CN110484620B

CN110484620B - Biomarker and application thereof in preparation of product for diagnosing PTMC (ptm-associated tumor cell)

Info

Publication number: CN110484620B
Application number: CN201910735625.5A
Authority: CN
Inventors: 唐爱发; 戴道凤; 赵永胜
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2019-08-09
Filing date: 2019-08-09
Publication date: 2023-09-19
Anticipated expiration: 2039-08-09
Also published as: CN110484620A

Abstract

The application discloses a biomarker and application thereof in preparation of products for diagnosing PTMC, wherein the biomarker is selected from one or more of hsa-miR-7-5p, hsa-miR-376a-3p, hsa-miR-4306 and hsa-miR-183-5p. By the biomarker, noninvasive screening and identification of PTMC can be realized, and noninvasive diagnosis of PTMC can be realized.

Description

Biomarker and application thereof in preparation of product for diagnosing PTMC (ptm-associated tumor cell)

Technical Field

The application relates to the fields of inspection medicine, clinical medicine, biotechnology, biochemistry and molecular biology, in particular to a biomarker for screening and identifying papillary thyroid carcinoma (papillary thyroid microcarcinoma, PTMC) and application thereof.

Background

Establishing a non-invasive diagnostic method for PTMC is always an ideal target for continuous pursuit. The method has important significance for establishing an early noninvasive diagnosis method by screening biomarkers for early diagnosis in serum and plasma of patients.

Disclosure of Invention

The application provides a novel biomarker and application thereof in preparation of products for diagnosing PTMC.

The application provides the use of a reagent for detecting a biomarker in a sample, the biomarker being selected from one or more of hsa-miR-7-5p, hsa-miR-376a-3p, hsa-miR-4306 and hsa-miR-183-5p, in the preparation of a product for diagnosing PTMC. The plural kinds refer to at least two kinds.

The biomarker is selected from one of hsa-miR-7-5p, hsa-miR-376a-3p, hsa-miR-4306 and hsa-miR-183-5p.

The biomarker is hsa-miR-183-5p.

The expression level of the biomarker is up-regulated compared to the reference.

A kit for diagnosing PTMC comprising a biomarker selected from one or more of hsa-miR-7-5p, hsa-miR-376a-3p, hsa-miR-4306, and hsa-miR-183-5p.

The beneficial effects of the application are as follows: by the biomarker, noninvasive screening and identification of PTMC can be realized, and noninvasive diagnosis of PTMC can be realized.

Drawings

FIG. 1 is a volcanic and Wen plot of exosome miRNAs differentially expressed in patients with papillary thyroid carcinoma (tumor size. Gtoreq.1 cm) and papillary thyroid carcinoma (PTMC) by small RNA sequencing analysis;

FIG. 2 is a graph of results of RT-qPCR validation of candidate exosome miRNAs;

FIG. 3 is a graph of ROC analysis results for 7 candidate miRNAs;

FIG. 4 is a volcanic and Wen plot of exosome miRNAs differentially expressed in papillary thyroid carcinoma (WLNM) and papillary thyroid carcinoma (LLNM) patients analyzed by small RNA sequencing;

FIG. 5 is a graph of results of validating expression of exosomes miR-4433a-5p in validation cohort patient plasma using RT-qPCR and diagnosing value of papillary thyroid carcinoma (LLNM) using ROC analysis of miR-4433a-5 p.

Detailed Description

The application will be described in further detail below with reference to the drawings by means of specific embodiments.

Experimental method

Patient' s

The application comprises 182 thyroid disease patients who receive thyroidectomy in Shenzhen second people hospital from 2017 1 to 2019, including PTC patients and benign thyroid nodule patients. These patients were divided into a screening cohort (n=27) and a validation cohort (n=155). Patient details are given in the table below.

The population in the above table is the PTC patient population with benign thyroid nodule patients subtracted, wherein the screening cohort includes 10 benign thyroid nodules and the validation cohort includes 59 benign thyroid nodules. In the screening and validation cohorts, group A refers to benign thyroid nodule patients, group B refers to PTMC patients, group C refers to PTC patients with tumor diameters of 1cm or more, group D refers to PTC patients without lymph node metastasis, and group E refers to PTC patients with lateral cervical lymph node metastasis. P in each group refers to plasma samples and S refers to serum samples.

