Application of ZDHHC22 as marker in preparation of high-risk neuroblastoma evaluation kit

Abstract

The invention discloses application of ZDHHC22 as a marker in preparation of a kit for evaluating high-risk neuroblastoma, and belongs to the technical field of biology. The invention discloses that abnormally high expression of ZDHHC22 is closely related to high-risk neuroblastoma for the first time, provides a novel high-risk neuroblastoma prediction biomarker, and develops a high-risk neuroblastoma diagnosis or prognosis evaluation kit by taking ZDHHC22 as a detection marker to realize layered diagnosis of neuroblastoma. The kit has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, good repeatability and the like, and is easy to popularize to a clinical sample precise detection scene.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of ZDHHC22 as a biomarker in the preparation of diagnostic or prognostic kits for high-risk neuroblastoma. Background Technology

[0002] Neuroblastoma (NB) is a highly heterogeneous extracranial solid tumor originating from the sympathetic nervous system, accounting for 8% to 10% of all childhood malignancies, and is considered one of the deadliest solid tumors in children (Rickman DS, et al.). Cancer Discovery 2018, 8(2):150-163. Approximately 50% of childhood neuroblastomas are high-risk, and MYCN gene amplification is a key oncogenic factor that can lead to chemotherapy resistance and poor prognosis through transcriptional activation (Huang M, et al.). Cold Spring Harbor Perspectives in Medicine 2013;3(10):a014415.). Although treatment regimens combining high-dose chemotherapy with autologous stem cell transplantation and anti-GD2 monoclonal antibodies have improved survival rates, the prognosis for high-risk neuroblastoma patients (commonly seen in children over 18 months of age, presenting as INSS4 stage and / or with MYCN amplification) remains poor, with a five-year survival rate of less than 50% (Irwin MS, et al.). J Clin Oncol .2021,39(29):3229-3241.).

[0003] Overall, this type of tumor is highly malignant and has a low survival rate, but children under one year old with early-stage tumors have a better prognosis. Therefore, developing new molecular markers for the diagnosis of high-risk neuroblastoma is of great significance for improving the early diagnosis and prognostic assessment of high-risk groups.

[0004] The ZDHHC family of enzymes, as palmitoyltransferases, catalyzes palmitoylation of proteins (Linder ME, et al.). Biochemical Society Transactions (2013, 41: 29-34.), plays a key regulatory role in the localization, stability and interaction of substrate proteins (Ko PJ, et al.). Embo Reports . 2018,19(10):e46666.). Recent studies have shown that members of the ZDHHC family, such as ZDHHC9 (Zhang ZX, et al.), Nature Communications .2021,12(1):5872.), ZDHHC14 (Yao H, et al. Nature Biomedical Engineering . 2019,3(5):414-414.), ZDHHC12 (Yuan M, et al. Acta Pharmaceutica Sinica B. 2020,10(8):1426-1439.), ZDHHC21 (Shao XJ, et al. Blood Abnormal expression of tumor markers such as . 2023,142(4):365-381. plays an important role in tumor progression and chemotherapy resistance. It is not only related to tumor proliferation, metastasis and drug resistance, but also has the potential to serve as a molecular marker for tumor diagnosis, subtyping or prognostic assessment.

[0005] However, there are currently no reports on ZDHHC22 as a molecular marker for the diagnosis or subtyping of neuroblastoma (especially high-risk types), and its clinical application value needs to be explored. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker for the diagnosis, classification, or prognostic assessment of high-risk neuroblastoma, which can be used to develop precision diagnostic products for high-risk neuroblastoma, thereby improving the ability to stratify the diagnosis of neuroblastoma and thus improving the prognosis and survival of patients.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of palmitoyltransferase ZDHHC22 as a detection biomarker in the preparation of diagnostic or prognostic assessment kits for high-risk neuroblastoma.

[0008] This invention has been discovered through research. ZDHHC22 The gene is specifically highly expressed in neuroblastoma, and relatively highly expressed in high-risk patients. However, it is expressed at low levels in other tumor types and in normal tissues adjacent to neuroblastoma, suggesting that ZDHHC22 plays a unique role in neuroblastoma.

[0009] Survival prognostic analysis revealed ZDHHC22 High gene expression is closely associated with poor prognosis in high-risk neuroblastoma patients. Database analysis revealed... ZDHHC22 Genes at ST3 and ST4 stages (International Neuroblastoma Staging System (INSS) stages), recurrence, or malignant progression. MYCN It is highly expressed in high-risk neuroblastoma classifications such as amplification and age ≥18 months.

