Application of ZDHHC9 gene in the preparation of drugs for treating lung adenocarcinoma

Abstract

This invention relates to the field of biomedical technology, and discloses the application of the ZDHHC9 gene in the preparation of a drug for treating lung adenocarcinoma. The drug contains an active component; the active component reduces the protein expression level of ZDHHC9 by silencing the ZDHHC9 gene. Research in this invention has found that knocking down ZDHHC9 with small interfering RNA in LUAD cells, thereby reducing ZDHHC9 expression, can inhibit LUAD cell proliferation, migration, and tumorigenicity in vivo. These results suggest that the ZDHHC9 gene plays an important role in controlling LUAD development, providing a new target for the treatment of LUAD, and has potential application value in the preparation of drugs for treating lung adenocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of the ZDHHC9 gene in the preparation of drugs for treating lung adenocarcinoma. Background Technology

[0002] Lung cancer is one of the leading causes of death and morbidity among malignant tumors worldwide. Global projections indicate that by 2050, lung cancer will cause 3.8 million new cases and nearly 3.2 million deaths. Lung adenocarcinoma is the most common subtype of lung cancer, accounting for approximately 40% of all cases, and its incidence continues to rise globally. In recent years, the treatment of lung adenocarcinoma has evolved from traditional surgery, chemotherapy, and radiotherapy to precision molecular targeted therapy and immunotherapy, achieving significant breakthroughs. However, some patients still lack a clearly identified driver gene, and suitable drugs are unavailable. Therefore, elucidating the pathogenesis of lung adenocarcinoma, discovering novel and effective therapeutic targets, and improving precision targeted therapy are of significant clinical value and scientific importance for the treatment of lung adenocarcinoma.

[0003] DHHC9 belongs to the DHHCs palmitoyltransferase family. Its cysteine-rich domain contains a DHHC motif and also binds two Zn groups. 2+ Therefore, it is also known as ZDHHC palmitoylation transferase. The human DHHC family has 23 members, primarily responsible for palmitoylation of proteins in cells. Palmitoylation is a reversible post-translational modification of proteins and is crucial for the dynamic regulation of protein localization, diffusion, and stability. Recent studies have shown that the proliferation, migration, and invasion of various cancer cells depend on palmitoylation of cancer-related proteins. Summary of the Invention

[0004] This invention provides the application of the ZDHHC9 gene in the preparation of drugs for treating lung adenocarcinoma. By silencing the ZDHHC9 gene, the protein expression level of ZDHHC9 is reduced, thereby inhibiting the proliferation, migration and in vivo growth of lung adenocarcinoma cells, providing a target for the treatment of lung adenocarcinoma.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] As a first aspect of the present invention, the application of the ZDHHC9 gene in the preparation of a drug for treating lung adenocarcinoma, the drug containing an active component, the active component reducing the protein expression level of ZDHHC9 by silencing the ZDHHC9 gene, thereby inhibiting the proliferation, migration and in vivo growth of lung adenocarcinoma cells.

[0007] A further improvement is that the NCBI number of the ZDHHC9 gene is 51114, and the sequence of the ZDHHC9 protein it encodes is shown in SEQ ID No. 1.

[0008] A further improvement is that the knockdown of the ZDHHC9 gene is either a transient knockdown or a stable knockdown of the ZDHHC9 gene.

[0009] As a second aspect of the present invention, a knockout-reducing interfering RNA for ZDHHC9 is also disclosed, the sequence of which is shown in SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0010] As a third aspect of the present invention, the use of a knockdown RNA of the ZDHHC9 gene as described above in the preparation of a product for treating lung adenocarcinoma is also disclosed.

[0011] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0012] This invention demonstrates through experiments that knocking down ZDHHC9 with small interfering RNA in LUAD cells, thereby reducing ZDHHC9 expression, can inhibit LUAD cell proliferation, migration, and tumorigenesis. These results suggest that the ZDHHC9 gene plays an important role in controlling LUAD development, providing a new target for the treatment of LUAD and having potential application value in the preparation of drugs for the treatment of lung adenocarcinoma. Attached Figure Description

[0013] Figure 1 The transcriptional and protein expression levels of ZDHHC9 in LUAD are shown. (A represents the transcriptional level of ZDHHC9 in the GEPIA database, B represents the transcriptional level of ZDHHC9 in lung adenocarcinoma tissue and adjacent normal tissue, C represents the transcriptional level of ZDHHC9 in different LUAD cells detected by PCR, and D represents the expression level of ZDHHC9 in different LUAD cells detected by Western blotting).