Extraction of plasma and serum exosomes

5ml of plasma and serum were collected for exosome extraction, respectively, per sample. The exosomes were separated using a combination of centrifugation and ultracentrifugation. The plasma or serum samples were first centrifuged at 17,000Xg for 30 minutes at 4℃to remove cell debris. The supernatant was collected and the exosomes were centrifuged at 120,000Xg for 2 hours at 4℃using an Optima XPN-100 ultracentrifuge (Beckman Coulter, USA). The precipitate was washed with 11ml of phosphate buffer. The ultracentrifugation step is repeated again. Finally, the exosome precipitate is dissolved in deionized water.

Exosome RNA extraction

To the extracted exosomes was added 1ml TRIzol (Invitrogen, USA) and left at room temperature for 3 minutes. 200 μl of chloroform was added, and the mixture was vigorously shaken by hand for 15 seconds, left at room temperature for 3 minutes, and centrifuged at 13,000Xg for 15 minutes at 4 ℃. The chloroform treatment step was repeated once to remove the protein as much as possible. The supernatant was transferred to a new centrifuge tube, 500. Mu.l of isopropyl alcohol and 5. Mu.l of glycogen were added, and the mixture was allowed to stand at 4℃for 30 minutes to precipitate RNA as much as possible. Centrifuge at 13,000Xg for 15 minutes at 4 ℃. The supernatant was decanted and 1ml of 75% ethanol was added to wash the RNA pellet. Standing for 10 min, and centrifuging at 13,000Xg for 5 min at 4deg.C. The ethanol was removed, the tube cap was opened, and the mixture was dried at room temperature for about 10 minutes, and 30. Mu.l of nuclease-free water was added to dissolve RNA. RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific, USA).

Sequencing of Small RNAs

A small RNA sequencing library was constructed using 0.2-1. Mu.g of RNA samples. RNA fragments of different sizes were separated by PAGE gels and 18-30nt bands were selected. The 3 'adapter, reverse transcription primer and 5' adapter are added sequentially to the selected RNA. Single-stranded cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, USA). PCR amplification was performed to enrich for cDNA fragments. PCR products of 100-120bp were purified by PAGE gels, followed by thermal denaturation and cyclization. Single-stranded circular DNA was used as the final library. The quality of the library was verified on a Agilent Technologies 2100 Bioanalyzer. Sequencing was performed on BGISEQ-500.

RT-qPCR

Because RT-qPCR in plasma lacks proper miRNA reference, synthesized cel-miR-39 (Ruibo) is used as reference of plasma exosome miRNART-qPCR, and cel-miR-39 with a final concentration of 10nM is added to RNA before cDNA synthesis.

Adding Poly (A) tail and cDNA to synthesize:

1) Adding the following solution

Reagent(s)	Volume (mul)
		mRQBuffer(2x)	5
RNA	3.75
		mRQEnzyme	1.25

2) The reaction was stopped by inactivating the enzyme by reacting at 37℃for 1 hour and then at 85℃for 5 minutes.

3) To the above solution was added 90. Mu.l of ddH ₂ O makes the total volume 100. Mu.l. This cDNA solution can be used directly in downstream qPCR reactions.

qPCR

Taking cel-miR-39 as an internal reference, the reaction systems of the target miRNA and the cel-miR-39 are respectively configured. The target miRNA reaction system is shown in the following table:

reagent(s)	Volume (mul)
		ddH2O	9
SYBR Advantage Premix(2X)	12.5
		ROX Dye(50X)	0.5
Target miRNA specific primer (10 mu M)	0.5
		mRQ 3’Primer	0.5
cDNA	2.0

The cel-miR-39 reaction system is shown in the following table:

reagent(s)	Volume (mul)
		ddH2O	9
SYBR Advantage Premix(2X)	12.5
		ROX Dye(50X)	0.5
cel-miR-39 primer (10 mu M)	0.5
		mRQ 3’Primer	0.5
cDNA	2.0

The reaction conditions were as follows:

Denaturation

95℃10sec

qPCR x 40 Cycles

95℃5sec

60℃20sec

Dissociation Curve

95℃60sec

55℃30sec

95℃30sec

using equation 2 ^-ΔCt To relatively quantify the expression of exosome mirnas. Delta ct=ct (miRNA of interest) -Ct (cel-miR-39).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0. Student's t-test was used to analyze statistically significant differences between control and experimental groups. P values < 0.05 were considered to be significantly different. ROC analysis was performed using miRNA expression levels of RT-qPCR data (receiver operating characteristic curve analysis).