[0010] This invention has been confirmed through research to utilize lentivirus packaging. ZDHHC22 After infecting neuroblastoma cell lines, cell clonogenic assays showed that... ZDHHC22 Upregulation of its expression can significantly promote the clonogenic ability of neuroblastoma.

[0011] Therefore, this invention uses ZDHHC22 as a detection biomarker to develop corresponding diagnostic or prognostic assessment products. By detecting the expression level of ZDHHC22 in tumor tissues or cells, the malignancy of neuroblastoma and the prognostic risk of patients can be assessed.

[0012] Furthermore, the high-risk characteristics of the aforementioned high-risk neuroblastoma include tumor stage 3 or 4, tumor recurrence or malignant progression, MYCN Positive amplification and decreased sensitivity to chemotherapy drugs are among the following. Studies have found that ZDHHC22 shows a high expression trend in various high-risk subtypes of neuroblastoma tissues, and is associated with poor clinical prognosis in stage, disease progression, and other factors. MYCN It is closely related to high-risk factors such as amplification status and age.

[0013] In this invention, the kit contains a detection... ZDHHC22 Reagents for detecting gene expression or ZDHHC22 protein levels can also be biochips.

[0014] Furthermore, the kit contains an antibody for detecting the ZDHHC22 protein. The antibody is used to detect the level of ZDHHC22 protein in tumor tissues or cells.

[0015] Furthermore, the amino acid sequence of the ZDHHC22 protein is shown in SEQ ID NO.1. This invention is not limited to the detection of human ZDHHC22 protein.

[0016] Furthermore, the kit contains a detection... ZDHHC22 Specific primers for gene expression. This invention can utilize techniques such as PCR to... ZDHHC22 Gene expression levels were detected.

[0017] Furthermore, ZDHHC22 The coding sequence of the gene is shown in SEQ ID NO.2. This invention is not limited to human-derived genes. ZDHHC22 Genetic testing was performed.

[0018] Furthermore, the nucleotide sequences of the specific primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0019] Furthermore, the kit also includes a premixed solution for constructing a real-time RT-PCR reaction system. This invention allows for the use of real-time RT-PCR to... ZDHHC22 Gene expression levels were detected.

[0020] Furthermore, the kit also includes a detection internal reference gene. GAPDHThe primers, whose nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0021] Furthermore, the kit also includes a control, which is a known low-risk neuroblastoma sample. This invention has found that the expression level of ZDHHC22 in high-risk neuroblastoma tumor tissues or cells is significantly higher than that in low-risk neuroblastoma.

[0022] The criteria for determining low-risk neuroblastoma are any one of the following: ① localized tumor and MYCN No amplification; ② Localized regional lesions, age less than 18 months and MYCN No amplification; ③ Any primary tumor with distant lymph node, bone marrow, liver, skin and / or other organ dissemination, age less than 18 months and MYCN No amplification.

[0023] The present invention also provides a method for diagnosing or assessing the prognosis of high-risk neuroblastoma using the kit, comprising the following steps: a) selecting in vitro tumor tissue samples from 20 clinically diagnosed low-risk patients, detecting the expression of ZDHHC22 using the kit, and calculating the average expression value as a control; b) detecting the expression level of ZDHHC22 in the tumor tissue sample to be tested; c) comparing the expression level of ZDHHC22 in the sample to be tested with that in the control, and if the expression level of ZDHHC22 in the sample to be tested is significantly higher than that in the control, it indicates that the sample to be tested is a high-risk neuroblastoma sample.

[0024] The beneficial effects of this invention are as follows: This invention discloses for the first time the close association between abnormally high expression of ZDHHC22 and high-risk neuroblastoma, providing a novel predictive biomarker for high-risk neuroblastoma. A diagnostic or prognostic assessment kit for high-risk neuroblastoma was developed using ZDHHC22 as a detection biomarker, enabling stratified diagnosis of neuroblastoma. This kit has advantages such as high sensitivity, strong specificity, ease of operation, and good reproducibility, making it easily applicable to precise clinical sample testing. Attached Figure Description

[0025] Figure 1 For database-based data analysis ZDHHC22 Gene expression across pan-cancer species. Results showed... ZDHHC22 The gene is specifically highly expressed in neuroblastoma.

[0026] Figure 2 For database-based data analysis ZDHHC22 Gene expression in neuroblastoma tissue and adjacent normal tissue.

