Application of PSMD14 gene inhibitor in preparation of anti-cervical cancer drugs

Abstract

The invention provides an application of a PSMD14 gene inhibitor in preparation of an anti-cervical cancer drug. The high expression of the PSMD14 in cervical cancer tissues is found by retrieving a TCGA database, and then the proliferation, migration and invasion ability of cervical cancer can be remarkably inhibited by knocking down the PSMD14 gene through experiments. Meanwhile, the PSMD14 gene inhibitor can increase the sensitivity of cervical cancer to cis-platinum, and the PSMD14 inhibitor and cis-platinum are combined to serve as a novel multi-target combined treatment medicine and have the effects of enhancing efficiency and reversing drug resistance. The invention provides a new drug design target, and has great clinical transformation and application effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of a PSMD14 gene inhibitor in the preparation of an anti-cervical cancer drug. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Cervical cancer is the second most common malignancy in women and the fourth leading cause of cancer-related death in women worldwide.

[0004] Treatments for cervical cancer include surgery, platinum chemotherapy drugs, and radiotherapy. The latter two are currently the most commonly used treatments for advanced and recurrent cervical cancer. Both kill tumor cells by causing DNA damage. Platinum drugs mainly cause DNA cross-linking and DNA replication fork obstruction; radiation directly causes single-strand or double-strand breaks in DNA. Serious drug side effects, tumor resistance, and poor selectivity of chemotherapy drugs are the main reasons for treatment failure. Therefore, finding effective targeted therapeutic molecules to enhance the sensitivity of cervical cancer to radiotherapy and chemotherapy is of great significance for improving the efficacy of cervical cancer and improving the prognosis of patients.

[0005] The ubiquitin proteasome system (UPS) can mediate the degradation of more than 80% of proteins in eukaryotic cells, and plays a key role in the protein stability and function of the substrate. As an important ubiquitination modification regulatory factor, deubiquitinating enzymes can cleave ubiquitin from substrates, modify ubiquitin chains and process ubiquitin precursors, and are closely related to a variety of physiological and pathological processes. Among them, the deubiquitinating enzyme PSMD14 (proteasome 26S subunit, non-ATPase 14) is located in the proteasome 26S subunit and is a non-ATPase component. At present, it is disclosed that PSMD14 is associated with the occurrence and development of various tumors, but the role and mechanism of PSMD14 in cervical cancer have not yet been clarified. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides the use of a PSMD14 gene inhibitor in the preparation of an anti-cervical cancer drug.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides the use of a PSMD14 gene inhibitor in the preparation of an anti-cervical cancer drug.

[0009] In one or more embodiments, the PSMD14 gene inhibitor comprises shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, a low molecular compound, a peptide or an antibody.

[0010] Preferably, the low molecular weight compound includes thiolutein (THL).

[0011] In one or more embodiments, the anti-cervical cancer drug can inhibit the proliferation of cervical cancer cells.

[0012] In one or more embodiments, the anti-cervical cancer drug can inhibit the migration and / or invasion of cervical cancer cells.

[0013] The second aspect of the present invention provides an anti-cervical cancer drug, including a PSMD14 gene inhibitor.

[0014] In one or more embodiments, the PSMD14 gene inhibitor comprises shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, a low molecular compound, a peptide or an antibody.

[0015] Preferably, the low molecular weight compound includes thiolutein (THL).

[0016] The third aspect of the present invention provides an anti-cervical cancer combined drug, the active ingredients of which include a PSMD14 gene inhibitor and a platinum drug.

[0017] In one or more embodiments, the platinum drug includes cisplatin, a first-line chemotherapy drug.

[0018] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0019] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy. Moreover, according to the usual methods, the drug can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0020] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0021] Preferably, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0022] Preferably, the medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. It can be applied optionally via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0023] Preferably, the drug administration subjects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0024] A fourth aspect of the present invention provides the use of the PSMD14 gene as a marker in the preparation of cervical cancer detection and / or efficacy evaluation products.

[0025] A fifth aspect of the present invention provides the use of the PSMD14 gene as a marker in the preparation of a reagent for evaluating the sensitivity of cervical cancer to chemotherapeutic drugs.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides the use of a PSMD14 gene inhibitor in the preparation of an anti-cervical cancer drug. By searching the TCGA database, it was found that PSMD14 was highly expressed in cervical cancer tissues, and then experimentally verified that knocking down the PSMD14 gene can significantly inhibit the proliferation, migration and invasion of cervical cancer. At the same time, the PSMD14 gene inhibitor can increase the sensitivity of cervical cancer to cisplatin. The PSMD14 inhibitor combined with cisplatin as a new multi-target combined therapeutic drug has synergistic and reversal effects of drug resistance. The present invention provides a new drug design target with greater clinical transformation and application effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1is the expression level of PSMD14 gene in cervical cancer; A is the expression level of PSMD14 gene in cervical cancer shown in TCGA database; B is the mRNA level of PSMD14 detected by q-PCR in 23 normal cervical tissues and 29 cervical cancer tissues; C is the TCGA database showing that the higher the grade of cervical cancer, the higher the expression of PSMD14;