Results

Screening and identifying thyroid papillary carcinoma (papillary thyroid microcarcinoma, PTMC) exosome miRNA markers

The screening cohort included 10 patients with benign thyroid nodules and 7 patients with PTMC, and plasma and serum exosome samples from these patients were used for small RNA sequencing to screen for exosome miRNA markers of PTMC, respectively. Patients with benign thyroid nodules were designated as group a and patients with PTMC as group B. Comparing small RNA sequencing data from plasma exosomes from group a and group B patients, 132 up-regulated mirnas and 500 down-regulated mirnas were found (as shown in figure 1A). Volcanic images showed that the expression of serum exosome mirnas was significantly different for group a and group B patients, including 230 up-regulated mirnas and 302 down-regulated mirnas (as shown in figure 1B). Of the upregulated mirnas, only 23 were present in both plasma and serum exosomes (as shown in fig. 1C). These 23 mirnas are listed in the following table. However, the expression of many exosome mirnas is low and unsuitable as a biomarker due to possible undetectability. Only 4 mirnas (expression values >3 in plasma and group B serum samples) were selected for further validation (see table below).

Note that: AP and BP represent plasma samples from group a and group B patients, respectively; AS and BS represent serum samples from group a and group B patients, respectively. Groups a and B represent patients with benign thyroid nodules (n=10) and PTMC (n=7) contained in the screening group (i.e., screening cohort), respectively. Possible biomarkers in group B are hsa-miR-7-5p, hsa-miR-376a-3p, hsa-miR-4306 and hsa-miR-183-5p.

The validation cohort included 59 patients with benign thyroid nodules (group a) and 40 patients with PTMC (group B). The 4 selected mirnas described above were validated in patients in a validation cohort using RT-qPCR. The expression levels of plasma exosomes miR-7-5P (P=0.0011), miR-376a-3P (P=0.0262) and miR-4306 (P < 0.0001) were significantly increased in group B patients compared to group A patients (as shown in FIGS. 2A, C and D). The highest increase in miRNA was miR-183-5p, which increased 1.89-fold (as shown in FIG. 2B). ROC analysis (receiver operating characteristic curve analysis) was performed to investigate the diagnostic value of miR-7-5p, miR-376a-3p, miR-4306 and miR-183-5p for PTMC. The AUC (area under the curve) range for the four mirnas was 0.6345 to 0.825 (as shown in fig. 3A-D). Wherein miR-183-5p exhibits the greatest AUC (0.825; 95% CI, 0.7344-0.9156) (as shown in FIG. 3B), followed by miR-4306 (0.753; 95% CI, 0.6536-0.8523) (as shown in FIG. 3D). Sensitivity and specificity of miR-183-5p were 82.5% and 74.58%, respectively. For miR-4306, the sensitivity and specificity are 70% and 64.41%, respectively. These results indicate that plasma exosome miR-183-5p has relatively high diagnostic accuracy for PTMC.

In fig. 1, volcanic plot A, B, D, E shows the distribution of differentially expressed exosome mirnas for AP vs BP, AS vs BS, AP vs CP and AS vs CS, respectively. AP, BP and CP represent plasma samples of A, B and group C patients, respectively; AS, BS and CS represent serum samples from group a, group B and group C patients, respectively. A. B and C are patients with benign thyroid nodules (n=10), PTMC (n=7) and papillary thyroid carcinoma (tumor size ≡1cm, n=10) in the screening group, respectively. Wien panel C shows significantly elevated exosome mirnas at AP vs BP and AS vs BS. Wien panel F shows significantly elevated exosome mirnas at AP vs CP and AS vs CS.

