Use of idi1 as a target in diagnosis and / or treatment of ovarian cancer disease

Abstract

The application provides application of IDI1 as a target point in diagnosis and / or treatment of ovarian cancer, and relates to the technical field of biological medicine.The application finds that IDI1 is highly expressed in an ovarian cancer cell line through expression analysis and detection of IDI1 in the human ovarian cancer cell line, and the survival prognosis analysis result of patients shows that high expression of IDI1 is accompanied by poor survival prognosis of patients, and after knockdown of IDI1 in the ovarian cancer cell line, the stemness maintenance of ovarian cancer stem cells can be significantly inhibited, and the growth and proliferation of ovarian cancer cells are inhibited, which provides a train of thought for exploring the mechanism research and treatment target point in the occurrence and development of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of IDI1 as a target in the diagnosis and / or treatment of ovarian cancer. Background Technology

[0002] Ovarian cancer is one of the most common malignant tumors of the female reproductive system. In 2005, Bapat et al. first isolated self-renewing cell clones from the ascites fluid of patients with advanced ovarian serous adenocarcinoma. Two of these clones could form spheroids in a suspension culture system and form tumors in mice for several generations. Subsequently, multiple studies reported the presence of ovarian cancer stem cells (OCSCs) in ovarian cancer tissue. These stem cells possess characteristics similar to stem cells, such as self-renewal, unlimited proliferation, strong tumorigenicity, and the ability to generate all cell types within tumor tissue, and have been shown to be involved in tumorigenesis. Several related studies have demonstrated that OCSCs are associated with chemotherapy resistance in ovarian cancer. The use of platinum-based chemotherapy induces treatment-mediated selection, leading to the enrichment of OCSCs. High expression of drug efflux pumps was detected in OCSCs, confirming their strong resistance to chemotherapy and radiotherapy. In addition, compared with other tumor cells, CSCs are better able to adapt to the ever-changing tumor microenvironment. Furthermore, due to their effective DNA repair capabilities and ability to evade host immune surveillance, CSCs are able to survive treatment and play a key role in tumor recurrence. Therefore, these properties also make them the biggest obstacle to radical tumor cure and a therapeutic target.

[0003] Currently, the main treatment for ovarian cancer involves cytoreductive surgery followed by chemotherapy with paclitaxel or platinum-based drugs. Most patients respond well to initial platinum- or paclitaxel-based treatment, but relapse occurs after a few months, after which different series of targeted therapies are initiated. Current strategies for treating cystic ovarian cancer (CSC) primarily focus on identifying targets expressed in CSCs or molecules involved in their self-renewal pathways, as well as defining characteristics of CSCs, such as cell surface markers, metabolic alterations, and signaling pathway changes. Furthermore, regulatory factors in the tumor microenvironment that maintain CSC stemness may also be targets for effective treatment. Therefore, exploring key genes for maintaining CSC stemness and researching treatment strategies for ovarian cancer patients are essential for their survival.

[0004] Isopentenyl-Diphosphate Delta Isomerase 1 (IDI) is located in intracellular peroxisomes and participates in the interconversion of DMAPP and IPP within cells. Multiple polymerization reactions can occur between IPP and DMAPP, as well as between intermediates generated from the condensation of IPP and DMAPP, ultimately synthesizing various isoprene-like substances, including sterols, steroid hormones, and other important intracellular metabolic substances. Literature reports that IDI1 plays a role in the development of decreased ovarian reserve (DOR) and Lewy bodies, and may be involved in the development of diabetic nephropathy (DKD), cervical squamous cell carcinoma, and other diseases. Summary of the Invention

[0005] The purpose of this invention is to provide an application of IDI1 as a target in the diagnosis and / or treatment of ovarian cancer.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of an IDI1 inhibitor in the preparation of a drug for treating ovarian cancer.

[0008] This invention also provides the application of an IDI1 inhibitor in the preparation of a stem cell maintenance drug for ovarian cancer stem cells.