[0030] Figure 2 The proliferation and migration ability of cervical cancer cells after downregulating the PSMD14 gene by small interfering agent; A is Western-blot to verify the siRNA knockdown efficiency of PSMD14; B is flow cytometry apoptosis experiment to verify the increase of cell apoptosis after knocking down PSMD14 (the horizontal axes in the left first, second and third figures in B are 0, 10 from left to right). 3 , 10 4 , and 10 5 , the vertical coordinates are 0 and 10 from bottom to top 2 , 10 3 , 10 4 , and 10 5 ); C is the CCK-8 method to verify that the cell proliferation is weakened after interfering with PSMD14; D is the plate clone formation experiment to verify that the cell plate clone formation ability is reduced after interfering with PSMD14; E: The scratch experiment detects the cell migration ability, and it is found that the migration ability of cervical cancer cells Hela after interfering with PSMD14 is significantly weakened after interfering with PSMD14 to inhibit the clone formation of cells;

[0031] Figure 3 The proliferation, migration and invasion abilities of cervical cancer cells after overexpression of PSMD14 gene; A is the verification of PSMD14 overexpression efficiency by qPCR and Western-blot; B is the verification of enhanced cell proliferation after overexpression of PSMD14 by CCK-8 method; C is the enhanced clone formation ability of cells after overexpression of PSMD14; D is the scratch test to detect cell migration ability, and it is found that the migration ability of cervical cancer cell Hela is significantly enhanced after overexpression of PSMD14; E is the Transwell experiment to find that the migration and invasion abilities of cells are significantly increased after overexpression of PSMD14;

[0032] Figure 4 The PSMD14 small molecule inhibitor has the effect of inhibiting the proliferation and migration of cervical cancer cells and promoting cell apoptosis; A shows that after the cervical cancer cells were treated with the PSMD14 small molecule inhibitor thiocarbazine, the cell spheroid formation was significantly reduced; B shows that after the cervical cancer cells were treated with the PSMD14 small molecule inhibitor thiocarbazine, cell apoptosis was significantly increased, and with the increase of the concentration of the inhibitor, the number of apoptotic cells increased (the horizontal axes in the left first, second and third figures of Caski in B and the left first, second and third figures of Hela are 0, 10 from left to right 4 , 105 , and 10 6 , the vertical coordinates are 0 and 10 from bottom to top 4 , 10 5 , 10 6 , and 10 7 ); C is that the PSMD14 small molecule inhibitor significantly inhibited the cloning ability of cervical cancer cells; D is the scratch test to detect the cell migration ability, and it was found that after the cervical cancer cells Hela were treated with thiocarbazine, the cell migration ability was significantly weakened;

[0033] Figure 5 This is the effect of the PSMD14 small molecule inhibitor THL and cisplatin on cervical cancer cells; CCK-8 experiments found that cisplatin combined with THL can enhance the sensitivity of cisplatin to cervical cancer cells. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0037] In the following embodiments, the experimental methods and operation steps involved specifically include:

[0038] (1) CCK-8 assay to detect cell proliferation

[0039] Collect cells in logarithmic growth state, digest with trypsin, and then count with a cell counter. 2-3×10 cells per well 3 Cells were inoculated into a 96-well plate. At a defined time point, 20 μL CCK-8 detection reagent was added to the 96-well plate. The cells were cultured at 37°C in a cell culture incubator for 1 h, and the absorbance was measured at 450 nm using an enzyme reader.

[0040] (2) Cell clone formation experiment

[0041] Cells in logarithmic growth were collected and digested with trypsin. 500 to 1000 cells were plated on a six-well plate and cultured in a 37°C incubator containing 10% fetal bovine serum and 5% carbon dioxide for 1 to 2 weeks. Subsequently, the cells were washed with PBS, fixed with paraformaldehyde for 10 minutes at room temperature, washed three times with PBS, and finally stained with 0.1% crystal violet for 20 minutes. The crystal violet stain was washed off with tap water and photographed.

