Novel prostate cancer treatment target and application thereof in prostate cancer treatment

Abstract

The invention relates to the technical field of medicines, in particular to NUDT5 and SEPT8 serving as novel prostate cancer treatment targets and application of the NUDT5 and the SEPT8 in prostate cancer treatment. The invention provides a novel targeted therapy scheme for prostatic cancer, the prostatic cancer cells are specifically killed in vivo by inhibiting prostatic cancer cell proliferation by simultaneously targeting NUDT5 and SEPT8 proteins with relatively high expression quantity in high-risk prostatic cancer, and a new thought is provided for clinical treatment of prostatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a new type of prostate cancer treatment target and its application in the treatment of prostate cancer. Background Art

[0002] Prostate cancer is the second most common solid tumor in men worldwide. The continued increase in its incidence and cancer-related mortality seriously threatens the lives and health of middle-aged and elderly men.

[0003] For decades, the standard treatment for most locally advanced or metastatic prostate cancer has been androgen deprivation therapy (ADT). Endocrine therapy uses the androgen-dependent property of prostate cancer to treat prostate cancer by depleting the patient's own endogenous androgens or directly targeting the patient's androgen receptors. Up to now, new endocrine drugs commonly used in clinical practice to achieve the effect of androgen blockade include: Bicalutamide, Flutamide, Abiraterone acetate, Enzalutamide, Darolutamide and Apalutamide.

[0004] However, after androgen deprivation therapy (ADT) with chemical or surgical castration, castration-resistant prostate cancer (CRPC) always relapses within a median of 14-20 months. Alterations in androgen receptor signaling are an important factor in the resistance of prostate cancer to androgen deprivation therapy. Metastatic castration-resistant prostate cancer (mCRPC) usually manifests alterations in the AR pathway due to amplification or gain-of-function mutations, increased AR transcription, or increased AR cells. Approximately 15% to 20% of advanced resistant prostate cancers lose their dependence on AR signaling and manifest as a transformation to the highly incurable castration-resistant neuroendocrine prostate cancer (NEPC).

[0005] Precision gene therapy is the research trend of future translational medicine. There is already gene editing technology that can edit multiple sites of the genome at the same time. Finding a gene combination that is universal in prostate cancer will be of great help in the development of precision gene therapy. At the same time, the protein product corresponding to the gene also has the potential to become a new therapeutic target, and the combined drug effect is good.

[0006] The SEPTIN family is a GTP-binding protein. In metaphase of mitosis, knocking out SEPTIN will lead to the loss of chromosomes on the metaphase plate; lack of chromosome separation and spindle elongation, and incomplete cell division due to delayed exit from mitosis. During mitosis, SEPTIN protein may form a scaffold structure for checkpoint regulation of centromere-associated protein E (CENP-E) and other effectors to coordinate cell separation and chromosome aggregation and separation. In this process, its ATP source may be produced by NUDT5 hydrolyzing ADP-ribose in the presence of pyrophosphate. ATP from the nucleus is crucial in the process of chromatin sufficiency, transcriptional regulation and cell proliferation. The present invention speculates that the place where NUDT5 and SEPT8 proteins play a common role should be in the nucleus. In DNA replication and mitosis engineering, NUDT5 provides a key energy source, and SEPT8 provides the scaffold required for mitosis, so knocking out these two proteins at the same time can greatly prevent the proliferation process of prostate cancer cells. There is no report on the application of simultaneous targeting of NUDT5 and SEPT8 in the treatment of prostate cancer. Summary of the invention

[0007] The purpose of the present invention is to provide a new targeted treatment for prostate cancer, which can simultaneously target NUDT5 and SEPT8 proteins, which are highly expressed in high-risk prostate cancer, and specifically kill prostate cancer cells in vivo by inhibiting the proliferation of prostate cancer cells, thereby providing a new idea for the clinical treatment of prostate cancer.