[0027] Figure 3To investigate the relationship between ZDHHC22 expression and prognosis in neuroblastoma patients using the Kaplan-Meier survival analysis database. Results showed no significant difference in prognosis between patients with high and low ZDHHC22 expression in st1, st2, and st4s stages of neuroblastoma. However, in st3 and st4 stages of neuroblastoma, patients with high ZDHHC22 expression had a significantly worse prognosis compared to those with low ZDHHC22 expression.

[0028] Figure 4 To analyze the expression of ZDHHC22 in high-risk neuroblastoma subtypes based on the GEO dataset, the high-risk features from left to right in the figure are: different stages, whether there is recurrence or malignant progression, and whether... MYCN Amplification, age > 18 months. Results showed that ZDHHC22 was present in ST3 and ST4 phases, relapsed or malignant progression. MYCN It is highly expressed in neuroblastoma patients with high-risk characteristics such as amplification and age >18 months.

[0029] Figure 5 To assess the clonogenic ability of ZDHHC22-overexpressing cells in different neuroblastoma cell lines, A shows the effect of ZDHHC22 overexpression detected by Western blotting; B shows representative clone images; and C shows the relative clonogenic rate. Results showed that overexpression of ZDHHC22 significantly enhanced clonogenic ability in SK-N-BE(2), SK-N-DZ, CHP126, and SH-SY5Y cells.

[0030] Figure 6 To detect the expression of ZDHHC22 in different tumor cell lines using RT-PCR with ZDHHC22 primers.

[0031] Figure 7 To detect the expression of ZDHHC22 in tumor tissues derived from neuroblastoma patients using RT-PCR with ZDHHC22 primers. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Experimental methods not specified in the examples are generally performed under standard conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0034] The application of the diagnostic reagent for detecting ZDHHC22 expression described in this invention can be referenced from conventional primer preparation methods and actual development.

[0035] The amino acid sequence of the ZDHHC22 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0036] Example 1 1. Based on The Protein Atlas dataset (https: / / www.proteinatlas.org / ), the expression level of ZDHHC22 in different cancer types was analyzed and sorted from high to low according to the mean expression of ZDHHC22.

[0037] See results Figure 1 ZDHHC22 is specifically highly expressed in neuroblastoma.

[0038] 2. Sequencing data from 88 neuroblastoma patients (GSE16476) and 13 normal adrenal tissue samples (GSE3526, GSE7307, GSE8514) were downloaded from the GEO dataset to analyze the expression of ZDHHC22 in neuroblastoma patients and normal adrenal tissues.

[0039] See results Figure 2 ZDHHC22 was significantly overexpressed in patients with neuroblastoma, suggesting that high expression of ZDHHC22 is closely related to neuroblastoma.

[0040] Example 2 Based on the GEO database GSE85047 dataset (n=283), Kaplan-Meier survival analysis was used to assess the correlation between ZDHHC22 expression level and prognosis in patients with neuroblastoma at different stages.

[0041] According to the International Neuroblastoma Staging System (INSS), patients were divided into high-risk groups (st3, st4) and low-risk groups (st1, st2, st4s). Based on this, patients were further divided into four subgroups according to the expression level of ZDHHC22 (high expression / low expression), and survival curves were plotted for each subgroup.

[0042] See results Figure 3 In patients with low-risk stages (st1, st2, st4s), there was no statistically significant difference in prognosis between the high and low ZDHHC22 expression groups; however, in patients with high-risk stages (st3, st4), the survival rate of the high ZDHHC22 expression group was significantly lower than that of the low expression group, and the difference was statistically significant.

[0043] The above results suggest that high expression of ZDHHC22 is closely related to poor prognosis in patients with high-risk neuroblastoma.

[0044] Example 3 Based on the GEO database GSE85047 dataset (n=283), we further analyzed the expression level of ZDHHC22 in neuroblastoma patients with different clinical high-risk characteristics. The results are shown in [link to results]. Figure 4 .

[0045] According to the International Neuroblastoma Staging System (INSS) results, ZDHHC22 expression was significantly higher in patients with high-risk staging (st3 and st4) than in patients with low-risk staging (st1, st2, st4s). P <0.001).

[0046] Furthermore, compared with patients who were initially diagnosed without progression, patients with relapse or malignant progression had significantly higher levels of ZDHHC22 expression. P <0.01).

[0047] At the molecular subtyping level, it has MYCN The amplified patients had significantly higher ZDHHC22 expression levels than MYCN Non-amplified patients ( P <0.001).

