A novel endocrine therapy resistance marker for advanced prostate cancer and its application

Abstract

The present invention relates to the field of medicine, and discloses a novel endocrine therapy resistance marker for advanced prostate cancer and its application, wherein the marker is DENND3. The present invention first discovered that the expression level of the DENND3 gene in prostate cancer abiraterone-resistant cells is significantly higher than that in parental prostate cancer cells. Therefore, the determination of the expression level of the DENND3 gene can be used as a basis for judging the drug resistance / sensitivity of the user to abiraterone; the DENND3 gene-specific siRNAs provided by the present invention can efficiently inhibit or knock down the expression of the DENND3 gene in target cells, thereby promoting cell apoptosis, inhibiting the proliferation of abiraterone-resistant prostate cancer cells, and further inhibiting the growth of prostate cancer cells. Therefore, it can be used to prepare a drug that improves the resistance of prostate cancer to abiraterone, and has great clinical and economic value in the treatment of prostate cancer.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a novel endocrine therapy resistance marker for advanced prostate cancer and application thereof. Background Art

[0002] Prostate cancer (PCa) is the second most common male malignant tumor in the world. In my country, most patients with prostate cancer are in the locally advanced stage or with extensive metastasis. Patients with extensively metastatic prostate cancer cannot receive local radical treatment and can only receive endocrine therapy or chemotherapy.

[0003] The survival and evolution of PCa cells depend on androgens, and androgen deprivation therapy (ADT) is an important means of treating PCa. ADT is effective in the early stage of treating PCa and can significantly reduce the level of prostate-specific antigen in patients, but after about 18-24 months, PCa cells gradually become resistant to ADT, and the disease enters the castration-resistant prostate cancer (CRPC) stage. CRPC is characterized by the activation or overexpression of androgen receptor (AR) under low testosterone levels, so that AR downstream target genes can still be transcribed and translated at low testosterone levels, resulting in PCa cells tolerating ADT treatment.

[0004] After PCa enters the CRPC stage, the continuous activation of the AR signaling axis is still an important pathway for tumor survival and progression. Targeting the AR signaling axis is an important direction for the treatment of CRPC. The new endocrine therapeutic drugs currently used in clinical practice are mainly abiraterone and enzalutamide. Abiraterone can effectively inhibit the production of androgens, while enzalutamide is a potent AR antagonist. Both can inhibit the activity of the AR pathway, thereby playing a role in the treatment of CRPC.

[0005] Although new endocrine therapy can effectively prolong the survival of patients, studies have found that 1 / 3 and 1 / 4 of CRPC patients treated with abiraterone and enzalutamide, respectively, had radiological progression of lesions 3 months after treatment, and all patients had disease progression and drug resistance after 24 months of treatment. The emergence of drug resistance has seriously affected the efficacy of new endocrine therapy drugs and is an important cause of disease progression and even death in CRPC patients.

[0006] Therefore, studying new endocrine therapy resistance markers is of great significance for guiding the subsequent treatment of CRPC.

[0007] Prior art CN 117344019 discloses that the expression of ACHIP is significantly upregulated in docetaxel-resistant prostate cancer cells, and CHIP can enhance the resistance of prostate cancer cells to docetaxel, while knocking down CHIP can reverse the resistance of docetaxel-resistant prostate cancer cells, indicating that measuring the expression level of CHIP gene or CHIP protein can be used as a basis for judging the drug resistance / sensitivity of the user to docetaxel.

[0008] CN 114164270 B discloses a molecular marker that can be used to detect the resistance of prostate cancer to docetaxel, wherein the molecular marker is CRIP2 gene or CRIP2 protein; the expression level of CRIP2 gene or CRIP2 protein in DTX-resistant prostate cancer cells is significantly higher than that in normal prostate cancer cells, and the difference is statistically significant. Therefore, the determination of the expression level of CRIP2 gene or CRIP2 protein can be used as one of the bases for judging the resistance / sensitivity of the user to DTX.

[0009] However, the prior art only involves two markers, whose sensitivity and specificity need to be improved, and neither involves resistance to new endocrine therapy for advanced prostate cancer. Based on this, this application will explore new markers for resistance to new endocrine therapy for advanced prostate cancer to fill the research gap. Summary of the invention

[0010] In order to solve the above problems, the present invention first provides a novel endocrine therapy resistance marker for advanced prostate cancer, wherein the marker is DENND3.

[0011] The present invention also provides the use of the above marker in preparing a novel endocrine therapy resistance reagent for detecting advanced prostate cancer, preparing a drug for treating prostate cancer, or preparing a drug for inhibiting the proliferation of abiraterone-resistant prostate cancer cells.

