kit. Of course, the reagent for extracting the urine miRNA may be purchased separately.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the melanoma biomarker of the application is expressed at a high level in the urine of a melanoma patient, and the expression level in the urine of a normal healthy person is significantly lower; through detecting the melanoma biomarker, the method can realize that the melanoma is detected by directly adopting a urine sample, provides a new scheme and way for melanoma detection, and is particularly suitable for early screening and large-scale screening of the melanoma.
Drawings
FIG. 1 shows the comparative analysis results of the expression levels of miRNA in the urine samples of melanoma patients and healthy human in the examples of the present application.
Detailed Description
The existing detection technology mainly detects the biomarker of melanoma through blood samples such as plasma or serum; however, early screening or large-scale screening of melanoma using blood samples has the problem of inconvenient sampling.
According to the application, a plurality of groups of miRNAs which are theoretically related to melanoma and express up-regulation or down-regulation are screened through existing literature data to serve as a target miRNA library. Then, urine of healthy people and melanoma patients is collected to carry out miRNA extraction separation and sequencing analysis, the sequencing result is compared with the target miRNA library and the miRbase database for analysis, the difference of the expression level of each miRNA in normal people and melanoma patients is compared, and a proper miRNA melanoma early screening biomarker, namely the miRNA with the sequence shown in Seq ID No.1, in the urine sample is screened out.
By adopting the melanoma biomarker, melanoma can be directly detected through a urine sample; the urine has the advantages of non-invasive collection, easy acquisition, convenient mass adoption and the like; provides a new biomarker for early screening and large-scale screening of melanoma.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
In this embodiment, multiple sets of mirnas theoretically related to melanoma and having up-regulated or down-regulated expression are screened as a target miRNA library according to existing literature data. 171 miRNA sequences were co-screened in this example, with the names shown in Table 1.
TABLE 1 target miRNA libraries
let-7a mir-182-5p mir-335 mir-138-5p mir-23a-5p mir-637
let-7a-1 mir-182 mir-337 mir-141 mir-27a mir-661
let-7a-2 mir-182-3p mir-33a mir-142-5p mir-27b mir-663a
let-7a-3 mir-186 mir-340 mir-143 mir-29a mir-663b
let-7b mir-18a mir-345-5p mir-144 mir-29b mir-675
let-7c mir-191 mir-34a mir-145 mir-29b-1 mir-7
let-7d mir-192 mir-34b mir-146a mir-29b-2 mir-7-5p
let-7e mir-193a-3p mir-34c mir-148a mir-29c mir-708-5p
let-7f-1 mir-194 mir-367 mir-148b mir-300 mir-744
let-7f-2 mir-195 mir-369-5p mir-150 mir-301a mir-873
let-7g mir-196a mir-373 mir-150* mir-301a-3p mir-876-3p
let-7i mir-196b mir-374a mir-150-5p mir-301b mir-9
mir-101 mir-198 mir-375 mir-152 mir-30a mir-9-5p
mir-107 mir-19b-3p mir-376a mir-15a mir-30b-5p mir-92
mir-10a mir-200a mir-377 mir-15b mir-31 mir-940
mir-10b mir-200b mir-378a-3p mir-16 mir-320a mir-96
mir-1178 mir-200c mir-410 mir-17-5p mir-323-3p mir-98
mir-1181 mir-200c-3p mir-424-5p mir-181a mir-329 mir-98-5p
mir-124 mir-203 mir-429 mir-181c mir-331-3p mir-99a
mir-1247 mir-205 mir-431 mir-1297 mir-216b-5p mir-506
mir-125a-3p mir-21-5p mir-448 mir-132 mir-217 mir-509-5p
mir-125a-5p mir-212 mir-449a mir-133a mir-218 mir-539
mir-126 mir-214 mir-451 mir-135a mir-221 mir-541
mir-126-3p mir-215 mir-451a mir-135a-1 mir-221-3p mir-613
mir-127 mir-216a mir-455-3p mir-135a-2 mir-222 mir-615-5p
mir-1271 mir-216a-3p mir-4787-5p mir-135b mir-223 mir-629
mir-128 mir-216a-5p mir-493 mir-135b-5p mir-224 mir-630
mir-1290 mir-216b mir-494 mir-137 mir-23a mir-634
mir-1291 mir-216b-3p mir-497
The miRbase database and the target miRNA library shown in Table 1 are used as reference sequences, the sequencing results of the miRNAs extracted from the urine sample are compared and analyzed, and the miRNAs with the expression levels being significantly different in the urine of a melanoma patient and normal healthy people are used as the melanoma biomarkers of the embodiment.
miRbase database website: https:// mirbase. org
Urine samples of 10 healthy people and 10 melanoma patients were collected for miRNA extraction and sequencing in each example. All urine samples were provided and stored by Shenzhen Heluos Biotech Limited.
Separating urine extracellular vesicles and extracting miRNA by using a trizol reagent, which comprises the following specific steps:
1) configuring 3 × Buffer EI: the method comprises the steps of preparing a solution with the concentration of polyethylene glycol of 450g/L and the concentration of sodium chloride of 1.5mol/L by using polyethylene glycol with the relative molecular mass of 6000-10000, sodium chloride and water. Configure 20 × Buffer NP: comprises the steps of preparing a 3mol/L sodium acetate solution with pH value of 5.2 by using sodium acetate, acetic acid and water.
2) A urine sample (15 mL) was centrifuged at 2000g at 4 ℃ for 15min, and the supernatant was used as the first supernatant.
3) The first supernatant was centrifuged at 4 ℃ at 20000g for 15min and the supernatant was taken as the second supernatant.
