Application of PCYOX1L as ovarian cancer platinum resistance diagnostic marker in preparation of diagnostic kit

Abstract

The invention discloses application of PCYOX1L as an ovarian cancer platinum resistance diagnostic marker in preparation of a diagnostic kit. Research finds that high expression of the isopentene cysteine oxidase 1-like protein (PCYOX1L) gene is related to ovarian cancer platinum resistance, and the expression level of the PCYOX1L remarkably affects proliferation and apoptosis processes of ovarian cancer cells, is remarkably and negatively related to prognosis of patients, can be used as a potential independent prognosis factor and a treatment target of the ovarian cancer, and can be used for preparing an ovarian cancer drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant drug-resistant gene. And the marker is also a diagnostic marker for platinum resistance of ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, and in particular to an application of PCYOX1L as a diagnostic marker for platinum resistance in ovarian cancer in the preparation of a diagnostic kit. Background Art

[0002] Ovarian cancer, as the gynecological malignancy with the highest mortality rate, is characterized by high morbidity and insidious onset. Due to the lack of effective early screening and diagnostic methods, most patients are already in the advanced stage when diagnosed, accompanied by local or distant metastasis, resulting in a five-year survival rate of only about 46%. At present, the standard treatment for ovarian cancer is mainly tumor reduction surgery combined with platinum-based chemotherapy. However, the widespread occurrence of chemotherapy resistance often leads to high recurrence rates and poor prognosis in advanced patients after treatment, making the clinical treatment of ovarian cancer face severe challenges. Although targeted therapies represented by PARP inhibitors have made breakthrough progress in the treatment of advanced ovarian cancer, other effective molecular therapeutic targets are still very scarce. Therefore, in-depth exploration of the molecular mechanisms of ovarian cancer development and chemotherapy resistance, and the discovery of new chemotherapy resistance-related prediction and treatment targets, are the key to improving its clinical efficacy. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the present invention aims to provide an application of PCYOX1L as a diagnostic marker for platinum resistance in ovarian cancer in the preparation of a diagnostic kit.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: an application of PCYOX1L as a diagnostic marker for platinum resistance in ovarian cancer in the preparation of a diagnostic kit.

[0005] After research, the applicant found that high expression of the prenylcysteine ​​oxidase 1-like protein (PCYOX1L) gene can be associated with platinum resistance in ovarian cancer. The expression level of PCYOX1L significantly affects the proliferation and apoptosis of ovarian cancer cells, and is significantly negatively correlated with the patient's prognosis. It can be used as a potential independent prognostic factor and therapeutic target for ovarian cancer, and is also a diagnostic marker for platinum resistance in ovarian cancer.

[0006] Sequence analysis revealed that PCYOX1L is highly conserved across species and possesses important biological functions. UniProt structural analysis revealed that it possesses typical oxidase domain characteristics and oxidoreductase function. Transcriptome sequencing analysis revealed that differentially expressed genes after PCYOX1L knockdown were significantly enriched in oxidative stress response pathways.

[0007] Analysis of differentially expressed genes related to cellular metabolism revealed that knockdown of PCYOX1L significantly reduced glucose-6-phosphate dehydrogenase (G6PD) and affected intracellular reactive oxygen species (ROS) levels. This suggests that PCYOX1L promotes G6PD expression and inhibits ROS production, thereby promoting ovarian cancer cell proliferation, migration, and platinum resistance, leading to poor patient prognosis. Therefore, PCYOX1L could be used as a diagnostic marker for platinum resistance in ovarian cancer and in the preparation of diagnostic kits. Furthermore, this study could provide a theoretical basis and companion diagnostic tools for the development of drugs targeting the PCYOX1L / G6PD-ROS pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Figure 3. Knockdown of PCYOX1L inhibits the proliferation and migration of ovarian cancer cells.

[0009] Figure 2 Overexpression of PCYOX1L promotes the proliferation, colony formation and invasion ability of ovarian cancer cells Caov3.

[0010] Figure 3 PCYOX1L expression is associated with cisplatin resistance and poor prognosis in ovarian cancer cells.

[0011] Figure 4 GO and KEGG analysis of significantly differentially expressed genes in ovarian cancer cells knocked down PCYOX1L.

[0012] Figure 5 Knockdown of PCYOX1L increases ROS levels in ovarian cancer cells without affecting normal ovarian epithelial cells.

[0013] Figure 6 PCYOX1L regulates the level of reactive oxygen species in ovarian cancer cells through G6PD.

[0014] Figure 7 PCYOX1L regulates ovarian cancer cell proliferation and cisplatin resistance through G6PD. DETAILED DESCRIPTION

[0015] The present invention will be further described in detail below through specific embodiments:

[0016] Example 1

[0017] 1. We downloaded the TCGA dataset and performed quality control and standardization screening. The final analysis included PCYOX1L expression profiles from 351 tumor tissues and 61 normal tissues. We then used this filtered dataset to analyze the association between PCYOX1L expression levels and ovarian cancer patient survival curves.