[0008] In order to achieve the purpose of the above invention, the present invention adopts the following technical solutions:

[0009] The present invention first detected the expression levels of NUDT5 and SEPT8 in different prostate cancer cell lines, and clarified that NUDT5 and SEPT8 are universal as therapeutic targets for prostate cancer. Then the PC3 prostate cancer cell line was selected, and the PC3 cell lines with NUDT5 single knockout, SEPT8 single knockout and NUDT5+SEPT8 double knockout were constructed using CRISPR / Cas9 gene editing technology. On the successfully constructed cell lines, the cell cycle of different cell lines was detected by flow cytometry. In order to further explore the effect of NUDT5 and SEPT8 on the proliferation of prostate cancer cells, the present invention used the ZenCell OWL live cell imaging system combined with the drawing of cell proliferation curves, and the EdU staining method to detect the cell proliferation rate. At the same time, the effect of SEPT8 and NUDT5 on cell migration ability was detected by cell scratch experiment, and the effect of SEPT8 and NUDT5 on cell invasion ability was detected by Transwell experiment. The reduction in cancer cell proliferation rate, migration and invasion ability represents a decrease in the malignancy of cancer cells, indicating that gene knockout can inhibit the activity of cancer cells and that this gene can be used as a target for targeted therapy of prostate cancer.

[0010] In a first aspect of the present invention, there is provided the use of NUDT5 and SEPT8 as targets in the preparation of drugs for treating prostate cancer.

[0011] The second aspect of the present invention provides the use of an agent for inhibiting the expression of NUDT5 and SEPT8 in the preparation of a drug for treating prostate cancer.

[0012] Furthermore, the reagent that inhibits the expression of NUDT5 and SEPT8 is an sgRNA, siRNA, shRNA, miRNA or antisense nucleotide that specifically interferes with the expression and processing of NUDT5 and SEPT8 genes, or a recombinant vector containing siRNA, shRNA, miRNA or antisense nucleotide, or an inhibitor that specifically inhibits the normal biological function of NUDT5 and SEPT8 proteins.

[0013] Furthermore, the reagent for inhibiting the expression of NUDT5 and SEPT8 is a reagent for inhibiting the expression of NUDT5 and SEPT8 by gene knockout technology, and the gene knockout technology is CRISPR-Cas9 technology.

[0014] Furthermore, the nucleotide sequences of the sgRNA used in the reagent for inhibiting the expression of NUDT5 and SEPT8 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0015] In a third aspect of the present invention, a recombinant vector is provided for use in preparing a drug for treating prostate cancer, wherein the recombinant vector comprises sgRNA that specifically interferes with the expression of NUDT5 and SEPT8 genes.

[0016] Furthermore, the nucleotide sequences of the sgRNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0017] In a fourth aspect, the present invention provides a drug for treating prostate cancer, wherein the drug comprises an agent that inhibits the expression of NUDT5 and SEPT8 as an active ingredient.

[0018] The present invention has the advantages that:

[0019] Compared with existing prostate cancer treatment methods, the present invention can reduce the off-target effects of targeted drug delivery for prostate cancer by targeting specific target proteins with high expression levels in prostate cancer. At the same time, in in vitro experiments, the present invention found that the simultaneous knockout of these two target proteins has a very ideal inhibitory effect on the proliferation, migration and invasion of prostate cancer cells. In addition, combined target therapy can reduce the concentration of targeted drugs required for each target, and is relatively safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Western Blot was used to detect the expression levels of NUDT5 and SEPT8 in different prostate cancer cell lines.

[0021] Figure 2 SEPT8 and NUDT5 were knocked out in PC3 cell lines using CRISPR-cas9 technology; Surveyor Assay was used to detect the gene editing efficiency of sgRNA for SEPT8 (A) and NUDT5 (B), and lentiCRISPR v2-Scramble was used as a control group. (C) Western Blot was used to detect the knockout efficiency of SEPT8 or NUDT5 in PC3 stable cell lines; (D) Western Blot was used to detect the knockout efficiency of SEPT8 and NUDT5 in PC3 SEPT8 and NUDT5 double knockout stable cell lines.

