Application of DHHC19 in evaluation of ovarian cancer IDO activity

Abstract

The invention relates to application of DHHC19 in evaluation of ovarian cancer IDO activity, and belongs to the technical field of biological medicine. The reagent for detecting DHHC19 contains a primer, and the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2. In malignant progression of ovarian cancer, tumor tissue and plasma exosome DHHC19 of an ovarian cancer patient are in progressive high expression and are significantly related to IDO activity. By acquiring whole blood of an ovarian cancer patient, extracting plasma exosomes and detecting DHHC19 expression, real-time evaluation of IDO activity in malignant development of ovarian cancer can be realized, and clinical diagnosis and treatment of ovarian cancer can be further guided. The method is effective and convenient. By detecting the relative expression level of the plasma exosome DHHC19 of an ovarian cancer patient, the IDO activity in the ovarian cancer progress is sensitively and quickly evaluated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of DHHC19 in evaluation of IDO activity in ovarian cancer. BACKGROUND

[0002] Indoleamine-2,3-dioxygenase (IDO) is a rate-limiting enzyme that catalyzes the first step of tryptophan catabolism along the kynurenine pathway and inhibits the immune function of T lymphocytes in the tumor microenvironment. Therefore, the ratio of kynurenine to tryptophan (K / T) can be detected to evaluate the IDO activity, and then the immunosuppression state of tumor patients can be judged. At present, the ways to detect the activity of IDO include: (1) detecting the expression of IDO protein: the expression of IDO protein in tumor tissue is directly detected by immunohistochemical method, but real-time detection cannot be achieved; (2) measuring IDO metabolites: the activity of IDO is reflected by the ratio of K / T, but the detection is complex and the false positive rate is high. Therefore, how to efficiently and accurately detect IDO is crucial to evaluate the clinical immunosuppression state of tumor patients. SUMMARY

[0003] The present application aims at overcoming the deficiencies of the prior art and providing application of DHHC19 in evaluation of IDO activity in ovarian cancer.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a reagent for detecting DHHC19, wherein the reagent contains primers, and the nucleotide sequences of the primers are shown in SEQ ID NO: 1 and 2.

[0005] The present application can sensitively and rapidly evaluate the IDO activity in ovarian progression by detecting the relative expression level of DHHC19 in plasma exosomes of ovarian cancer patients, and further guide the clinical diagnosis and treatment of ovarian cancer.

[0006] In a second aspect, the present application provides application of the reagent for detecting DHHC19 in preparation of a product for detecting ovarian cancer. In a third aspect, the present application provides application of the reagent for detecting DHHC19 in preparation of a product for detecting the activity of indoleamine-2,3-dioxygenase.

[0007] Further, the product is a product for detecting the activity of indoleamine-2,3-dioxygenase in ovarian cancer.

[0008] Still further, the reagent includes a reagent for detecting DHHC19 in plasma exosomes.

[0009] Still further, the reagent includes primers for detecting DHHC19 or immunohistochemical reagents for detecting DHHC19.

[0010] Further, the nucleotide sequences of the primers for detecting DHHC19 are shown as SEQ ID NO: 1 and 2.

[0011] In a fourth aspect, the present application provides a kit for detecting ovarian cancer, wherein the kit contains reagents for detecting DHHC19.

[0012] Further, the reagents contain primers, and the nucleotide sequences of the primers are shown as SEQ ID NO: 1 and 2.

[0013] Further, the reagents include reagents for detecting DHHC19 in plasma exosomes.