4) 10mL of the second supernatant was mixed with 5mL of 3 XBuffer EI, mixed well and left to stand at 4 ℃ for 16 h.
5) Centrifuging the mixed solution after standing in the step 4) at 4 ℃ and 20000g for 30min, removing supernatant, and obtaining precipitate, namely the extracellular vesicles.
6) To the above extracellular vesicles, 1mL of Trizol reagent was added, sufficiently shaken, and allowed to stand at room temperature for 20 min.
7) Adding 200 μ L of 1-bromine and 3-chloropropane, shaking, standing for 10min, centrifuging at 4 deg.C and 20000g for 20min, and collecting supernatant as third supernatant.
8) To the third supernatant was added 700. mu.L of-20 ℃ pre-cooled isopropanol, 70. mu.L of 20 XBuffer NP, and 7. mu.L of polyacrylic acid.
9) The mixture of step 8) was gently inverted and mixed, and left at-20 ℃ for 30 min.
10) Centrifuging at 4 deg.C for 20min at 20000g, removing supernatant, and retaining precipitate.
11) Adding 1mL of precooled 75% ethanol into the precipitate obtained in the step 10), and shaking and whirling to suspend the precipitate.
12) Centrifuging at 4 deg.C for 10min at 20000g, removing supernatant, and retaining precipitate.
13) 1mL of precooled 75% ethanol was added to the precipitate from step 12), and the precipitate was suspended by vortexing.
14) Centrifuging at 4 deg.C 20000g for 10min, removing supernatant, and retaining precipitate.
15) And (3) drying the precipitate obtained in the step 14) at room temperature, adding 30 mu L of RNase-free water, and uniformly mixing and dissolving to obtain the miRNA solution.
And (3) controlling the quality of the extracted miRNA solution by using an Invitrogen Qubit microRNA detection kit, wherein the miRNA concentration is more than or equal to 10,000ng/mL and the miRNA solution is a qualified sample. The results show that the miRNA concentrations of the urine samples of 20 healthy persons and 20 melanoma patients extracted in this example are all acceptable, and can be used for subsequent detection.
miRNA library preparation was performed on miRNA extracted from urine samples of 10 healthy persons and 10 melanoma patients using small RNA library preparation kit for Illumina library preparation from Vazyme.
Then, the prepared miRNA library was subjected to high-throughput sequencing using Illumina Novaseq 6000 sequencer, with a sequencing data volume of 3Gb, and high-throughput sequencing data analysis was performed using fastq program.
Analysis results show that the expression level of miR-21-5p in the urine of a melanoma patient is obviously higher than that of normal human urine. miR-21-5p is miRNA with the sequence shown in Seq ID No. 1.
Seq ID No.1：5’-UAGCUUAUCAGACUGAUGUUGA-3’。
The miRNA biomarker of the sequence shown in the Seq ID No.1 is generated by processing miRNA of the sequence shown in the Seq ID No.2 as a precursor in cells.
Seq ID No.2：
5’-UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA-3’。
The analysis results are shown in table 2, table 3 and fig. 1.
TABLE 2 mean comparison of miR-21-5p expression levels in healthy humans and melanoma patients
Sample(s) Mean value N Standard deviation of
Health sample 4258.1516 20 344.95199
Melanoma sample 416668.7811 20 20858.35344
TABLE 3 expression level one-way analysis of variance of miR-21-5p in healthy humans and melanoma patients
Sum of squares df Mean square F P
miR-21-5p 1700825272614.049 1 1700825272614.049 7816.474 ＜0.01
The results in table 2, table 3 and figure 1 show that the expression level of miR-21-5p in the urine of melanoma patients is significantly higher than that of healthy human urine, while the precursors thereof show no significant expression difference. Therefore, miRNA of the sequence shown in Seq ID No.1, namely miR-21-5p, is used as a melanoma biomarker of the urine sample.
According to the results, miR-21-5p can be used as a melanoma biomarker of a urine sample. If the expression level of miR-21-5p in the urine sample of the object to be detected is obviously higher than that of the urine sample of a healthy person as a control, the object to be detected can be judged to have melanoma.
Using the melanoma biomarkers of the urine samples obtained in this example, 54 urine samples of early-screened subjects with family history of melanoma, previous history of excessive uv exposure, or the presence of suspected melanoma nevi were tested according to the above method. And simultaneously performing biopsy to diagnose whether the detected object has melanoma. The results showed that 9 subjects had melanoma and the remaining 45 subjects did not have melanoma, as judged by the analysis of the melanoma biomarkers in the urine samples of this example; the biopsy results showed that 8 of the 9 patients who were positive in the biomarker of this example were positive, and 45 patients who were negative. Compared with the result of tissue biopsy, the detection result has the sensitivity of 100 percent and the specificity of 88.9 percent.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen Shanpulos Biotech Co., Ltd
<120> melanoma biomarker of urine sample and application thereof
<130> 22I33818
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> RNA
<213> miR-21-5p sequences
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uagcuuauca gacugauguu ga 22
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<212> RNA
<213> miR-21 sequences
<400> 2
ugucggguag cuuaucagac ugauguugac uguugaaucu cauggcaaca ccagucgaug 60
ggcugucuga ca 72