[0014] Figure 2 To detect the proliferation capacity of LUAD cells after ZDHHC9 knockdown;

[0015] Figure 3 To detect the migration ability of LUAD cells after ZDHHC9 knockdown;

[0016] Figure 4 To ensure stable knockdown of ZDHHC9, the transcriptional level (B) and expression level (C) of ZDHHC9 were measured.

[0017] Figure 5 To stabilize the tumorigenicity of LUAD in vivo after ZDHCC9 knockdown. Detailed Implementation

[0018] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0019] I. Materials and Reagents

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional biochemical methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0021] 1. Cell line

[0022] The normal human lung epithelial cells Beas-2B and human LUAD cell lines PC-9, H460, H520, A549, H1299, and H1975 were all purchased from the Cell Bank of the Chinese Academy of Sciences.

[0023] 2. Interfering RNA

[0024] Interfering RNA was synthesized by a contract biotechnology company.

[0025] II. Methods

[0026] Example 1: ZDHHC9 Expression Analysis

[0027] The expression level of ZDHHC9 in different tumors was detected using the standard procedure included in the GEPIA2 database. The results showed that the transcriptional level of ZDHHC9 was abnormally elevated in LUAD. Figure 1 A). Further, 18 pairs of clinical LUAD tissues and adjacent normal tissues were collected, and differential RNA expression was detected by RNA-seq. The results showed that ZDHHC9 expression was abnormal in LUAD tissues ( Figure 1 B). Further analysis of whole protein lysates from Beas-2B, PC-9, H460, H520, A549, H1299, and H1975 cells by Western blotting revealed significantly elevated ZDHHC9 expression in PC-9, A549, and H1975 cells. Further RNA extraction from Beas-2B, PC-9, H460, H520, A549, H1299, and H1975 cells, followed by QT-PCR detection of ZDHHC9 mRNA levels, showed higher levels than in Beas-2B cells. This indicates abnormal transcriptional and expression levels of ZDHHC9 in LUAD cells.

[0028] Example 2: Colony formation assay to analyze the proliferative capacity of LUAD cells after ZDHHC9 knockdown.

[0029] Human PC-9, A549, and H1975 cells in logarithmic growth phase and in good condition were seeded into 6-well plates at a density of 1000 cells per well. After transfection with ZDHHC9 small interfering RNA using LIP3000 transfection reagent, the cells were cultured for 7-14 days, fixed with paraformaldehyde, and stained with crystal violet. The results confirmed that the proliferation capacity of LUAD cells was significantly reduced after the use of ZDHHC9 small interfering RNA (see results below). Figure 2 (Relative proliferation rate data are shown in Table 1).

[0030] Table 1. Relative proliferation rate of LUAD cells after ZDHHC9 silencing

[0031]

[0032] The small interfering RNA sequences SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4 are as follows:

[0033] si / sh ZDHHC9#1: 5'-GGCCCUCAAAUCUUUGAAATT-3'

[0034] si / sh ZDHHC9#2: 5'-GGGACUGACUGGAUUUCAUTT-3'

[0035] si / sh ZDHHC9#3: 5'-CCUACAGCCAUGGCAAAUTT-3'

[0036] Example 3: Transwell assay analysis of LUAD cell migration ability after ZDHHC9 knockdown

[0037] Human PC-9, A549, and H1975 cells in logarithmic growth phase and in good condition were transfected with ZDHHC9 small interfering RNA using LIP3000 transfection reagent and then seeded into the upper chamber of a Transwell (2-4 × 10⁻⁶ cells). 4 Cells were cultured in a well containing 10% serum. 700-800 μL of serum-free medium was added to the lower chamber. Cells were cultured overnight, then the medium in the lower chamber was replaced with 10% serum. After 24 hours of culture, cells were fixed with paraformaldehyde and stained with crystal violet. The results showed that the use of ZDHHC9 small interfering RNA significantly reduced the migration ability of LUAD cells (see results below). Figure 3 (Relative mobility data are shown in Table 2).

