Use of prpf19 in treating lung cancer

Abstract

The application relates to application of an agent for inhibiting PRPF19 expression and / or activity in preparation of a drug for treating lung cancer. The application provides a new use of PRPF19 as a target point in treatment of lung cancer, and provides a new strategy for prevention and treatment of lung cancer in clinic.

Description

Technical Field

[0001] This invention relates to the application of PRPF19 as a target in the treatment of lung cancer, and belongs to the field of biomedical technology. Background Technology

[0002] Lung cancer is one of the most common malignant tumors. Based on histopathological morphology, lung cancer can be classified into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and small cell lung cancer, among which lung adenocarcinoma is the most common subtype, accounting for more than 40% of all lung cancer cases. Most patients with lung adenocarcinoma are diagnosed at an advanced stage, having lost the opportunity for surgical treatment. Treatment typically involves radiotherapy and chemotherapy, with a 5-year survival rate of less than 15%. Since the 21st century, targeted therapy and immunotherapy for lung cancer have made significant progress. Multiple studies have confirmed that targeted therapy is more effective than traditional chemotherapy for NSCLC patients with driver gene-positive tumors, but only about 50% of patients show the highest mutation probability in epidermal growth factor receptor (EGFR); while immunotherapy only shows good efficacy in patients with PD-L1 expression. Therefore, a large proportion of patients do not benefit from targeted therapy and immunotherapy, and both targeted and immunotherapies eventually develop resistance. Therefore, for the treatment of lung adenocarcinoma patients, there is an urgent need to find new targets and treatment regimens to prolong patient survival and improve quality of life.

[0003] Pre-messenger RNA processing factor PRPF19 is a highly conserved gene from yeast to eukaryotes. It is a key component of the NTC complex, consisting of an N-terminal U-box domain, a central coiled-coil domain, and seven C-terminal WD40 repeat domains. The U-box domain is a conserved ubiquitin ligase domain through which PRPF19 performs its E3 ubiquitin ligase function. The central coiled-coil domain mediates the formation of the PRPF19 tetramer and is crucial for structural stability. The C-terminal WD40 repeat domains play a role in substrate recognition and recruitment. PRPF19 is a multifaceted protein. On one hand, it stabilizes the U4 / U6-U5 ternary snRNP complex by mediating K63 ubiquitination of the U4 snRNP component Prp3, participating in the assembly of the pre-messenger RNA splicing complex. On the other hand, it also promotes ATR-mediated DNA damage repair by ubiquitinizing members of the RPA complex. Furthermore, cells can also promote phosphorylation of PRPF19 at S149 in an ATM-dependent manner to respond to stresses such as hydrogen peroxide, thereby inhibiting apoptosis and prolonging cell lifespan. Studies have shown that PRPF19 plays an important role in multiple biological processes.

[0004] Existing literature reports that mRNA precursor splicing and DNA damage repair are closely related to tumorigenesis and development. Although the role of PRPF19 in mRNA precursor splicing and DNA damage repair is relatively well understood, research on PRPF19 in tumors, especially lung cancer, is still limited. In hepatocellular carcinoma, UCH37 promotes cell invasion and metastasis by deubiquitinizing PRPF19 to stabilize its protein expression. Further research shows that PRPF19 promotes invasion and metastasis by activating the p38 MAPK signaling pathway through ubiquitination of TAK1. Additionally, PRPF19 can regulate the cell cycle by modulating CDC5L expression. In oral cancer, PRPF19 can promote cell proliferation and radiotherapy / chemotherapy resistance. Furthermore, PRPF19 can promote neuroblastoma metastasis by upregulating YAP expression through mRNA precursor splicing. However, in lung cancer, studies have reported that PRPF19 causes cell cycle arrest by upregulating p21 expression. These studies indicate that PRPF19 is involved in the process of tumorigenesis and development.

[0005] Therefore, there is an urgent need in this field to find new targets and treatment options to prolong patients' survival and improve their quality of life. Summary of the Invention

[0006] The problem that this invention aims to solve is that there are currently no new targeted drugs for the treatment of lung cancer.

[0007] The present invention solves the above-mentioned technical problems through the following technical solutions:

[0008] This invention provides the application of a reagent that inhibits PRPF19 expression and / or activity in the preparation of a drug for treating lung cancer.

[0009] Preferably, the reagent for inhibiting PRPF19 expression and / or activity includes an inhibitor of PRPF19.

[0010] Preferably, the drug comprises a medically acceptable carrier and an effective amount of an active ingredient, said active ingredient being a reagent that inhibits PRPF19 expression and / or activity.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention provides a novel application of PRPF19 as a target in the treatment of lung cancer, offering a new strategy for the prevention and treatment of lung cancer in clinical practice. Attached Figure Description

[0013] Figure 1The following graphs show the results of screening CRISPR libraries for targeted oncogenes: A shows the in vitro screening results in the KP(KRASG12D,P53- / -) lung cancer model; B shows the expression results of sgRNA targeting Prpf19; C shows the comparison of PRPF19 expression in lung cancer tissue and normal lung tissue in the TCGA database; D shows the comparison of PRPF19 expression in lung cancer tissue and normal lung tissue in the GEO database; E shows the survival curves of the relationship between PRPF19 expression levels and overall survival in lung cancer patients in the database; F shows the survival curves of the relationship between PRPF19 expression levels and progression-free survival in lung cancer patients in the database; G shows the immunohistochemical images of patients with high and low PRPF19 expression; and H shows the survival curves of the relationship between PRPF19 expression levels and progression-free survival in lung cancer patients in our research group.