[0009] Preferably, the inhibitor is selected from IDI1 gene antagonists, shRNAs that inhibit IDI1 gene expression, or gene editing vectors that inhibit IDI1 gene expression.

[0010] Preferably, the shRNA is synthesized containing sequences SEQ ID NO.1 to SEQ ID NO.4.

[0011] Preferably, the inhibitor can target and suppress IDI1 gene expression, significantly inhibiting the growth and proliferation of ovarian cancer cells.

[0012] This invention also provides the application of a reagent for detecting IDI1 expression in the preparation of diagnostic products for ovarian cancer.

[0013] Preferably, the reagent is used to diagnose the development of ovarian cancer by detecting the expression level of IDI1 in the ovarian cancer tissue cells of the subject and comparing it with that in normal ovarian tissue cells.

[0014] Preferably, the expression level of IDI1 in ovarian cancer tissue cells of different subtypes is higher than that in normal ovarian epithelial cells.

[0015] The present invention also provides the application of a reagent for detecting IDI1 expression in the preparation of products for assessing the survival time of ovarian cancer patients.

[0016] This invention also provides the application of the IDI1 molecular marker in the preparation of diagnostic reagents for diagnosing the prognosis and progression of ovarian cancer.

[0017] Compared with the prior art, the present invention has the following technical effects:

[0018] This invention analyzes and detects the expression of IDI1 in human ovarian cancer cell lines, and finds that IDI1 is highly expressed in ovarian cancer cell lines. At the same time, the patient survival prognosis analysis results show that high expression of IDI1 is associated with poor patient survival prognosis. Knocking down IDI1 in ovarian cancer cell lines can significantly inhibit the maintenance of stemness of ovarian cancer stem cells and inhibit the growth and proliferation of ovarian cancer cells.

[0019] This invention utilizes a suspension culture method to induce ovarian cancer stem cells in vitro. These 3D cultured ovarian cancer cells exhibit tumor stem cell characteristics. The expression of IDI1 in normally cultured ovarian cancer cells (2D) and 3D ovarian cancer stem cells was detected. Western blotting results showed high expression of stem cell markers CD133, ALDH, and Oct4 in the 3D cultured A2780 and SKOV3 ovarian cancer stem cells constructed in this invention, indicating stem cell characteristics. Furthermore, IDI1 was highly expressed at the protein level in ovarian cancer stem cells. Real-time quantitative PCR was used to detect the expression of these genes at the mRNA level, and the results also showed a significant increase in IDI1 expression at the mRNA level in ovarian cancer stem cells, suggesting that IDI1 is involved in maintaining the stemness of ovarian cancer stem cells. This invention provides insights into the mechanisms of ovarian cancer development and progression, and identifies therapeutic targets. Attached Figure Description

[0020] Figure 1 This is a graph showing the expression analysis of IDI1 in ovarian cancer cell lines.

[0021] Figure 2 This is a graph showing the expression analysis of IDI1 in various ovarian cancer subtypes.

[0022] Figure 3 The Kaplan-Meier Plotter analysis plot shows the correlation between high IDI1 expression and poor patient survival prognosis. The left plot shows the overall survival (OS) of patients, and the right plot shows the progression-free survival (PPS) of patients.

[0023] Figure 4 Figure showing the protein expression and quantification results of IDI1 in ovarian cancer cell lines and immortalized normal ovarian epithelial cells;

[0024] Figure 5 Figure showing the protein expression results of IDI1 in ovarian cancer stem cells;

[0025] Figure 6Figure showing the expression of IDI1 at the mRNA level in ovarian cancer stem cells;

[0026] Figure 7 Image showing the spheroidization of A2780 and SKOV3 ovarian cancer stem cells after IDI1 knockdown;

[0027] Figure 8 Figure 1 shows the spheroidization rate and spheroid diameter of A2780 cells after IDI1 knockdown.

[0028] Figure 9 Figure 1 shows the spheroidization rate and spheroid diameter of SKOV3 cells after IDI1 knockdown.