[0018] 2. Cell lines and cell culture

[0019] This example uses four ovarian cancer cell lines (Caov3, A2780, Skov3, and OVCAR3) and one ovarian epithelial cell line, ISOE, to detect PCYOX1L expression. The cells were cultured in DMEM, RPMI-1640, and McCoy's 5A medium, respectively. A2780 and OVCAR3 were cultured in RPMI-1640, Caov3 and ISOE were cultured in DMEM, and Skov3 was cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cultures were performed in a 5% CO2, 37°C incubator.

[0020] 3. Cell transfection

[0021] Cells were transfected with shRNA targeting PCYOX1L (shPCYOX1L) or a negative control (NC) using LipoFiter 3.0 transfection reagent. Cells were used for subsequent assays or replated for other experiments 40 hours after transfection.

[0022] 4. RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis

[0023] Total cellular RNA was extracted according to the TRIZOL reagent instructions and reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit. For qRT-PCR analysis, the relative expression levels of PCYOX1L and G6PD were calculated using the comparative Ct value method (ΔΔCt), using β-actin as an internal reference gene.

[0024] 5. Western blotting analysis

[0025] Cells were lysed on ice for 20 min in RIPA lysis buffer containing a cocktail of phosphatase and protease inhibitors; the lysate was centrifuged at 12,000 rpm for 20 min at 4°C and centrifuged using Pierce TM Protein concentration was determined using a BCA protein quantification kit. 20 μg of protein was loaded per well for 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with an optimal blocking buffer for 1–2 hours, then incubated with primary antibodies (PCYOX1L, β-actin, G6PD) overnight at 4°C, followed by incubation with an HRP-conjugated secondary antibody for 1 hour at room temperature. Protein detection was performed using ECL luminescence solution, and chemiluminescence signals were acquired using a ChemiDoc MP imaging system. Grayscale analysis was performed using ImageLab software on the Thermo Fisher Connect platform. β-Actin was used as an internal control.

[0026] 6. Cell proliferation assay

[0027] Forty hours after shRNA transfection, cells were harvested and counted, then seeded at a density of 5,000 cells / well in a 96-well plate and cultured in a CO2-containing incubator. Cell viability was assessed using CCK-8 reagent after 6, 24, 48, 72, and 96 hours of culture, with absorbance at 450 nm measuring the cell number. All experiments were repeated three times.

[0028] 7. Clone formation experiment

[0029] Cells were seeded at a density of 500 cells / well in 12-well plates, with triplicate wells per sample. Complete medium was replaced every three days and cultured at 37°C, 5% CO₂ for 7 days or until the number of colonies exceeded 50 cells. Cells were fixed with paraformaldehyde and stained with crystal violet for 1 hour at room temperature. After rinsing with PBS, colonies were observed and counted under a microscope (>50 cells were considered a colony).

[0030] 8. Cell migration assay

[0031] Cell migration experiments were performed in 24-well Transwell chambers. Transfected cells were seeded in the upper chamber containing serum-free medium; 300 μL of medium supplemented with 20% fetal bovine serum was added to the lower chamber. After 24 hours of culture, non-invading cells in the upper chamber were gently removed with a cotton swab. Cells in the lower chamber were fixed with 4% paraformaldehyde for 15 minutes and then stained with crystal violet. Five randomly selected fields of view were counted under a microscope.

[0032] 9. Reactive oxygen species (ROS) level detection

[0033] Total intracellular ROS levels were measured using a reactive oxygen species detection kit. Tumor cells were harvested and suspended in diluted DCFH-DA, an oxidation-sensitive fluorescent probe that is converted to DCF (fluorescent dichlorofluorescein) within the cells. The cells were incubated at 37°C for 20 minutes. After washing with serum-free medium, cell fluorescence was measured by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, and green fluorescence was observed using a fluorescence microscope. ROS levels were quantified using FlowJo software.

[0034] 10. Statistical analysis

[0035] Data were processed using SPSS 26.0 software. Data distribution was verified using the Kolmogorov-Smirnov test. Measurement data are presented as mean ± standard deviation (SD). Comparisons between the two groups were performed using the Student's t-test, with p < 0.05 considered statistically significant.

[0036] Experimental results

[0037] 1. Knockdown of PCYOX1L significantly inhibits the proliferation and migration of ovarian cancer cells

[0038] A2780 and OVCAR cell lines with high expression of pcyox1L gene were selected for PCYOX1L knockdown phenotype study (e.g. Figure 1 A), the results showed that both shRNAs (shPCYOX1L-1 and shPCYOX1L-2) could significantly reduce the expression of pcyox1L gene and protein ( Figure 1 Further cell proliferation experiments showed that knocking down PCYOX1L significantly reduced the proliferation capacity of two ovarian cancer cell lines ( Figure 1 EF). The results of cell clone formation experiments showed that knockdown of PCYOX1L significantly reduced the clone formation ability and proliferation ability of two ovarian cancer cells ( Figure 1 G). Transwell assay results showed that PCYOX1L knockdown significantly reduced the invasive ability of two ovarian cancer cell lines ( Figure 1 H).