[0014] Figure 2 To verify the experimental results of Prpf19 knockdown inhibiting lung cancer progression, the diagrams are as follows: A shows the validation of Prpf19 knockdown in mouse lung cancer cells KP-1; B shows the inhibition of Prpf19 knockdown on the proliferation of mouse lung cancer cells KP-1; C shows the inhibition of Prpf19 knockdown on the colony formation of mouse lung cancer cells KP-1; D shows the quantitative statistical results of diagram C; and E shows human lung cancer cells H2009. PRPF19 knockdown validation graph; F shows the inhibition of H2009 human lung cancer cell proliferation by PRPF19 knockdown; G shows the inhibition of H2009 human lung cancer cell colony formation by PRPF19 knockdown; H represents the quantitative statistical results of graph G; I shows the inhibition of KP-1 mouse lung cancer cell tumor growth by PRPF19 knockdown; J shows the inhibition of H2009 human lung cancer cell tumor growth by PRPF19 knockdown; all data are expressed as mean ± SD; ns, no statistical difference, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Detailed Implementation

[0015] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0016] Example 1

[0017] This embodiment verifies that PRPF19 can treat non-small cell lung cancer.

[0018] CRISPR screening identifies therapeutic targets for non-small cell lung cancer.

[0019] Step 1: Construct a CRISPR-sgRNA library targeting 524 epigenetic regulatory genes;

[0020] Step 2: Package the CRISPR-sgRNA library into a viral library;

[0021] Step 3: Infect mouse lung cancer model KP(KRASG12D,P53- / -) cells with the virus library;

[0022] Step 4: Establish a cell bank with different epigenetic regulatory genes knocked out;

[0023] Step 5: In vitro cell culture;

[0024] Step 6: Perform sequence alignment analysis of sgRNA in cells before and after culture.

[0025] Example 2

[0026] In vitro proliferation experiments confirmed that knocking down PRPF19 can inhibit the proliferation of non-small cell lung cancer.

[0027] Step 1: Constructing non-small cell lung cancer cell lines

[0028] A plasmid system of pLKO-Tet-on-shRNA was used to construct doxycycline-induced knockdown of PRPF19 in mouse and human non-small cell lung cancer cell lines.

[0029] Step 2: CCK-8 proliferation experiment

[0030] 500 cells were seeded in 96-well plates. From day 0 to day 5, an equal amount of CCK-8 was added to each well at fixed time points each day. The absorbance at OD450 was measured 2 hours later, and growth curves were plotted based on the absorbance values. The effects of knocking down PRPF19 on cell proliferation rate were then compared.

[0031] Step 3: CCK-8 Cloning Experiment

[0032] 500 control cells or stable PRPF19 knockdown cells were seeded into 6-well plates and cultured for 8 days. The cells were then stained with 0.5% crystal violet, photographed, and plotted. The area of ​​the clones was counted using ImageJ software to compare the effect of PRPF19 knockdown on cell proliferation rate.

[0033] Example 3: In vivo animal experiments to verify the inhibitory effect of PRPF19 knockdown on non-small cell lung cancer tumor growth

[0034] 1. Subcutaneous tumor-bearing experiment using mouse cell lines: Each B6 mouse was inoculated with 1×10⁶ KP cells with Prpf19 knockdown (KP-shPrpf19) or control cells (KP-shLacZ), and tumor growth was monitored. After a certain period of time, the tumors were removed, and their size and weight were recorded to analyze the effect of Prpf19 knockdown on tumor proliferation.

[0035] 2. Subcutaneous tumor-bearing experiment using human cell lines: Each nude mouse was inoculated with 5 × 10⁶ PRPF19-knockdown lung adenocarcinoma cells (A549-shPRPF19) or control cells (A549-shLacZ), and tumor growth was monitored. After a certain period of time, the tumors were removed, and their size and weight were recorded to analyze the effect of PRPF19 knockdown on tumor proliferation.

Claims

1. The application of reagents that inhibit PRPF19 expression and / or activity in the preparation of drugs for treating lung cancer, wherein the reagents that inhibit PRPF19 expression and / or activity are reagents containing shPRPF19 of lung adenocarcinoma cells with knocked-down PRPF19; specifically including murine shPrpf19-1, murine shPrpf19-2, human shPRPF19-1, and human shPRPF19-2; the reagents are constructed using the pLKO-Tet-on-shRNA plasmid system and verified by CCK-8 proliferation assays, clonogenic assays, and in vivo animal tumor-bearing experiments; the lung cancer includes non-small cell lung cancer, specifically murine KRASG12D, P53- / - lung cancer and human H2009 lung adenocarcinoma.

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