[0014] Compared with the prior art, the present application has the following beneficial effects: Tumor cell-derived exosomes are rich in tumor cell characteristic substances, especially proteins, and are widely present in patient body fluids, which can transfer these genetic information or tumor signaling pathways to the tumor microenvironment, promote tumor metastasis, and have significant advantages in early screening and clinical diagnosis of tumors. In the malignant progression of ovarian cancer, DHHC19 in tumor tissues and plasma exosomes of ovarian cancer patients is progressively highly expressed, and is significantly related to IDO activity. By obtaining whole blood of ovarian cancer patients, extracting plasma exosomes and detecting DHHC19 expression, real-time evaluation of IDO activity in the malignant progression of ovarian cancer can be achieved, further guiding the clinical diagnosis and treatment of ovarian cancer. The method is effective and convenient. By detecting the relative expression level of DHHC19 in plasma exosomes of ovarian cancer patients, the IDO activity in the progression of ovarian cancer is sensitively and rapidly evaluated. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 DHHC19 expression, IDO expression and ovarian malignant progression in ovarian cancer are positively correlated. Among them, A is the correlation of different DHHC family proteins palmitoyltransferases with ovarian cancer; B is the detection result of DHHC19 and IDO in different stage ovarian cancer tissues; C is the expression amount of DHHC19 in different stage ovarian cancer; D is the expression amount of IDO in different stage ovarian cancer; E is the pearson correlation analysis of DHHC19 and IDO expression in ovarian cancer.

[0016] Figure 2DHHC19 relative expression in plasma exosomes of ovarian cancer patients was used to evaluate IDO activity. A, relative expression of DHHC19 in plasma exosomes of patients at different stages; B, pearson correlation analysis of DHHC19 expression in plasma exosomes of ovarian cancer patients and IDO expression in tumor sections; C, critical value of DHHC19 expression obtained by ROC curve analysis; D, pearson correlation analysis of DHHC19 expression in plasma exosomes of 126 ovarian cancer patients and IDO expression in tumor sections; E, relationship between DHHC19 expression and clinical prognosis. DETAILED DESCRIPTION

[0017] To better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples. Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial channels.

[0018] Example 1: DHHC19 expression and IDO expression in cancer nests are positively correlated with ovarian malignant progression I. Experimental methods DHHC family proteins, palmitoyl transferases, are key enzymes for catalyzing protein S-palmitoylation modification, widely involved in protein stability and functional regulation, and closely related to tumor occurrence and development. To determine the correlation between DHHC family and ovarian cancer, 12 normal ovarian tissues and 24 ovarian cancer tissues were collected, and RNA was extracted for qRT-PCR detection.

[0019] Total RNA was extracted from normal tissues and ovarian tissues by TRIzol method, and was reverse transcribed into cDNA after DNase I treatment; primers were designed for human ZDHHC1-ZDHHC23 main transcripts, with product length of 80-160 bp, spanning exon junction sites, GC content of 40%-60%, and annealing temperature of 58-62℃; reference gene was TBP, which was used for normalization after stability screening.

[0020] DHHC1 (mRNA NCBI accession number: NM_001323627.2, protein NCBI accession number: NP_001310556.1) forward sequence: 5'-GATAGGCATAAGAAGGAGCA-3'; reverse sequence: 5'-CGGGCCAATTACCTGTCTTA-3'.

[0021] DHHC2 (NCBI Accession No. NM_001362988.2 for mRNA, NP_001349917.1 for protein) Forward sequence: 5'-GGCATTCAATTTACTCGACC-3'; Reverse sequence: 5'- CCCATATATCTCTTCCGGAT-3'.

[0022] DHHC3 (NCBI Accession No. NM_001135179.2 for mRNA, NP_056192.1 for protein) Forward sequence: 5'-TGGCAGATGTTCATCCAATC-3'; Reverse sequence: 5'- GAGTTCCATTACGTGGTAAC-3'.

[0023] DHHC4 (NCBI Accession No. NM_024645.5 for mRNA, NP_078995.2 for protein) Forward sequence: 5'-TGTGCTGATGCTGATGTTGA-3'; Reverse sequence: 5'- CCTTGAATGGAAGCATTGTA-3'.

[0024] DHHC5 (NCBI Accession No. NM_015457.3 for mRNA, NP_056272.2 for protein) Forward sequence: 5'-AATGGCATCTTCTGGAACAA-3'; Reverse sequence: 5'- CTGGAACCTTGATGGAATGA-3'.

[0025] DHHC6 (NCBI Accession No. NM_001303134.2 for mRNA, NP_001290063.1 for protein) Forward sequence: 5'-GCTTCAAGGATGCTGATGAA-3'; Reverse sequence: 5'- TAGGCTTCACTGGTGATGGA-3'.