Screening and identifying thyroid papillary carcinoma (tumor diameter is more than or equal to 1 cm) exosome miRNA markers

The screening cohort included 10 patients with benign thyroid nodules and 10 patients with papillary thyroid carcinoma (tumor diameter. Gtoreq.1 cm), and plasma and serum exosome samples from these patients were used for small RNA sequencing to screen for exosome miRNA markers of PTMC, respectively. Patients with benign thyroid nodules were designated as group A, and patients with papillary thyroid carcinoma (tumor diameter. Gtoreq.1 cm) were designated as group C. Comparing small RNA sequencing data from plasma exosomes from group a and group C patients, 165 up-regulated mirnas and 396 down-regulated mirnas were found (as shown in figure 1D). Volcanic images showed that the expression of serum exosome mirnas was significantly different for group a and group B patients, including 207 up-regulated mirnas and 416 down-regulated mirnas (as shown in fig. 1E). Of the upregulated mirnas, only 28 were present in both plasma and serum exosomes (as shown in fig. 1F). The 28 mirnas are listed in the following table. However, the expression of many exosome mirnas was low, so only 3 mirnas (expression values >3 in plasma and group B serum samples) were selected for further validation (see table below).

Note that: AP and CP represent plasma samples from group a and group C patients, respectively; AS and CS represent serum samples from group a and group C patients, respectively. Groups a and C represent patients with benign thyroid nodules (n=10) and thyroid milky carcinoma heads (tumor diameter. Gtoreq.1 cm, n=7) contained in the screening group, respectively. Possible biomarkers in group C are hsa-miR-204-3p, hsa-miR-323b-3p, hsa-miR-654-5p.

The validation cohort included 59 patients with benign thyroid nodules (group A) and 56 patients with thyroid milky carcinoma heads (tumor diameter. Gtoreq.1 cm) (group C). The 3 selected mirnas described above were validated in patients in a validation cohort using RT-qPCR. Plasma exosomes miR-204-3P (P < 0.0001), miR-323b-3P (p=0.0141) and miR-654-5P (p=0.0014) were significantly elevated in group C patients compared to group a patients (as shown in fig. 2E-G). The expression of miR-204-3p is increased by 1.66 times, and the miRNA is the most increased of three miRNAs. ROC curve analysis showed AUCs for miR-204-3p, miR-323b-3p, and miR-654-5p to be 0.7415 (95% ci, 0.6499-0.8331) (shown in fig. 3E), 0.6161 (95% ci, 0.5111-0.721) (shown in fig. 3F), and 0.6524 (95% ci, 0.5495-0.7553) (shown in fig. 3G). Sensitivity and specificity of miR-204-3p were 66.07% and 76.27%, respectively. For miR-654-5p, the sensitivity and specificity are 58.93% and 72.88%, respectively. These results indicate that exosome miR-204-3p has relatively high diagnostic value for PTC (tumor size not less than 1 cm).

In FIG. 2, the patient plasma used for the validation cohort included 59 benign thyroid nodule patients (group A), 40 PTMC patients (group B) and 56 papillary thyroid carcinoma (tumor size. Gtoreq.1 cm) patients. In FIG. 3, ROC analysis was performed using RT-qPCR data of the validation queue.

Screening and verifying exosome miRNA markers of patients with lateral cervical lymph node metastasis thyroid papillary carcinoma

The screening cohort patients included 10 patients with benign thyroid nodules (group a), 8 patients without lymph node metastasis (without lymph node metastasis, WLNM, group D), and 3 patients with lateral lymph node metastasis (lateral lymph node metastasis, LLNM, group E). To identify exosome miRNA markers for patients with LLNM papillary thyroid cancer, the small RNA sequencing data of groups D and E were compared to that of group a, respectively. Comparing small RNA sequencing data from plasma exosomes from group a and group D patients, 78 up-regulated mirnas and 619 down-regulated mirnas were found (as shown in figure 4A). Volcanic images showed that the expression of serum exosome mirnas was significantly different for group a and group D patients, including 156 up-regulated mirnas and 432 down-regulated mirnas (as shown in fig. 4B). Of the upregulated mirnas, only 14 were present in both plasma and serum exosomes (as shown in fig. 4C). The expression of these 14 mirnas is shown in the table below.

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Note that: AP and DP represent plasma samples from group a and group D patients, respectively; AS and DS represent serum samples from group a and group D patients, respectively. Groups a and D represent patients with benign thyroid nodules (n=10) and thyroid milky carcinoma heads (WLNM, n=8) contained in the screening group, respectively. The biomarkers present in group D, E were hsa-miR-219b-5p, hsa-miR-376a-3p, hsa-miR-4433a-5p and hsa-miR-7705.