[0022] Figure 3 Flow cytometry was used to detect the effects of SEPT8 and NUDT5 knockout on PC3 cell cycle.

[0023] Figure 4 The figure shows the effect of knocking out SEPT8 or NUDT5 alone on the proliferation rate of PC3 cells. (A) The figure shows the cell status recorded in real time by ZenCell OWL, and (B) The figure shows the statistical graph of cell proliferation rate.

[0024] Figure 5 The effect of knocking out SEPT8 and NUDT5 simultaneously on the proliferation rate of PC3 cells. (A) The figure shows the cell status recorded in real time by ZenCell OWL, and (B) the figure shows the statistical graph of cell proliferation rate.

[0025] Figure 6 The EdU method was used to detect the effect of SEPT8 and NUDT5 knockout on the proliferation rate of PC3 cells. (A) The figure shows the EdU staining results in cells with single knockout of SEPT8 or NUDT5, or simultaneous knockout of SEPT8 and NUDT5, as well as control cells. The fluorescent signal in the Azide-488 channel in the figure is the EdU-positive cell, that is, the cell in the process of DNA replication. The more fluorescent signals, the faster the cell proliferation rate. Hoechst 33342 is a nuclear dye. (B) The figure shows the statistical graph of the proportion of EdU-positive cells in the corresponding cell lines.

[0026] Figure 7 Knockout of SEPT8 and / or NUDT5

[0027] Effects on PC3 cell migration. (A) Knockout of SEPT8 or NUDT5 alone in PC-3 cells has no effect on PC3 cell migration. (B) Knockout of SEPT8 and NUDT5 simultaneously can reduce PC3 cell migration. The two figures on the left are real-time cell photos, and the two figures on the right are PC3 cell migration rate statistics.

[0028] Figure 8 The results of the Transwell experiment showed that the simultaneous knockout of SEPT8 and NUDT5 could inhibit the cell invasion ability of the PC3 cell line. (A) The transwell experiment of simultaneous knockout of SEPT8 and NUDT5 in PC-3 cells showed that the invasion ability of PC-3 cells with simultaneous knockout of SEPT8 and NUDT5 was significantly reduced (B) The figure is a statistical chart. DETAILED DESCRIPTION

[0029] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0030] Example:

[0031] 1. Experimental methods:

[0032] Cell culture: All cell lines used in the present invention were cultured under the constant temperature conditions of 5% CO2 and 37°C. The culture medium was RPMI 1640 culture medium (10% FBS + 1% penicillin / streptomycin double antibody).

[0033] Western Blot: Equal amounts of protein (about 40 μg) were separated by SDS-PAGE electrophoresis and transferred to a 0.22 μm pore size PVDF membrane. The primary antibody was diluted 1:1000 with TBST and incubated overnight at 4°C. The HRP-labeled secondary antibody was diluted 1:5000 with TBST and the WB results were detected by chemiluminescent colorimetric reaction.

[0034] Primary Antibody:

[0035]

[0036] Secondary Antibody:

[0037]

[0038] CRISPR-Cas9 gene knockout:

[0039]

[0040] The guide was constructed on the lentiCRISR v2 plasmid and packaged as lentivirus in 293T cells; the cleavage efficiency of the guide RNA was tested by a surveyor assay. The prostate cancer cell line was infected with lentivirus by the polybrene method, the cells were screened by puromycin 1.0 ng / μL, and the knockout efficiency was identified by WB.

[0041] ZenCell OWL cell proliferation assay: Prostate cancer cells were plated at 5,000 cells / well in a 24-well plate. The ZenCell OWL live cell assay system was used to record cell proliferation in real time, and the cell proliferation rate was calculated using GraphPad.

[0042] EdU method to detect cell proliferation: Prostate cancer cells were cultured at a rate of 2×10 5 The cells were plated in 6-well plates with 100 cells / well. After culturing for 24 h, the cells were fixed and stained with the EdU-488 cell proliferation detection kit from Bio-Time. The cells were photographed with the Mica laser confocal system, and the proportion of EdU-positive cells was counted using GraphPad.