[0029] Figure 10 Image showing the spheroidization of A2780 second-generation ovarian cancer stem cells after IDI1 knockdown;

[0030] Figure 11 Figure 1 shows the spheroidization rate and spheroid diameter of A2780 second-generation cells after IDI1 knockdown.

[0031] Figure 12 Figure showing the protein expression levels of stem cell markers in SKOV3 and A2780 ovarian cancer cells after IDI1 knockdown;

[0032] Figure 13 Figure showing the mRNA expression levels of stem cell markers in SKOV3 and A2780 ovarian cancer cells after IDI1 knockdown;

[0033] Figure 14 Figure showing the expression levels of IDI1 in the ovarian cancer cell lines SKOV3 and A2780 after IDI1 knockdown.

[0034] Figure 15 Figure 1 shows the growth count results of SKOV3 and A2780 cells after IDI1 knockdown.

[0035] Figure 16 The image shows the results of the SKOV3 cell cloning experiment after IDI1 knockdown. Detailed Implementation

[0036] This invention analyzes the expression of IDI1 in ovarian cancer tissue cells. The results show that IDI1 is highly expressed in ovarian cancer cells compared with normal ovarian cells. The ovarian cancer cells are classified and statistically analyzed according to their respective ovarian cancer subtypes. The results show that IDI1 is highly expressed in ovarian cancer tissues of different subtypes, suggesting that IDI1 is involved in the occurrence and development of ovarian cancer.

[0037] This invention provides the application of an IDI1 inhibitor in the preparation of a drug for treating ovarian cancer.

[0038] This invention also provides the application of an IDI1 inhibitor in the preparation of a stem cell maintenance drug for ovarian cancer stem cells. Ovarian cancer, as the leading cause of death among malignant tumors of the female reproductive system, seriously threatens women's lives and health. Ovarian cancer stem cells, present within ovarian cancer, play a crucial role in the recurrence and metastasis of ovarian cancer. However, currently there are few therapeutic targets for ovarian cancer stem cells, making it impossible to effectively eliminate them to achieve therapeutic goals. This invention aims to explore the key gene IDI1 involved in maintaining the stemness of ovarian cancer stem cells, hoping to use it as a therapeutic target for ovarian cancer patients and inhibit the occurrence and development of ovarian cancer.

[0039] In this invention, the inhibitor is selected from IDI1 gene antagonists, shRNA that inhibits IDI1 gene expression, or gene editing vector that inhibits IDI1 gene expression.

[0040] In this invention, the shRNA is synthesized using sequences SEQ ID NO.1 to SEQ ID NO.4.

[0041] In this invention, the inhibitor can target and suppress the expression of the IDI1 gene, significantly inhibiting the growth and proliferation of ovarian cancer cells.

[0042] This invention also provides the application of a reagent for detecting IDI1 expression in the preparation of diagnostic products for ovarian cancer.

[0043] In this invention, the reagent is used to diagnose the development of ovarian cancer by detecting the expression level of IDI1 in the ovarian cancer tissue cells of a subject and comparing it with that of normal ovarian tissue cells.

[0044] In this invention, the expression level of IDI1 in different subtypes of ovarian cancer tissue cells is higher than that in normal ovarian epithelial cells.

[0045] The present invention also provides the application of a reagent for detecting IDI1 expression in the preparation of products for assessing the survival time of ovarian cancer patients.

[0046] This invention also provides the application of the IDI1 molecular marker in the preparation of diagnostic reagents for diagnosing the prognosis and progression of ovarian cancer. This invention utilizes the Kaplan-Meier Plotter assay to analyze the correlation between IDI1 expression and patient survival prognosis. Overall survival (OS) and progression-free survival (PPS) results showed that high IDI1 expression was associated with poor patient prognosis.

[0047] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0048] Example 1

[0049] Constructing the pGreenPuro-IDI1-sh knockdown vector:

[0050] The pGreenPuro lentiviral vector suitable for expressing shRNA was selected, and the shRNA sequence corresponding to IDI1 was cloned into the vector. The primers for IDI1-sh were designed according to the primer design principles and the vector instructions, as shown in Table 1.