[0012] The present invention also provides a primer pair for detecting the marker, and the sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0013] The present invention also provides a kit for detecting resistance to a novel endocrine therapy for advanced prostate cancer, the kit comprising the primer pair according to claim 3.

[0014] The present invention also provides an application of the above primer or the above kit in detecting drug resistance of a new endocrine therapy for advanced prostate cancer, wherein the application is for non-diagnostic purposes.

[0015] The present invention also provides a method for detecting drug resistance to a new endocrine therapy for advanced prostate cancer, which comprises the step of using the above-mentioned primers or the above-mentioned kit to detect the DENND3 expression level of the sample to be tested. When the DENND3 expression level increases, it indicates that the sample to be tested has developed drug resistance.

[0016] The present invention also provides a pharmaceutical composition for treating prostate cancer, the pharmaceutical composition comprising siRNA-1 as described in SEQ ID NO.5 and SEQ ID NO.6, and / or siRNA-2 as described in SEQ ID NO.7 and SEQ ID NO.8.

[0017] In certain embodiments, the pharmaceutical composition further comprises abiraterone.

[0018] The present invention also provides an application of the above-mentioned pharmaceutical composition, which is used for treating prostate cancer or inhibiting the proliferation of prostate cancer abiraterone-resistant cells.

[0019] Finally, the present invention provides a method for treating prostate cancer, which comprises the step of administering an appropriate amount of the above-mentioned pharmaceutical composition.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] (1) The present invention first discovered that the expression level of the DENND3 gene in prostate cancer cells resistant to abiraterone is significantly higher than that in parental prostate cancer cells. Therefore, the determination of the expression level of the DENND3 gene can be used as a basis for judging the resistance / sensitivity of the user to abiraterone.

[0022] (2) The DENND3 gene-specific siRNAs provided by the present invention can effectively inhibit or knock down the expression of the DENND3 gene in target cells, thereby promoting cell apoptosis, inhibiting the proliferation of abiraterone-resistant prostate cancer cells, and further inhibiting the growth of prostate cancer cells. Therefore, it can be used to prepare drugs for improving the resistance of prostate cancer to abiraterone, and has great clinical and economic value in the treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the results of drug resistance testing of prostate cancer cell lines and drug-resistant cells.

[0024] Figure 2 Expression of DENND3 in prostate cancer cells and drug-resistant cells.

[0025] Figure 3 Effect of siRNA on the expression of DENND3 in drug-resistant prostate cancer cells.

[0026] Figure 4The results of siRNA affecting the drug resistance of prostate cancer resistant cells.

[0027] Figure 5 Effect of siRNA on the proliferation of drug-resistant prostate cancer cells. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0029] Example 1 Construction of abiraterone-resistant prostate cancer cell model

[0030] Cell source: Prostate cancer C4-2 cell line, purchased from the American Type Culture Collection (ATCC). The induction method of abiraterone-resistant prostate cancer cells (C4-2-AbiR) can be found in "Lu Dingheng, Study on the effect and mechanism of KAT2A-mediated androgen receptor nuclear translocation on abiraterone resistance in castration-resistant prostate cancer cells". The specific induction steps are as follows:

[0031] (1) C4-2 cells were cultured in RPMI-1640 complete medium at 37°C and 5% CO2 for 3-4 generations according to the following steps and then frozen for subsequent experiments.

[0032] (2) Cell passaging: When the confluence of C4-2 cells reaches about 90%, discard the culture medium and gently rinse twice with PBS. After discarding the PBS, add 1 ml of 0.25% trypsin solution and gently shake until the trypsin is evenly distributed and fully in contact with the cells. Observe the C4-2 cells at any time during digestion. When the cell morphology changes and is about to fall off, add complete culture medium to stop digestion. Fully blow the suspension to mix and separate the cells, transfer the suspension into a centrifuge tube, and centrifuge for 5 minutes (4°C, 1000rpm). Carefully remove the upper layer of liquid, add complete culture medium and blow to resuspend. Add the cell suspension to a new culture bottle, shake evenly and return it to the cell culture incubator.

[0033] (3) Cryopreservation of cells: Immediately after centrifugation and discarding the upper liquid in the previous step, add 1 ml of the prepared cell freezing solution, blow the cells thoroughly to mix them, and then transfer them into the cryopreservation tube. Cryopreservation is performed according to the gradient freezing method: room temperature → 4°C, 30 minutes → -20°C, 30 minutes → -80°C, 48 hours.