[0043] Flow cytometry detection of cell cycle: Prostate cancer cells were divided into 2×10 5 The cells were plated in 6-well plates with 100 cells / well. After culturing for 24 h, the cells were fixed and stained using the Lianke Biotechnology Cell Cycle Detection Kit, and the number and proportion of cells in different cell cycles were detected using MACSQuant.

[0044] Cell scratch healing experiment: Prostate cancer cells were pressed at 1×10 5Cells were plated per well in a 12-well plate. After culturing for 24 h, a pipette tip was used to make straight scratches of appropriate width on the bottom of the culture dish. The cells were washed with 1×PBS and then switched to RPMI 1640 medium without FBS to continue culturing the cells. Photos were taken at 0 h, 24 h, and 48 h, and the scratch width was calculated using Image Pro Plus software. GraphPad was used to calculate the cell migration rate.

[0045] Cell Transwell experiment: Matrigel diluted in RPMI 1640 medium at a ratio of 1:8 was added to the Transwell chamber and incubated in a cell culture incubator at 37°C and 5% CO2 for 3 h; 5×10 prostate cancer cells resuspended in RPMI 1640 medium without FBS were added to the upper chamber. 5 0.5 mL of RPMI 1640 medium containing 30% FBS was added to the lower chamber and cultured for 24-48 h. The chamber was removed and the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet stain. Photos were taken and the percentage of cells that successfully penetrated the membrane was calculated using GraphPad.

[0046] 2. Experimental results

[0047] First, in four common prostate cancer cell lines, PC3, DU145, LNCap and C4-2, and a prostate hyperplasia epithelial cell line, BPH 1, the present invention detected the expression levels of NUDT5 and SEPT8 by Western Blot. Figure 1 As shown in Figure 2, NUDT5 and SEPT8 have relatively high expression levels in almost all cell lines, so the subsequent validation experiments were performed on the PC3 cell line.

[0048] Using CRISPR-Cas9 technology, the present invention constructed stable cell lines of NUDT5 single knockout, SEPT8 single knockout and NUDT5+SEPT8 double knockout in PC3 cell line, such as Figure 2 The detailed process is as follows: the present invention designs a series of sgRNAs and screens out active sgRNAs through Surveyor Assay, such as Figure 2 A and 2B. Subsequently, two sgRNAs, SEPT8-g1 and NUDT5-g1, were selected to perform gene knockout in PC3 using the CRISPR-Cas9 system. The results are shown in Figure 2As shown in C, SEPT8 and NUDT5 genes were successfully knocked out in PC3 cell lines (using puromycin at a concentration of 1.0 μg / mL to screen stable cell lines). Then, based on these two stable cell lines PC3-SEPT8 KO and PC3-NUDT5 KO, NUDT5 and SEPT8 genes were knocked out again (using Blasticidin at a concentration of 10 μg / mL to screen stable cell lines). The results are shown in Figure 2 As shown in D, two SEPT8 and NUDT5 double knockout cell lines were successfully constructed in the PC3 cell line, namely, PC3-S+N KO and PC3-N+S KO stable cell lines.

[0049] Next, flow cytometry was used to detect the cell cycle changes of PC3 cell lines with SEPT8 and NUDT5 knockout, respectively. Figure 3 , knocking out SEPT8 or NUDT5 alone in PC3 cells had no effect on the cell cycle of PC3 cells.

[0050] The present invention further observed and recorded the cell proliferation process of different PC3 stable cell lines by using ZenCell OWL live cell imaging system, and drew the corresponding cell proliferation curves. Figure 4 A and 4B. Knockout of SEPT8 or NUDT5 alone did not affect the proliferation rate of the PC3 cell line. Therefore, the present invention used the ZenCell OWL live cell imaging system to observe and record the cell proliferation process of double knockout of SEPT8 and NUDT5 in the PC3 cell line, and drew the corresponding cell proliferation curve, as shown in FIG. Figure 5 A and 5B, it can be seen that the proliferation rate of the two SEPT8 and NUDT5 double knockout cell lines was significantly slowed down, suggesting that SEPT8 and NUDT5 of the present invention have a certain degree of synergistic effect, and together affect the cell proliferation process of PC3 cells.