[0051] Table 1 Primer sequence information

[0052]

[0053] Example 2

[0054] Lentiviral packaging:

[0055] The HIV lentiviral expression vector designed and used in this study was pGreenPuro, and the HIV viral packaging elements were provided by psPAX2 and pMD2.G plasmids.

[0056] (1) Cell plating: Place 1.5-2.0×10 6 293TN cells were evenly seeded into 6cm culture dishes, and the cell growth density was increased to 50-70% after 24 hours.

[0057] (2) When the 293TN cells reach the required density after 24 hours of growth, the following operations are performed in the biosafety cabinet to obtain viruses containing pGreenPuro empty vector and pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 vectors for subsequent experiments.

[0058] (3) Take two 1.5ml sterile EP tubes, label them A and B respectively. Add 2.5μg psPAX, 2.5μg pMD2.G, and 3μg of the plasmid to be transfected (pGreenPuro empty vector, pGreenPuro-IDI1-sh1, or pGreenPuro-IDI1-sh2 vector) to tube A, and add 250μl opti-MEM. Add 24μl of PEI at a concentration of 1μg / μl and 250μl of opti-MEM to tube B. Vortex to mix and incubate at room temperature for 5min.

[0059] (4) Mix tubes A and B, vortex to mix, and incubate at room temperature for 20 minutes.

[0060] (5) Remove the culture medium from the 293TN cells and add the mixture evenly to a culture dish. Incubate at room temperature for 5 minutes, then carefully and slowly add 2.5 ml of DMEM culture medium and place in a cell culture incubator to continue culturing.

[0061] (6) 24 hours after transfection, replace with 3 ml of fresh DMEM medium; 48 hours later, collect the supernatant and add 3 ml of fresh DMEM medium. Store the supernatant in a 4°C refrigerator. 72 hours later, collect the supernatant again, mix the two supernatants and centrifuge, and aliquot and store at -80°C for later use.

[0062] Example 3

[0063] Construction of pGreenPuro-IDI1-sh1, pGreenPuro-IDI1-sh2 and Ctrl-sh cell lines

[0064] (1) Digest the cells and place an appropriate amount of cells in a 6cm culture dish so that the cell density reaches 50%-80% during transfection. The same type of cells should be used to construct Ctrl-sh and pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cell lines. The treatment time, culture conditions and density should be kept as consistent as possible.

[0065] (2) Thaw the collected virus from the -80℃ freezer. Select SKOV3 and A2780 cells that are growing well in a 6cm culture dish. Discard the original culture medium in the cell culture dish. Add about 1ml of pGreenPuro-IDI1-sh1, pGreenPuro-IDI1-sh2, or pGreenPuro virus evenly to the culture dish, being as gentle as possible to avoid damaging the cells. After 6 hours, add 2ml of RPMI-1640 medium. After 24 hours, expand the culture to a density of 90%-100%. After 48 hours, replace the medium with RPMI-1640 medium containing 2μg / ml puromycin for cell selection. Culture for 2-3 days and then conduct subsequent experiments on IDI1 expression, stem cell stemness maintenance, and cell growth.

[0066] Example 4

[0067] IDI1 expression detection

[0068] (1) After 2-3 days of screening with puromycin, cells were observed to express green fluorescence under a microscope. Cells were collected, RNA was extracted, and reverse transcription was performed. GAPDH was used as an internal control and Ctrl-sh cells were used as a control. Real-time quantitative PCR was used to detect the mRNA expression level of IDI1 in pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells to confirm that the target gene IDI1 was knocked down.

[0069] (2) Collect cells after puromycin screening and extract total protein. Using Ctrl-sh cells as a control, Western Blot was used to detect the expression level of IDI1 protein in pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells to confirm that the target gene IDI1 was knocked down.