Comparing small RNA sequencing data from plasma exosomes from group a and group E patients, 200 up-regulated mirnas and 361 down-regulated mirnas were found (as shown in figure 4D). Volcanic images showed that the expression of serum exosome mirnas was significantly different for group a and group E patients, including 265 up-regulated mirnas and 350 down-regulated mirnas (as shown in figure 4E). Of the upregulated mirnas, only 66 were present in both plasma and serum exosomes (as shown in fig. 4F). The 66 miRNAs were expressed as shown in the following table.

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Note that: AP and EP represent plasma samples from group a and group E patients, respectively; AS and ES represent serum samples from group a and group E patients, respectively. Groups a and E represent patients with benign thyroid nodules (n=10) and thyroid milky carcinoma heads (LLNM, n=3) contained in the screening group, respectively. The biomarkers present in group D, E were hsa-miR-219b-5p, hsa-miR-376a-3p, hsa-miR-4433a-5p and hsa-miR-7705.

These results indicate that 66 possible exosome miRNA markers for LLNM and 14 possible exosome miRNA markers for WLNM's papillary thyroid cancer were determined. To obtain exosome miRNA markers that can distinguish between LLNM and WLNM papillary thyroid carcinomas, these possible biomarkers were analyzed in both groups, and only 4 exosome miRNAs (miR-219 b-5p, miR-376a-3p, miR-4433a-5p, and miR-7705) were found to be present in the biomarker list of both groups simultaneously. In addition, both miR-4433a-5p and miR-7705 were upregulated in group E plasma and serum exosomes compared to group D; the up-regulation of miR-4433a-5p is higher in multiple. These results indicate that exosomes miR-4433a-5p are likely to be biomarkers for papillary thyroid cancer that distinguish LLNM from WLNM.

The validation cohort included 59 patients with benign thyroid nodules (group a), 44 patients with WLNM thyroid milky cancer (group D), and 52 patients with LLNM thyroid milky cancer (group E). The expression level of plasma exosomes miR-4433a-5P was increased 1.43-fold in group D patients compared to group a patients (p=0.0014); up-regulated 1.91-fold in group E (P < 0.0001) (as shown in fig. 5A). miR-4433a-5P increased 1.33-fold in group E (P < 0.0001) compared to group D (as shown in figure 5A).

ROC curves distinguishing group a and group D showed AUCs of miR-4433a-5p of 0.7454 (95% ci, 0.6491-0.8417), sensitivity and specificity of 81.82% and 64.41%, respectively (as shown in figure 5B). ROC curves distinguishing between groups a and E showed AUCs of miR-4433a-5p of 0.8688 (95% ci, 0.7988-0.9388), sensitivity and specificity of 82.69% and 81.36%, respectively (as shown in figure 5C). ROC curves distinguishing between group D and group E showed AUCs of miR-4433a-5p of 0.7177 (95% ci, 0.6130-0.8223), sensitivity and specificity of 73.08% and 63.64%, respectively (as shown in figure 5D). These results indicate that the plasma exosome miR-4433a-5p has high diagnostic value and can be used for identifying papillary thyroid carcinoma with LLNM.

In fig. 4, volcanic plot A, B, D, E shows the distribution of differentially expressed exosome mirnas for AP vs DP, AS vs DS, AP vs EP and AS vs ES, respectively. AP, DP and EP represent plasma samples from patients in groups A, D and E, respectively; AS, DS and ES represent serum samples from group a, group D and group E patients, respectively. Groups a, D and E are patients with benign thyroid nodules (n=10), papillary thyroid carcinomas (WLNM, n=8) and papillary thyroid carcinomas (LLNM, n=3) in the screening group, respectively. Wien panel C shows significantly elevated exosome mirnas at AP vs. dp and AS vs. ds. Wien panel F shows significantly elevated exosome mirnas at AP vs. ep and AS vs. es.

The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.

Claims

1. Use of a reagent for detecting the expression level of a biomarker in blood exosomes for the preparation of a product for diagnosing PTMC, characterized in that the biomarker is hsa-miR-183-5p, the expression level of which is up-regulated compared to a reference.