[0026] DHHC7 (NCBI Accession No. NM_001145548.2 for mRNA, NP_001139020.1 for protein) Forward sequence: 5'-AGGCAATCTTCTGGTGAACA-3'; Reverse sequence: 5'- CTTGGTGATGGTGATGTTGA-3'.

[0027] DHHC8 (NCBI Accession No. NM_024503.5 for mRNA, NP_078853.2 for protein) Forward sequence: 5'-TGAAGGATGCTGGAATCATT-3'; Reverse sequence: 5'-GCTTCTGATGGAAGCTTCAA-3'.

[0028] DHHC9 (NCBI Accession No. NM_001008222.3 for mRNA, NP_001008223.1 for protein) Forward sequence: 5'-CAGGATGATGATGCTGATGA-3'; Reverse sequence: 5'-TTGACCTTGAAGGCTGATTA-3'.

[0029] DHHC10 (NCBI Accession No. NM_014761.5 for mRNA, NP_055536.1 for protein) Forward sequence: 5'-GATGCTGATGGAAGATGCTA-3'; Reverse sequence: 5'-CCAATTGGAAGTCTGATGGA-3'.

[0030] DHHC11 (NCBI Accession No. NM_001144998.2 for mRNA, NP_001138470.1 for protein) Forward sequence: 5'-TTGACGATGGAAGCTGATGA-3'; Reverse sequence: 5'-GGTTAAGGATGCTGGAATGA-3'.

[0031] DHHC12 (NCBI Accession No. NM_001318015.2 for mRNA, NP_001304944.2 for protein) Forward sequence: 5'-AAGAAGGATGCTGGAATGAA-3'; Reverse sequence: 5'-CTGGTTAAGGCTGATGGAAT-3'.

[0032] DHHC13 (NCBI Accession No. NM_018031.5 for mRNA, NP_060362.1 for protein) Forward sequence: 5'-GATGGAAGGCTTCAATGATT-3'; Reverse sequence: 5'-TTGACCTGATGGAAGCTTGA-3'.

[0033] DHHC14 (NCBI Accession No. NM_025111.4 for mRNA, NP_079282.1 for protein) Forward sequence: 5'-CTTGATGGAAGGCTGATGAT-3'; Reverse sequence: 5'-AGGCTTCAATGGAAGTCTTA-3'.

[0034] DHHC15 (NCBI Accession No. NM_001146256.2 for mRNA, NP_001139728.1 for protein) Forward sequence: 5'-GATGGAAGCTTCAATGGAAT-3'; Reverse sequence: 5'-CTTAAGGCTGATGGAAGTTA-3'.

[0035] DHHC16 (NCBI Accession No. NM_207170.3 for mRNA, NP_997298.1 for protein) Forward sequence: 5'-AAGGCTGATGGAATGCTTAA-3'; Reverse sequence: 5'-GGATGCTTAAGGCTGATGGA-3'.

[0036] DHHC17 (NCBI Accession No. NM_001359626.1 for mRNA, NP_060438.1 for protein) Forward sequence: 5'-TGGAATGCTGATGGAAGTTA-3'; Reverse sequence: 5'-CCTGATGGAAGCTTCAAGTA-3'.

[0037] DHHC18 (NCBI Accession No. NM_032283.3 for mRNA, NP_001346555.1 for protein) Forward sequence: 5'-GCTGGAAGTTAAGGCTGATT-3'; Reverse sequence: 5'-TTGGAAGCTTCAATGGAAGT-3'.

[0038] DHHC19 (NCBI Accession No. NM_001039617.2 for mRNA, NP_001034706.1 for protein) Forward sequence: 5'-CAAAGGTTGACATGCCCATCT-3'; Reverse sequence: 5'-CTTGCTGACATTGACGGGAA-3'.

[0039] DHHC20 (NCBI accession number of mRNA is NM_001286638.2, NCBI accession number of protein is NP_001273567.1) forward sequence: 5'-AAGGCTGATGGAATGCTTGA-3'; reverse sequence: 5'-GATGCTTCAAGGCTGATGGA-3'.