[0070] Example 5

[0071] Cell growth counting experiment

[0072] (1) The confirmed IDI1 knockout pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells and control Ctrl-sh cells were digested and counted, and then divided into groups of 1×10⁻⁶ cells. 4 / wells were inoculated into 24-well plates.

[0073] (2) Cell counting: On day 1 after cell inoculation, 1×10⁶ cells were counted. 4 The initial cell number was used as the starting point. Cell counts were performed on days 3, 5, and 7, and cell growth data were recorded.

[0074] (3) Growth curve plotting: Plot the growth curve based on the count data and perform error and significance analysis to confirm whether cell growth and proliferation are inhibited.

[0075] Example 6

[0076] Cloning experiment

[0077] (1) The pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells, which were confirmed to have IDI1 knockdown, and the control Ctrl-sh cells were digested and counted. 800 cells were seeded into 12-well plates, and the cells were dispersed by pipetting to ensure that they were evenly distributed in the wells to ensure that individual cells formed clones. The cell status was observed during the culture process, and the culture medium was changed in a timely manner.

[0078] (2) After culturing for about two weeks, the culture was terminated after observing significant differences in the formation of cell clones in different groups.

[0079] (3) Take the culture medium from the well plate and wash it with PBS 2-3 times. Add 1 ml of formaldehyde fixative to each well and fix it at room temperature for 20 min.

[0080] (4) After fixation, remove the fixative and add 500 μl of 0.1% crystal violet stain to each well. Stain at room temperature for 20 min.

[0081] (5) After staining, remove the staining solution and rinse with deionized water 2-3 times to remove excess staining solution. Place the plate in a dry place and let it air dry naturally before taking pictures and analyzing.

[0082] (6) Use ImageJ to analyze the final clone formation results and statistically analyze the error and significance.

[0083] Example 7

[0084] Stem cell spheroidization analysis

[0085] (1) The pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells confirmed to have IDI1 knockdown and the control Ctrl-sh cells were digested and collected in 1.5ml EP tubes. The cells were washed with PBS 2-3 times to completely remove residual culture medium.

[0086] (2) Count the washed cells according to a count of 1×10⁻⁶. 4 / wells were seeded into ultra-low adsorption six-well plates and cultured in stem cell culture medium. The culture medium was changed on day 3 and day 6.

[0087] (3) On the 7th day of culture, the cell spheres were photographed, and the sphere formation rate and cell sphere diameter were statistically analyzed to detect the cell sphere formation ability.

[0088] Example 8

[0089] Analysis of stem cell self-renewal capacity

[0090] (1) Collect the cell spheres formed 1 week after the confirmed knockdown pGreenPuro-IDI1-sh1 and pGreenPuro-IDI1-sh2 cells and control Ctrl-sh cells into 1.5ml EP tubes, add 80-100μl of 0.05% trypsin according to the number of cell spheres, and continuously pipette for 1min to completely digest the cell spheres into single cells.

[0091] (2) After digestion, add 200 μl of serum-containing RPMI-1640 medium to stop digestion, and then wash with PBS 2-3 times to completely remove the medium.

[0092] (3) Count the cells after washing, at a rate of 1 × 10⁻⁶ cells per well. 4The cells were re-passaged and seeded in 6-well plates with ultra-low adsorption. After 7 days of culture, the second-generation cell spheres were photographed and statistically analyzed to detect the self-renewal capacity of stem cells.

[0093] The results are analyzed as follows:

[0094] (1) According to Figures 1-2 It can be seen that the expression of IDI1 in ovarian cancer cells was analyzed ( Figure 1 The results showed that IDI1 was highly expressed in ovarian cancer cells compared to normal ovarian cells. Ovarian cancer cells were classified and statistically analyzed according to their respective ovarian cancer subtypes. Figure 2 The results showed that IDI1 was highly expressed in different subtypes of ovarian cancer tissues, suggesting that IDI1 is involved in the occurrence and development of ovarian cancer.