[0048] Furthermore, in the age-stratified analysis, patients older than 18 months at diagnosis had significantly higher ZDHHC22 expression levels than patients ≤18 months of age. P <0.001).

[0049] The above results suggest that ZDHHC22 shows a high expression trend in various high-risk subtypes of neuroblastoma tissues, and is associated with poor clinical prognosis, including stage, disease progression, and other factors. MYCN The amplification status and age are closely associated with high-risk factors, suggesting that ZDHHC22 may be closely related to the highly malignant biological behavior and disease progression of neuroblastoma.

[0050] Example 4 The sequence encoding ZDHHC22 (SEQ ID NO.2) was cloned into the lentiviral expression vector pCDH to construct the plasmid pCDH-ZDHHC22-Flag. Simultaneously, a control plasmid pCDH was constructed. The correctly identified plasmids, along with the packaging plasmid pRΔ8.9 and the envelope plasmid pMD.G, were packaged into lentiviruses and then introduced into SK-N-BE(2), SK-N-DZ, CHP126, and SH-SY5Y cells (purchased from the Cell Bank of the Chinese Academy of Sciences) via lentiviral infection. After 72 hours, the cells were digested and transfected into six-well plates for cloning. The remaining cells were lysed using RIPA lysis buffer, and the overexpression effect of ZDHHC22 was assessed by Western blotting (anti-Flag antibody purchased from Cell Signaling Technology). The plate clones were fixed with trichloroacetic acid after 14 days, stained with SRB, and images of the clones were taken and processed.

[0051] See results Figure 5 SK-N-BE(2), SK-N-DZ, CHP126 and SH-SY5Y cells showed significantly enhanced clonogenic ability after overexpression of ZDHHC22.

[0052] Example 5 1. Different tumor cell lines (n=20) were collected, and mRNA was extracted by lysing the cells with Trizol lysis buffer. Then, the mRNA level was detected by quantitative real-time RT-PCR. ZDHHC22 The primer pairs are 5'-CCTATTCCTCTCGGCCAACG-3' and 5'-TGGAAAGGACAGCGGAGATG-3', and the primer pairs for the internal reference GAPDH are 5'-GTCATCCATGACAACTTTGG-3' and 5'-GAGCTTGACAAAGTGGTCGT-3'.

[0053] See results Figure 6 The expression level of ZDHHC22 varies greatly among different tumor cell lines, and it is generally highly expressed in neuroblastoma cell lines.

[0054] 2. Tumor tissues from neuroblastoma patients (n=8) were collected. Cells were lysed using Trizol lysis buffer to extract mRNA, and then mRNA levels were detected by quantitative real-time RT-PCR. Based on clinical diagnosis, 3 cases were classified as low-risk patients and 5 cases as high-risk patients.

[0055] See results Figure 7 Compared to tumor tissues from low-risk neuroblastoma patients, ZDHHC22 was expressed at higher levels in tumor tissues from 5 high-risk neuroblastoma patients.

[0056] The above results suggest that the primer pair can reflect the expression level of ZDHHC22.

[0057] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of palmitoyltransferase ZDHHC22 as a detection biomarker in the preparation of diagnostic or prognostic assessment kits for high-risk neuroblastoma.

2. The application as described in claim 1, characterized in that, High-risk characteristics of neuroblastoma include tumor stage 3 or 4, tumor recurrence or malignant progression. MYCN At least one of the following: positive amplification, decreased sensitivity to chemotherapy drugs.

3. The application as described in claim 1, characterized in that, The kit contains an antibody for detecting the ZDHHC22 protein.

4. The application as described in claim 3, characterized in that, The amino acid sequence of the ZDHHC22 protein is shown in SEQ ID NO.

1.

5. The application as described in claim 1, characterized in that, The kit contains detection. ZDHHC22 Gene expression-specific primers.

6. The application as described in claim 5, characterized in that, ZDHHC22 The coding sequence of the gene is shown in SEQ ID NO.

2.

7. The application as described in claim 6, characterized in that, The nucleotide sequences of the specific primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The application as described in any one of claims 5-7, characterized in that, The kit also includes a premix for constructing a real-time RT-PCR reaction system.

9. The application as described in claim 8, characterized in that, The kit also includes a detection internal reference gene. GAPDH The primers, whose nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.

6.

10. The application as described in claim 1, characterized in that, The kit also includes a control, which is a known low-risk neuroblastoma sample.

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