[0034] (4) Cell recovery: Cell recovery should follow the principle of "slow freezing and fast thawing". After taking out the cryotube from the -80℃ refrigerator, quickly place it in a constant temperature water bath at 37℃ and shake it quickly and gently to dissolve the liquid. After dissolving, transfer the cell suspension into a centrifuge tube containing 10ml complete culture medium and centrifuge for 5 minutes (4℃, 1000rpm). Discard the supernatant, add new culture medium, and then transfer it to a new culture bottle or well plate according to the experimental needs, place it in a cell culture incubator for culture, change the liquid every 2~3 days, and observe the cell growth at any time.

[0035] (5) Construction of abiraterone-resistant cells: C4-2 cells were induced using a concentration gradient method. The culture medium used phenol red-free RMPI 1640 medium, and the serum used fetal bovine serum adsorbed with activated carbon. The initial concentration of abiraterone acetate solution was 0.5 μM. At this concentration, the cells were stably passaged three times after the confluence reached 90%. The drug concentration was then gradually increased in the following order: 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 14 μM, 16 μM, 20 μM, 22 μM and 25 μM.

[0036] When C4-2 cells can be stably grown and subcultured in a medium with high concentrations of abiraterone acetate, it indicates that C4-2-AbiR is established. After two weeks of normal subculture in a drug-free medium, the cells can be used for subsequent experiments.

[0037] (5) Cell resistance (IC50) analysis method: C4-2 cell suspension in the logarithmic growth phase was inoculated into a 96-well plate, with approximately 5,000 cells per well. After the cells adhered to the wall and restored their morphology, the supernatant was discarded, and the culture medium without abiraterone and containing different concentration gradients of abiraterone were added to the 96-well plate. Each group had 5 replicate wells, with 100 μl per well. After sufficient action, the liquid was discarded, and CCK-8 reagent was added, 100 μl per well, and then placed in a constant temperature incubator for incubation for approximately 2 hours. The 96-well plate was placed in a 450nm absorbance (optical density, OD) microplate reader for measurement, and the OD value of each well was recorded. The IC50 (50% inhibiting concentration, IC50) of abiraterone can be calculated based on the OD value. After obtaining the IC50 of C4-2-P and C4-2-AbiR, the resistance index (RI) can be calculated.

[0038] The C4-2-AbiR abiraterone-resistant cell line induced by the concentration increasing method was used to draw a dose-effect curve using the CCK-8 detection method. The IC50= 25.01μM and the IC50 of the C4-2-P parent cell line= 3.65μM were obtained using SPSS software (as shown in Figure 1). According to the "resistance index RI= IC50 resistance / IC50 parent", RI= 6.85 was obtained. The results show that C4-2-AbiR cells are more sensitive to abiraterone, indicating that the abiraterone-resistant cell model of prostate cancer was successfully constructed.

[0039] Example 2 qRT-PCR detection of DENND3 gene expression in cells

[0040] 1. RNA was extracted using an RNA extraction kit (QIAGEN).

[0041] 2. Reverse transcription:

[0042] 1 μg of total RNA template was mixed with 2 μL 10× buffer, 2 μL dATP (10 mM), 0.5 μL polyA polymerase, 0.5 μL RNase inhibitor and RNase free water to a final volume of 20 μL, and incubated at 37°C for 1 h; then 1 μL 0.5 μg / μL Oligo (dT) specific RT primer was added to the reaction tube, incubated at 70°C for 5 min, and immediately incubated on ice for at least 2 min, and then the reaction mixture was mixed with 4 μL 5× buffer, 1 μL dNTP (10 mM), 0.5 μL M-MLV reverse transcriptase, 0.5 μL RNase inhibitor, 10 μL polyA reaction mixture and 4 μL RNase free water, and incubated at 42°C for 1 h.

[0043] 3. QPCR amplification primers were designed based on the coding sequences of DENND3 gene and GAPDH gene in Genebank and synthesized by Wuhan Qingke Biological Company. The specific primer sequences are as follows:

[0044] 1) Primer design:

[0045] The primer sequences for the DENND3 gene are:

[0046] Forward primer: 5'-GTCTCCGAAGTCTCGAGCAG-3' (SEQ ID NO. 1);

[0047] Reverse primer: 5'-GACCTCTGGATCAAGAAGAGCA-3' (SEQ ID NO. 2);

[0048] Primer sequences for GAPDH:

[0049] Forward primer: 5'-GCTCCCTCTTTCTTTGCAGC-3' (SEQ ID NO. 3);

[0050] Reverse primer: 5'-GTTGTCATGGATGACCTTGGC-3' (SEQ ID NO. 4);

[0051] 2) Reaction system

[0052] SYBR Green polymerase chain reaction system 12.5 μL, forward and reverse primers (5 μM) 1 μL each, template cDNA 2.0 μL, ddH2O 8.5 μL.