[0042] (3) Transwell assay

[0043] Remove the culture medium from cells in good growth state and starve them for 24 hours by adding serum-free culture medium. Place the matrix gel on ice and melt overnight at 4°C. Dilute the matrix gel to 1 mg / mL with serum-free culture medium, add it vertically to the Transwell chamber, and incubate it in a 37°C, 5% carbon dioxide incubator for 2 hours. Take 700 μL of complete culture medium containing 10% FBS and add it to the lower chamber of the 24-well plate. Use tweezers to place the Transwell chamber in the 24-well plate. Digest the starved cells, resuspend them with serum-free culture medium, and add them to the chamber at a ratio of sample: cell suspension = 1:1. Place the 24-well plate at 37°C and culture for 24 hours. Take out the Transwell chamber, remove the culture medium, add 600 μL of 4% paraformaldehyde fixative to the clean wells of the 24-well plate, and place the chamber in fixation for 30 minutes. Discard the fixative and wash the inside and outside of the chamber once with PBS. Add 600 μL of crystal violet staining solution to the clean wells of the 24-well plate and place the chamber in staining for 10 minutes. The chamber was removed and the inside and outside of the chamber were washed with PBS for 3 times. After proper air drying, the chamber was observed under a microscope for qualitative research.

[0044] (4) Scratch test

[0045] After trypsin digestion, the cells in the experimental group and the control group were counted at 2×10 5 Cells / well were placed in a six-well plate, which was placed in a 37°C incubator containing 5% carbon dioxide. After 24 hours, a 200 μL pipette tip was used to scratch the bottom of the six-well plate, and the width of the scratch was kept consistent as much as possible. The scratches were photographed under a microscope at 0 hours, 24 hours, and 48 hours after the scratches, and the changes in the scratches were analyzed.

[0046] (5) Cell apoptosis detection

[0047] Flow cytometry was used to analyze the effect of PSMD14 on cell apoptosis at different stages.

[0048] After knocking down the PSMD14 gene for 48 hours, the cells were washed three times with PBS, and the cells were collected by trypsin digestion without EDTA. The centrifuge parameters were set to 1500 rpm for 5 min. The cell pellet was stained with an apoptosis kit for 15 min and analyzed by flow cytometry (BD LSR II).

[0049] (6) q-PCR detection

[0050] Total RNA was isolated using an RNA extraction kit (B004D, EZ Bioscience, USA), and then reverse transcribed into template cDNA using reverse transcriptase (HiScript III RT SuperMix for qPCR Kit (R323-01, Vazyme, China)). 100 ng of cDNA was taken as a template, and then the mRNA level was quantitatively amplified using real-time fluorescence quantitative PCR (RT-qPCR) (Hieff qPCR SYBR Green Master Mix (11201ES08, Easen Biotech, China) was used to detect the mRNA level. After the amplification, it was confirmed that there were no double peaks in the melting curve, and the expression level of the target gene was calculated according to the 2-ΔΔCT method.

[0051] (7) Protein immunoblotting

[0052] The cells were washed three times with PBS and then lysed on ice for about 30 min. The lysate consisted of immunoprecipitation assay buffer (RIPA), phenylmethylsulfonyl fluoride (PMSF), and NaF. The cells were then lysed by ultrasound, centrifuged at 12,000 rpm at 4°C, and the supernatant was collected and the protein concentration was determined using a BCA protein assay kit (Beyotime, Jiangsu, China). The loading buffer was added and the protein was denatured by heating in a metal bath for 5 min. Total protein (30-50 μg) was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% gel (Invitrogen) and transferred to a PVDF membrane (Immobilon P; Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1 h, the membrane was cut into strips and incubated with the designated primary antibodies overnight at 4°C. The next day, the cells were washed three times with TBS-T (20 mM Tris, pH 7.4, 137 mM NaCl, 0.05% Tween-20) and labeled with secondary antibodies at room temperature for 1 h. Finally, the immunoreactive protein bands were detected by enhanced chemiluminescence (ECL) using ImageQuant LAS 4000 (GE Healthcare Life Sciences, Logan, UT, USA).

[0053] (8) 3D spheroid formation experiment

[0054] The cells in the experimental and control groups were digested with trypsin and the culture medium completely terminated the reaction. The cells were counted using a cell counter, with approximately 2×10 5 The cells were placed in an ultra-low attachment dish and cultured in a cell culture incubator at 5% CO2 and 37°C. After 3 days of cell culture, the cell spheres were observed under a microscope and photographed.

[0055] Example 1

[0056] The TCGA database revealed that PSMD14 was highly expressed in cervical cancer tissues ( Figure 1 A), q-PCR was used to detect the mRNA level of PSMD14 in 23 normal cervical tissues and 29 cervical cancer tissues, and it was found that the mRNA level of PSMD14 was significantly increased in cervical cancer tissues ( Figure 1 B). At the same time, through the analysis of TCGA database, it was found that with the increase of cervical cancer grade, the higher the expression of PSMD14 ( Figure 1 C).