[0040] DHHC21 (NCBI accession number of mRNA is NM_001354118.2, NCBI accession number of protein is NP_001341047.1) forward sequence: 5'-TTAAGGCTGATGGAAGCTTA-3'; reverse sequence: 5'-GGAATGCTGATGGAAGCTTA-3'.

[0041] DHHC22 (NCBI accession number of mRNA is NM_001012729.3, NCBI accession number of protein is NP_001012747.1) forward sequence: 5'-CTGATGGAAGCTTAAGGCTT-3'; reverse sequence: 5'-AAGGCTTCAATGGAAGCTTA-3'.

[0042] DHHC23 (NCBI accession number of mRNA is NM_001320466.2, NCBI accession number of protein is NP_001307395.1) forward sequence: 5'-GGAAGCTTCAATGGAATGCT-3'; reverse sequence: 5'-TTGCTGATGGAAGGCTTCAA-3'.

[0043] qRT-PCR adopts SYBR Green method, reaction system 10 μL: 2 × SYBR Master Mix 5.0 μL, forward and reverse primers each 0.4 μL (10 μM), cDNA 1 μL, the rest is supplemented with water; the program is 95 ℃ pre-denaturation for 30 s, then 40 cycles: 95 ℃ 5 s, 60 ℃ 20-30 s, collect fluorescence, after amplification, perform 65-95 ℃ melting curve analysis. All samples are set with three duplicate holes and no template negative control (NTC) and no reverse transcriptase control (-RT), the amplification efficiency is controlled in 90-110%, the data is calculated by 2^-ΔΔCt method to calculate the relative expression amount.

[0044] Further, 35 early-stage ovarian cancer tissues (Figo 2018, stage I-II) and 23 advanced ovarian cancer tissues (Figo 2018, stage III-IV) were collected to prepare paraffin sections of ovarian cancer tissues, and the expression levels of DHHC19 and IDO (NCBI accession number of mRNA: NM_002164.6, NCBI accession number of protein: NP_002155.1) in the tissues were detected by immunohistochemical method.

[0045] After the paraffin sections of ovarian cancer tissues (4 μm) were routinely dewaxed, xylene and gradient ethanol hydrated, rehydrated sections were obtained. The rehydrated sections were treated with 0.3% (v / v) H2O2 at room temperature for 10 min to block endogenous peroxidase in the sections to obtain H2O2-treated sections. The H2O2-treated sections were subjected to microwave / pressure cooker antigen retrieval with 0.01 M citrate buffer (pH 6.0) for 20 min (or Tris-EDTA pH 9.0 can be used for antigen retrieval), cooled to room temperature and washed with PBS to obtain antigen-repaired sections. The antigen-repaired sections were blocked with 5% (v / v) normal serum or BSA for 30 min to reduce non-specific binding to obtain blocked sections. The diluted DHHC19 (Cat. M361929-2, DAKO) and IDO (Cat. 86630, CST) primary antibodies (dilution ratio of 1:400) were added, and the sections were incubated at 4°C overnight to obtain primary antibody-incubated sections. The primary antibody-incubated sections were washed with PBS, and the secondary antibody (ZSGB, BioTech) was added and incubated at room temperature for 60 min to obtain secondary antibody-incubated sections. The secondary antibody-incubated sections were washed with PBS, and diaminobenzidine (DAB) was used for color development for 1 min, and the color development was stopped by water washing, and then the sections were stained with hematoxylin for 30 s, washed with running water, and then the stained sections were dehydrated with alcohol and transparentized with xylene, and then mounted, observed under a microscope and scored, and a semi-quantitative score (H-score = Σ (cell staining intensity x percentage of positive cells) was used.

[0046] II. Experimental results Compared with normal ovarian tissues, only DHHC19 in the DHHC family was significantly highly expressed in ovarian cancer tissues Figure 1 A).