Claims (10)

1. A melanoma biomarker for a urine sample, characterized by: the melanoma biomarker is miRNA of a sequence shown in Seq ID No. 1;
Seq ID No.1：5’-UAGCUUAUCAGACUGAUGUUGA-3’。
2. a cloning vector, characterized in that: the cloning vector contains the melanoma biomarker according to claim 1 or an expression gene thereof.
3. A cell or vesicle, characterized by: the cell or vesicle comprising a melanoma biomarker according to claim 1 or an expression gene thereof, or comprising a cloning vector according to claim 2.
4. Use of a melanoma biomarker according to claim 1, a cloning vector according to claim 2, or a cell or vesicle according to claim 3 in the preparation of a reagent for detecting melanoma.
5. Use according to claim 4, characterized in that: the reagent for detecting the melanoma is a reagent for detecting the melanoma through a urine sample.
6. The application of miRNA or cloning vector, cell or vesicle containing the miRNA as a biomarker for detecting melanoma through a urine sample is characterized in that: the miRNA is a sequence shown in Seq ID No. 1.
7. A kit for detecting melanoma, which is characterized in that: the kit comprises reagents capable of specifically detecting the melanoma biomarker of claim 1.
8. The kit of claim 7, wherein: the reagent capable of specifically detecting the melanoma biomarker according to claim 1 is a specific primer and/or probe designed for miRNA of the sequence shown in Seq ID No. 1.
9. The kit according to claim 7 or 8, characterized in that: the kit further comprises a cloning vector of claim 2 and/or a cell or vesicle of claim 3.
10. The kit according to claim 7 or 8, characterized in that: the kit also comprises a reagent for extracting the urine miRNA.
CN202210626270.8A 2022-06-02 2022-06-02 Melanoma biomarker of urine sample and application thereof Pending CN114807375A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636308A (en) * 2015-10-30 2017-05-10 益善生物技术股份有限公司 Probe composition for detecting related marker of skin cancer and kit of probe combination
US20170175112A1 (en) * 2014-02-14 2017-06-22 Universite De Lausanne Mir-21-3p inhibitors in skin disorders
US20210254180A1 (en) * 2018-10-03 2021-08-19 Alma Mater Studiorum - Università di Bologna Method for the prognosis of melanoma

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US20170175112A1 (en) * 2014-02-14 2017-06-22 Universite De Lausanne Mir-21-3p inhibitors in skin disorders
CN106636308A (en) * 2015-10-30 2017-05-10 益善生物技术股份有限公司 Probe composition for detecting related marker of skin cancer and kit of probe combination
US20210254180A1 (en) * 2018-10-03 2021-08-19 Alma Mater Studiorum - Università di Bologna Method for the prognosis of melanoma

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Title
GIORGIO DURANTE等: "Circulating microRNA biomarkers in melanoma and non-melanoma skin cancer", EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, vol. 22, no. 3, 11 March 2022 (2022-03-11), pages 305 - 318 *
ZHAOHUI YANG等: "miR-21-5p promotes cell proliferation and G1/S transition in melanoma by targeting CDKN2C", FEBS OPEN BIO, vol. 10, 23 February 2020 (2020-02-23), pages 752 - 760 *
王理伟等: "肿瘤系列 胃癌 基础与临床的转化", 31 December 2020, 上海交通大学出版社, pages: 15 - 16 *
邓旭等: "hsa-miＲ-21-5p 靶基因预测及其在急性肾损伤中的调控机制", 儿科药学杂志, vol. 20, no. 7, 31 December 2014 (2014-12-31), pages 1 - 5 *

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