[0095] (2) The correlation between IDI1 expression and patient survival prognosis was analyzed using the Kaplan-Meier Plotter assay. Overall survival (OS) and progression-free survival (PPS) results showed... (See...) Figure 3 High expression of IDI1 is associated with poor patient survival prognosis, indicating that high IDI1 expression is related to poor patient survival.

[0096] (3) According to Figure 4 As can be seen, Western blot analysis of IDI1 expression in immortalized human normal ovarian epithelial cells IOSE80 and ovarian cancer cells showed that ( Figure 4 (Right image) Compared with normal ovarian epithelial cells IOSE80, IDI1 is highly expressed in various ovarian cancer cells such as A2780, SKOV3, and OVCAR5.

[0097] (4) To investigate the effect of IDI1 on the maintenance of ovarian cancer stem cell ... Figure 5 Real-time quantitative PCR was used to detect the expression of these genes at the mRNA level, and the results also showed that IDI1 expression was significantly increased at the mRNA level in ovarian cancer stem cells. Figure 6 The results suggest that IDI1 is involved in maintaining the stemness of ovarian cancer stem cells.

[0098] (5) To confirm the effect of IDI1 on the maintenance of ovarian cancer stem cell stemness, this invention used ovarian cancer cells A2780 and SKOV3 to knock down the endogenously expressed IDI1, and then evaluated the stemness of ovarian cancer cells from the following three aspects:

[0099] Ovarian cancer stem cell stemness maintenance capacity: By observing the spheroidization of ovarian cancer stem cells after A2780 and SKOV3 inoculation ( Figure 7 The study analyzed the final ovarian cancer stem cell spheroidization ability and the size of the spheroids. The results showed that after IDI1 knockdown, the spheroidization rate of A2780 and SKOV3 cells decreased significantly, and the diameter of the spheroids decreased significantly, indicating that the stemness of ovarian cancer stem cells was inhibited after IDI1 knockdown.

[0100] Ovarian cancer stem cell self-renewal capacity: A2780 ovarian cancer stem cells that had already formed spheroids were digested and passaged. The ability of these cells to reform into cell spheroids and the size of the cell spheroids were statistically analyzed. Figure 10 This study can assess the self-renewal capacity of ovarian cancer stem cells. Results showed that IDI1 knockdown significantly reduced the spheroidization ability of A2780 second-generation cells. Figure 11 ), and the diameter of the formed spheres decreased significantly ( Figure 12 This indicates that the stemness of ovarian cancer stem cells was suppressed after IDI1 knockdown.

[0101] Expression of stem cell markers: CD133, Oct4, and ALDH are markers of tumor stem cell stemness, and their expression can reflect cell stemness. The expression of stem cell markers in SKOV3 and A2780 ovarian cancer cells after IDI1 knockdown was detected. Western blotting results showed that the expression of stem cell markers was downregulated after IDI1 knockdown. Figure 12 ), and qPCR testing showed the same results ( Figure 13 This indicates that cell stemness is suppressed after IDI1 knockdown.

[0102] (6) According to Figures 13-16 It can be seen that knocking down IDI1 in ovarian cancer cell lines SKOV3 and A2780, which have high IDI1 expression, significantly inhibits the growth and proliferation of ovarian cancer cells through cell growth counting and colony formation experiments, indicating that IDI1-targeted therapy for ovarian cancer is feasible.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting IDI1 expression in the preparation of diagnostic products for ovarian cancer.

2. The application according to claim 1, characterized in that, The reagent is used to diagnose the development of ovarian cancer by detecting the expression level of IDI1 in the subject's ovarian cancer tissue cells and comparing it with that in normal ovarian tissue cells.

3. The application according to claim 2, characterized in that, The expression level of IDI1 in ovarian cancer tissue cells of different subtypes is higher than that in normal ovarian epithelial cells.

4. Application of reagents for detecting IDI1 expression in the preparation of products for assessing the survival time of ovarian cancer patients.

5. Application of reagents for detecting IDI1 molecular markers in the preparation of diagnostic reagents for diagnosing the prognosis and progression of ovarian cancer.

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