[0047] Compared with early-stage ovarian cancer tissues, the expression of DHHC19 and IDO in the cancer nest region of advanced tissues was significantly increased Figure 1 B, Figure 1 C and Figure 1 D). Spearman correlation analysis showed that the expression of DHHC19 and IDO was significantly positively correlated (r = 0.521, P < 0.001) Figure 1 E).

[0048] Example 2 Assessment of IDO activity by relative expression of DHHC19 in plasma exosomes of ovarian cancer patients 1. Extraction of human plasma exosomes (1) Collect whole blood samples from 35 patients with early stage ovarian cancer (Figo 2018, stage I-II) and 23 patients with advanced ovarian cancer (Figo 2018, stage III-IV), centrifuge at 3000g for 30 min at 4°C to obtain plasma.

[0049] (2) Remove residual blood cells by centrifuging the plasma from step (1) at 8000g for 30 min at 4°C.

[0050] (3) Remove cell debris by centrifuging the plasma from step (2) at 12000g for 30 min at 4°C.

[0051] (4) Dilute the plasma from step (3) with an equal volume of PBS, centrifuge at 160000g for 16 h at 4°C to enrich plasma exosomes.

[0052] (5) Exosome protein extraction and qRT-PCR detection.

[0053] 2. To ensure the accuracy and reproducibility of DHHC19 expression in qRT-PCR detection, three pairs of candidate primers (Primer A, B, C) were designed, optimized according to the principles of GC content, Tm value matching and avoidance of duplex structure, and the length of primer amplification product was controlled at 120-150 bp to adapt to the characteristics of exosome RNA fragments. The three pairs of primers are 20-22 nt, GC is 45-55%, the length of the product is 120-150 bp, and the candidate sequences are optimized by NCBIPrimer-BLAST specificity search and OligoAnalyzer duplex / hairpin prediction.

[0054] qRT-PCR was performed using SYBR Green method, the reaction system was 10 μL: 2xSYBR Master Mix 5.0 μL, forward and reverse primers 0.4 μL each (10 μM), exosome cDNA 1 μL, and the rest was made up with water; the program was 95°C pre-denaturation for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 20-30 s, fluorescence collection, and melting curve analysis at 65-95°C after amplification. All samples were set up in triplicate, with no template negative control (NTC) and no reverse transcriptase control (-RT), the amplification efficiency was controlled at 90-110%, and the relative expression was calculated by 2^-ΔΔCt method.

[0055] Primer A: Forward sequence: 5'-CAAAGGTTGACATGCCCATCT-3' (SEQ ID NO: 1); Reverse sequence: 5'-CTTGCTGACATTGACGGGAA-3' (SEQ ID NO: 2).

[0056] Primer B: Forward sequence: Forward 5'-GCTGATGACCTGGTGATGGA-3'; Reverse sequence: Reverse 5'-ACAGCCTTCTTGGGCTTCTT-3'.

[0057] Primer C: Forward sequence: Forward 5'-TGGACCGTATGCTGATGAGA-3'; Reverse sequence: Reverse 5'-GGATCAGGTGCAGGATGTTT-3'.

[0058] The miRNeasy Mini kit was used to extract plasma exosome RNA, and the expression level of DHHC19 was detected by qRT-PCR.

[0059] 3. In another group of 126 cases of ovarian cancer patient cohort, the correlation between the content of plasma exosome DHHC19 and the expression level of IDO in ovarian cancer tissue was further verified by Spearman correlation analysis and Kaplan-Meier prognosis analysis, and the value of plasma exosome DHHC19 in predicting the prognosis of ovarian cancer was evaluated.

[0060] II. Experimental results 1. The amplification efficiency of Primer A was 96.3% (R 2 = 0.998), which was better than that of Primer B (R 2 = 88.1%) and Primer C (R 2 = 103.4%). Melting curve analysis showed that Primer A had a single specific peak, while B and C groups had secondary peaks, suggesting that there might be non-specific amplification. In summary, the efficiency, linear relationship and specificity were selected, and Primer A was finally selected as the optimal primer group for qRT-PCR detection of DHHC19 expression.