[0057] Example 2

[0058] We selected Hela, a cervical cancer cell line with high expression of PSMD14 gene, and conducted siRNA interference experiment (siPSMD14-1: GACACAGCAGAACAAGTCT; siPSMD14-2: CACTCAGCAGAGCTTTGAA) to verify the effect of knocking down PSMD14 gene on the biological function of cervical cancer cells. First, we verified the interference efficiency of small interference on PSMD14 gene by WB ( Figure 2 A), flow cytometry apoptosis experiments found that knocking down the PSMD14 gene increased apoptosis in cervical cancer cells ( Figure 2 B). Whether it affects the cell proliferation ability, the effect of PSMD14 gene on the proliferation of cervical cancer cells was detected by CCK-8 and plate cloning experiments. The experimental results showed that the proliferation ability of cervical cancer cells was significantly reduced after knocking down the PSMD14 gene ( Figure 2 C and D). The scratch assay verified the effect of knocking down the PSMD14 gene on cell migration ability and found that the migration of cervical cancer cells was weakened after knocking down PSMD14 ( Figure 2 E).

[0059] Example 3

[0060] The biological function of PSMD14 gene on cervical cancer cells was observed by overexpressing it in cervical cancer cells, and the overexpression rate of PSMD14 in cervical cancer cells was verified by qPCR and WB. Figure 3A). The results are consistent with the hypothesis that overexpression of the PSMD14 gene enhances the proliferation and cloning ability of cervical cancer cells ( Figure 3 B and C); Overexpression of PSMD14 gene enhances cell migration ability ( Figure 3 D); Transwell experiments found that the migration and invasion abilities of cervical cancer cells were significantly enhanced after overexpression of PSMD14 gene ( Figure 3 E).

[0061] Example 4

[0062] Thioluteolin (THL), as a small molecule inhibitor of the PSMD14 gene, has an anti-tumor effect in head and neck cancer. Does THL play the same role in cervical cancer? Therefore, its biological function in cervical cancer was verified. The 3D spheroid experiment can simulate the state of tumor cells in the microenvironment, so a 3D spheroid experiment was conducted and it was found that the spheroidization ability of cervical cancer cells treated with THL was significantly weakened, and the diameter of the spheroids formed was significantly reduced ( Figure 4 A). Flow cytometry apoptosis experiments found that after THL treatment, cell apoptosis increased significantly and was concentration-dependent, indicating that small molecule inhibitors of the PSMD14 gene have the effect of promoting apoptosis of cervical cancer cells ( Figure 4 B). Plate cloning experiments have shown that as the concentration of THL increases, the plate cloning ability of cells gradually weakens ( Figure 4 C). The scratch test showed that after THL treatment of cervical cancer cells, cell migration was significantly weakened ( Figure 4 D) The above experiments prove that THL, a small molecule inhibitor of PSMD14 gene, inhibits the biological function of cervical cancer and has the effect of inhibiting tumor proliferation and metastasis.

[0063] Example 5

[0064] Cisplatin is the most common chemotherapy drug in the current clinical treatment of cervical cancer. Next, we will explore whether it has the effect of enhancing the sensitivity of cervical cancer to cisplatin. Under the premise of treating cervical cancer cells with cisplatin (1μg / ml), further treatment with THL (1μM) found that the killing effect on cervical cancer cells was significantly enhanced ( Figure 5 ), which means that THL promotes the sensitivity of cervical cancer cells to cisplatin and can be used as a combined inhibitor to inhibit cervical cancer.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of PSMD14 gene inhibitors in the preparation of anti-cervical cancer drugs.

2. The use according to claim 1, characterized in that The PSMD14 gene inhibitor includes shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides or antibodies; Preferably, the low molecular weight compound includes thiolutein.

3. The use according to claim 1, characterized in that Anti-cervical cancer drugs can inhibit the proliferation of cervical cancer cells.

4. The use according to claim 1, characterized in that Anti-cervical cancer drugs can inhibit the migration and / or invasion of cervical cancer cells.

5. An anti-cervical cancer drug, characterized in that: Including PSMD14 gene inhibitors.

6. The anti-cervical cancer drug according to claim 5, characterized in that: The PSMD14 gene inhibitor includes shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides or antibodies; Preferably, the low molecular weight compound includes thiolutein.

7. An anti-cervical cancer combined drug, characterized in that: Its active ingredients include PSMD14 gene inhibitors and platinum drugs.

8. The anti-cervical cancer combined drug according to claim 7, characterized in that: The platinum drugs include cisplatin, a first-line chemotherapy drug.

9. Application of PSMD14 gene as a marker in the preparation of cervical cancer detection and / or efficacy evaluation products.

10. Application of PSMD14 gene as a marker in the preparation of reagents for evaluating the sensitivity of cervical cancer to chemotherapeutic drugs.

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