[0053] 3) Reaction conditions

[0054] 95℃10min, (95℃15s, 60℃60s)×45 cycles. SYBR Green was used as a fluorescent marker and PCR reaction was performed on a Light Cycler fluorescent quantitative PCR instrument. ΔΔ The relative quantification was performed by CT method.

[0055] 4. Results

[0056] The results are as follows Figure 2 As shown, the expression level of DENND3 gene was upregulated in prostate cancer resistant cells.

[0057] Example 3 Silencing of DENND3 gene

[0058] 1. siRNA design

[0059] siRNA was designed for the sequence of DENND3 gene. The designed siRNA sequence is as follows:

[0060] SiRNA-1:

[0061] Sense strand: 5'-GGCUUUGUGUAGCUACUAACU-3' (SEQ ID NO. 5),

[0062] Antisense strand: 5'-UUAGUAGCUACACAAAGCCAA-3' (SEQ ID NO. 6);

[0063] SiRNA-2:

[0064] Sense strand: 5'-GGAUGUAAAGAAUCAUUUAAA-3' (SEQ ID NO. 7),

[0065] Antisense strand: 5'-UAAAUGAUUCUUUACAUCCCU-3' (SEQ ID NO. 8);

[0066] Cells in the logarithmic growth phase were digested and inoculated into 6-well plates at a rate of 200,000 per well. After spreading evenly, cells were incubated overnight and transfected when the cell confluence reached 70% to 80%.

[0067] The experiment was divided into a blank control group, a negative control group (siRNA-NC) and an experimental group (20nM) (SiRNA-1, SiRNA-2). The siRNA in the negative control group had no homology with the sequence of the DENND3 gene, and the concentration was 20nM / well. Transfection was performed separately at the same time. The volume was filled to 2mL with complete culture medium; 4) After 24h, fresh culture medium was replaced and routine culture was reserved for subsequent experiments.

[0068] 1. QPCR detected the expression level of DENND3 gene. The results are as follows Figure 3 It showed that the experimental group was able to reduce the expression level of DENND3.

[0069] Example 4 CHIP enhances the resistance of prostate cancer cells to abiraterone

[0070] (1) Experimental methods:

[0071] The transfection method of small interfering RNA is the same as that in Example 3, and the detection method of IC50 is the same as that in Example 1.

[0072] (2) Experimental results:

[0073] Figure 4 The results showed that compared with the control siRNA-NC group, the cells in the DENND3 knockdown group (SiRNA-1, SiRNA-2) had lower IC50 values ​​for abiraterone, suggesting that targeted inhibition of DENND3 expression can significantly improve the drug resistance of prostate cancer abiraterone-tolerant cells, making abiraterone have stronger tumor killing ability.

[0074] Example 5 Cell proliferation experiment

[0075] 1. Cell culture and transfection were the same as in Example 3. The experiment was divided into three groups: siRNA-NC, siRNA-1, and siRNA-2.

[0076] 2. Cell proliferation assay, the following experimental steps were repeated three times:

[0077] 1) With a cell density of 5x10 3 / well, inoculated in 96-well plates, 100 μL per well, and 3 replicate wells were set up for each group.

[0078] 2) After the cells are completely attached, transfection is performed according to the above steps. CCK8 detection is performed at 0h, 24h, 48h, 72h, and 96h after transfection. The CCK8 solution is replaced, incubated at 37°C for 2h in the dark, and the absorbance at a wavelength of 450nm is measured using an enzyme marker.

[0079] Results Figure 5 , indicating that inhibiting DENND3 expression can significantly inhibit the proliferation ability of prostate cancer abiraterone-resistant cells.

[0080] The above results indicate that DENND3 can be used as a new endocrine therapy resistance marker for advanced prostate cancer.

[0081] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting DENND3 in the preparation of a kit for detecting abiraterone resistance in advanced prostate cancer.

2. A pharmaceutical composition for treating abiraterone-resistant prostate cancer, characterized in that: The pharmaceutical composition comprises siRNA-1 as shown in SEQ ID NO.5 and SEQ ID NO.6, and / or siRNA-2 as shown in SEQ ID NO.7 and SEQ ID NO.

8.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also includes abiraterone.

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