[0061] 2. The results showed that the expression of DHHC19 in plasma exosomes of patients with advanced ovarian cancer was significantly higher than that of patients in early stage ( Figure 2 A). Spearman correlation analysis further showed that the expression of plasma exosome DHHC19 was significantly positively correlated with the expression of IDO (r = 0.702, P < 0.001) Figure 2 B).

[0062] 3、According to the immunohistochemical score of IDO (H-score =∑pi(i+1), wherein pi is the proportion of positive cells, and i is the staining intensity), H-score≥8 is defined as high IDO activity, and H-score<8 is defined as low activity. Combined with ROC curve analysis, it is found that the plasma exosome DHHC19 level can effectively predict the IDO activity, and the AUC is 0.899 (P<0.001). According to Youden's index, the optimal threshold for evaluating IDO activity is 0.802: when the relative expression of DHHC19 is ≥0.802, it indicates high IDO activity, and vice versa Figure 2 C).

[0063] In another group of 126 patients with ovarian cancer, the plasma exosome DHHC19 level was further verified to be significantly positively correlated with the expression of IDO (r=0.5361, P<0.0001) Figure 2 D). In addition, the clinical prognosis of patients with high expression of DHHC19 is significantly poorer, and the prognosis of patients with low expression is better Figure 2 E).

[0064] Example 3 A kit for detecting ovarian cancer and its IDO activity I. Composition 1. Primers, as shown in SEQ ID NO: 1 and 2.

[0065] 2. Other reagents: 2x SYBR Master Mix and water.

[0066] II. Method of use 1. Extraction of human plasma exosomes (1) Collect whole blood samples from patients to be tested, centrifuge at 3000g at 4°C for 30 min, and obtain plasma.

[0067] (2) Centrifuge the plasma of step (1) at 8000g at 4°C for 30 min to remove residual blood cells.

[0068] (3) Centrifuge the plasma treated in step (2) at 12000g at 4°C for 30 min to remove cell debris.

[0069] (4) Dilute the plasma treated in step (3) with the same volume of PBS, centrifuge at 160000g at 4°C for 16h to enrich plasma exosomes, extract RNA and reverse transcribe into cDNA.

[0070] 2. Extraction of plasma exosome RNA for qRT-PCR detection qRT-PCR adopts SYBR Green method, reaction system 10 μL: 2 x SYBR Master Mix 5.0 μL, forward and reverse primers each 0.4 μL (10 μM), cDNA 1 μL, the rest is supplemented with water; program is 95℃ pre-denaturation 30s, then 40 cycles: 95℃ 5s, 60℃ 20-30s, collect fluorescence, after amplification, perform 65-95℃ melting curve analysis. All samples are set with three duplicate holes and no template negative control (NTC) and no reverse transcriptase control (-RT), amplification efficiency is controlled in 90-110%, data is calculated with 2^-ΔΔCt method to calculate relative expression.

[0071] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A reagent for detecting DHHC19, characterized by, The reagent contains a primer, and the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and 2.

2. Application of the reagent for detecting DHHC19 in the preparation of a product for detecting ovarian cancer.

3. Application of the reagent for detecting DHHC19 in the preparation of a product for detecting indoleamine-2,3-dioxygenase activity.

4. Use according to claim 3, wherein the compound is ###0002### The product is a product for detecting indoleamine-2,3-dioxygenase activity in ovarian cancer.

5. Use according to any one of claims 2 to 4, wherein the compound is ###0002### The reagent includes a reagent for detecting DHHC19 in plasma exosomes.

6. Use according to any one of claims 2 to 4, wherein The reagent includes a primer for detecting DHHC19 or an immunohistochemical reagent for detecting DHHC19.

7. Use according to claim 6, wherein The nucleotide sequence of the primer for detecting DHHC19 is shown as SEQ ID NO: 1 and 2.

8. A kit for detecting ovarian cancer, characterized by, The kit contains a reagent for detecting DHHC19.

9. The kit of claim 8, wherein The reagent contains a primer, and the nucleotide sequence of the primer is shown as SEQ ID NO: 1 and 2.

10. The kit of claim 8, wherein The reagent includes a reagent for detecting DHHC19 in plasma exosomes.

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