[0039] 2. Overexpression of PCYOX1L promotes proliferation and migration of ovarian cancer cells

[0040] A PCYOX1L gene overexpression plasmid was constructed, and the ovarian cancer cell line Caov3 with a low PCYOX1L gene expression level was selected for overexpression, and the expression level was detected. Figure 2 As shown in Figure 2A and Figure 2C, PCYOX1L transcription and protein expression levels increased, indicating successful overexpression. Cell growth curve results showed that high expression of PCYOX1L significantly improved the proliferation ability of Caov3 cells ( Figure 2 B). The results of clone formation and Transwell experiments confirmed that PCYOX1L overexpression promoted the clone formation and migration ability of Caov3 cells ( Figure 2 DF)

[0041] 3. PCYOX1L expression is associated with cisplatin resistance and poor prognosis in ovarian cancer cells

[0042] Using the online database TCGA, we analyzed the effect of PCYOX1L expression on the prognosis of ovarian cancer patients at the RNA level. Figure 3 As shown in AB, high expression of PCYOX1L significantly reduces the disease-free survival of ovarian cancer patients ( Figure 3 A) and disease-free survival ( Figure 3 B) The relationship between PCYOX1L expression and cisplatin sensitivity in ovarian cancer cells was detected using A2780 cell line. Figure 3 As shown in Figure C, increasing concentrations of cisplatin treatment significantly increased the expression of PCYOX1L gene in A2780 cells. Figure 3 D shows that knockdown of the PCYOX1L gene significantly reduces the survival rate of A2780 cells under cisplatin-treated conditions.

[0043] 4. PCYOX1L participates in cell proliferation and drug resistance by affecting cellular redox levels

[0044] Transcriptome sequencing technology was used to analyze the differentially expressed genes in ovarian cancer cell A2780 after knockdown of PCYOX1L, and to explore the molecular mechanism by which PCYOX1L regulates ovarian cancer cell proliferation and cisplatin sensitivity. Subsequently, the GO ( Figure 4 A) and KEGG analysis ( Figure 4 Results from the study (B) showed that PCYOX1L primarily regulates biological processes such as cellular hypoxic stress; this regulatory process is closely linked to signaling pathways such as cellular metabolism. UniProt structural analysis revealed that PCYOX1L possesses typical oxidase domain characteristics, indicating that it is an oxidoreductase, potentially affecting cellular redox levels and contributing to cell proliferation and drug resistance.

[0045] 5. Knockdown of PCYOX1L affects the level of reactive oxygen species (ROS) in ovarian cancer cells

[0046] Since reactive oxygen species (ROS) play an important role in the development and progression of ovarian cancer, we first examined the effect of knocking down PCYOX1L on ROS levels in ovarian cancer cells. Both flow cytometry and fluorescence detection results showed that knocking down PCYOX1L significantly increased ROS levels in ovarian cancer cells A2780 ( Figure 5 A and C of 5), while the ROS level in normal ovarian epithelial cells IOSE-80 showed no significant changes ( Figure 5 B and 5D), demonstrating that PCYOX1L expression can specifically regulate the level of ROS in ovarian cancer cells.

[0047] 6. PCYOX1L regulates ROS levels in ovarian cancer cells through G6PD

[0048] To further analyze the mechanism of action of PCYOX1L, we analyzed the differentially expressed genes related to cell metabolism. The results showed that glucose-6-phosphate dehydrogenase (G6PD) was significantly reduced ( Figure 6 A), cell experiments also confirmed that G6PD expression was reduced when PCYOX1L was knocked down ( Figure 6B). Previous ovarian cancer studies have confirmed that high levels of G6PD expression are closely related to ovarian cancer metastasis and invasion, and that inhibiting G6PD expression can increase the oxidative stress sensitivity of ovarian cancer cells. Therefore, we speculate that PCYOX1L may regulate the level of reactive oxygen species in ovarian cancer cells by regulating the expression of G6PD, thereby affecting their cisplatin resistance. To confirm this speculation, we constructed a G6PD overexpression vector, and both real-time fluorescence quantitative PCR and protein immunoblotting experiments confirmed that G6PD expression was successfully restored in PCYOX1L knockdown cells ( Figure 6 C and D of 6). Both flow cytometry and fluorescence detection results showed that G6PD replenishment significantly reduced the ROS level in PCYOX1L knockdown ovarian cancer cells ( Figure 6 E and F ), confirming that PCYOX1L regulates ROS levels in ovarian cancer cells through G6PD.

[0049] 7. PCYOX1L regulates ovarian cancer cell proliferation and cisplatin resistance through G6PD

[0050] The clone formation experiment confirmed that G6PD replenishment improved the clone formation ability of PCYOX1L knockdown cells ( Figure 7 A), the cell growth curve also confirmed that G6PD replenishment promoted the proliferation of PCYOX1L knockdown cells ( Figure 7 B). This indicates that G6PD is the key to PCYOX1L regulating the proliferation of ovarian cancer cells. At the same time, G6PD re-expression can significantly improve the cisplatin resistance of ovarian cancer cells with PCYOX1L knockdown ( Figure 7 C).

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention 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 invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of PCYOX1L as a diagnostic marker for platinum resistance in ovarian cancer in the preparation of a diagnostic kit.

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