[0038] Table 2. Relative migration rate of LUAD cells after ZDHHC9 silencing

[0039]

[0040] Example 4: Construction of a stable ZDHHC9 knockdown cell line

[0041] Small interfering RNA was loaded into the LV3(H1 / GFP&Puro) lentiviral vector and added to H1975 cells. After co-culturing for 24 h, the cell culture medium was changed, and cells were screened with antibiotics and then continuously cultured. The transfection efficiency was observed using a fluorescence microscope. The results showed that most cells were infected. Figure 4 A). Further QT-PCR and WB were used to detect ZDHHC9 RNA levels, confirming the successful construction of a stable ZDHHC9 knockdown cell line. Figure 4 B, C).

[0042] Example 5: Analysis of the in vivo tumorigenicity of LUAD cells after ZDHHC9 knockdown in a xenograft tumor model

[0043] Stable ZDHHC9 knockdown H1975 cell lines in good growth condition and logarithmic growth phase were gradually expanded and passaged until the cell number met experimental requirements. The cells were then digested following the same procedure as in cell passage. A mixture of sterile PBS and matrix gel was added at a 1:1 ratio, and 0.1 mL of the H1975 cell suspension was injected subcutaneously into the right back of nude mice, resulting in a cell count of 5 × 10⁶ cells. 6 Each xenograft is removed after the xenograft reaches 5×5mm in size, measured every 2 days. After 7 measurements and 14 days, the xenograft is removed. Figure 5 A). The results showed that after stable ZDHHC9 knockdown, tumor growth was significantly slowed down ( Figure 5 B), weight decreased significantly ( Figure 5 C).

[0044] ZDHHC9 protein sequence SEQ ID No. 1:

[0045] MSVMVVRKKVTRKWEKLPGRNTFCCDGRVMMARQKGIFYLTLFLILGTCTLFAFAFECRYLAVQLSPAIPVFAAMLFLSMATLLRTSFSDPGVIPRALPDEAAFIEMEIEATNGAVPQGQRPPPRIKNFQINNQIVKLKYCYTCKIRPPRASHCSICDNCVERFDHHCPWVGNCVGKRNYRYFYLFILSLSLLTIYVFAFNIVYVALKSLKIGFLETLKETPGTVLEVLICFFTLWSVVGLTGFHTFLVALNQTTNEDIKGSWTGKNRVQNPYSHGNIVKNCCEVLCGPLPPSVLDRRGILPLEESGSRPPSTQETSSSLLPQSPASTEHMNSNEMAEDTSIPEEMPPPEPPEPPQEASEAEK.

Claims

1. The application of the ZDHHC9 gene in the preparation of drugs for treating lung adenocarcinoma, characterized in that, The drug contains an active ingredient; The active component reduces the protein expression level of ZDHHC9 by silencing the ZDHHC9 gene.

2. The application of the ZDHHC9 gene in the preparation of a drug for treating lung adenocarcinoma, characterized in that, The drug is used to inhibit the proliferation, migration, and growth of lung adenocarcinoma cells in vivo.

3. The application according to claim 1, characterized in that, The ZDHHC9 gene has an NCBI number of 51114, and the protein sequence it encodes is shown in SEQ ID No.

1.

4. The application according to claim 1, characterized in that, Silencing of the ZDHHC9 gene can be either transient or stable knockdown.

5. A knockout-reducing interfering RNA for ZDHHC9, characterized in that, Its sequence is shown as SEQ ID No2, SEQ ID No3 or SEQ ID No4.

6. The application of the ZDHHC9 knockout-reducing interfering RNA according to claim 5 in the preparation of a drug for treating lung adenocarcinoma, characterized in that, The drug contains an active ingredient; The active component includes knockout-reducing interfering RNA of ZDHHC9.

7. The use of the ZDHHC9 knockout-reducing interfering RNA according to claim 6 in the preparation of a drug for treating lung adenocarcinoma.

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