[0051] In order to more accurately verify the effect of SEPT8 and NUDT5 on the proliferation rate of PC3 cells, the present invention used EdU staining to detect 4 different PC3 cell lines, namely: PC3-Scramble stable cell line of the control group, PC3-SEPT8 KO and PC3-NUDT5 KO single gene knockout stable cell lines, and PC3-SEPT8KO+NUDT5K double gene knockout stable cell lines. Fluorescence images of the 4 PC3 cell lines were taken by MICA confocal microscope, and the proportion of EdU-positive cells was calculated by Image J software to determine their proliferation rates, such as Figure 6As shown. Among them, the number of EdU-positive cells in the two PC3 stable cell lines with double knockout of SEPT8 and NUDT5 was significantly less than that in other groups, indicating that the simultaneous knockout of SEPT8 and NUDT5 in PC3 cells can inhibit the proliferation rate of PC3 cells. Consistent with the previous results, the knockout of SEPT8 or NUDT5 alone did not affect the proliferation rate of PC3 cells.

[0052] In order to study the effects of SEPT8 and NUDT5 on the migration ability of PC3 cell lines, the present invention detected the effects of knocking out SEPT8 or NUDT5 alone or knocking out both SEPT8 and NUDT5 on the migration ability of PC3 cell lines by cell scratch assay. Figure 7 The results showed that knocking out SEPT8 or NUDT5 alone had no significant effect on the migration ability of the PC3 cell line, while knocking out SEPT8 and NUDT5 simultaneously significantly slowed down the speed of scratch healing in the PC3 cell line, indicating that the simultaneous loss of SEPT8 and NUDT5 significantly affected the migration ability of cells, suggesting the possibility that SEPT8 and NUDT5 need to work together in the process of cell migration.

[0053] Referring to the above experimental results, the present invention directly uses the PC3 cell line in which SEPT8 and NUDT5 proteins are knocked out to perform Transwell experiments to detect their effects on the invasive ability of the PC3 cell line. Figure 8 After 24 hours of Transwell experiment, the number of cells stained with crystal violet was observed under a microscope. It can be seen that the number of cells in the PC3 stable cell line with simultaneous knockout of SEPT8 and NUDT5 that can successfully penetrate the membrane is much lower than that in the control group, indicating that simultaneous knockout of SEPT8 and NUDT5 in the PC3 cell line can significantly inhibit the cell invasion ability of the PC3 cell line.

[0054] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of agents that inhibit the expression of NUDT5 and SEPT8 in the preparation of drugs for the treatment of prostate cancer.

2. The use according to claim 1, characterized in that: The reagents for inhibiting the expression of NUDT5 and SEPT8 are sgRNA, siRNA, shRNA, miRNA or antisense nucleotides that specifically interfere with the expression and processing of NUDT5 and SEPT8 genes, or recombinant vectors containing siRNA, shRNA, miRNA or antisense nucleotides, or inhibitors that specifically inhibit the normal biological functions of NUDT5 and SEPT8 proteins.

3. The use according to claim 1, characterized in that: The reagent for inhibiting the expression of NUDT5 and SEPT8 is a reagent for inhibiting the expression of NUDT5 and SEPT8 by gene knockout technology, and the gene knockout technology is CRISPR-Cas9 technology.

4. The use according to claim 3, characterized in that: The nucleotide sequences of the sgRNA used in the reagents for inhibiting the expression of NUDT5 and SEPT8 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

5. Use of a recombinant vector in the preparation of a drug for treating prostate cancer, characterized in that: The recombinant vector contains sgRNA that specifically interferes with the expression of NUDT5 and SEPT8 genes.

6. The use according to claim 5, characterized in that: The nucleotide sequences of the sgRNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

7. A drug for treating prostate cancer, characterized in that: The drug has an agent that inhibits the expression of NUDT5 and SEPT8 as an active ingredient.

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