Application of RBM15 inhibitors in paclitaxel chemotherapy resistance in ovarian cancer

Abstract

The present invention relates to the application of an inhibitor of RBM15 in paclitaxel chemotherapy resistance in ovarian cancer. The present invention designs a small interfering siRNARBM15 targeting RBM15, and prepares a lentiviral packaging plasmid sh-RBM15. Through cell viability, cell apoptosis and cell stemness detection, it is found that targeted knockdown of RBM15 can reverse the resistance of paclitaxel-resistant ovarian cancer cells, proving that targeting RBM15 can be used as a therapeutic target for reversing tumor paclitaxel chemotherapy resistance, so as to develop a small molecule inhibitor targeting RBM15, thereby improving the therapeutic effect of ovarian cancer and the prognosis of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of an inhibitor of RBM15 in paclitaxel chemotherapy resistance in ovarian cancer. Background Art

[0002] Ovarian cancer is the malignant tumor with the lowest five-year survival rate among gynecological tumors. The most common pathological type of ovarian cancer is epithelial ovarian cancer. The current standard first-line treatment is tumor reduction surgery followed by platinum-based chemotherapy combined with paclitaxel (PTX). However, most patients develop drug resistance after chemotherapy, leading to a poor prognosis. In epigenetics, m6A modification (i.e., N6-methyladenosine (m6A)) refers to a specific methylation modification of the sixth nitrogen atom (N) on adenine (A) catalyzed by a methyltransferase. It is one of the most common mRNA modifications and affects the transcription, stability, and translation of mRNA. RBM15 (RNAbinding motifprotein 15) is one of the components of the m6A methyltransferase complex.

[0003] Due to the inherent high recurrence rate and resistance to chemotherapy drugs of ovarian cancer, the patient's prognosis is poor, which greatly limits the clinical efficacy and application scope of chemotherapy, which is also the main reason for the failure of ovarian cancer treatment. The m6A methylation modification plays an important role in tumor chemotherapy resistance. Therefore, more and more studies are aimed at developing small molecule inhibitors of m6A methyltransferase for the treatment of recurrent tumors, but the current research is still in the animal experiment stage. The existing research mainly involves RBM15 in the occurrence and development of tumors, and its relationship with tumor resistance has not been reported. Through previous studies, we first discovered that RBM15 is closely related to PTX chemotherapy resistance in ovarian cancer. Targeted knockdown of RBM15 protein by RBM15 inhibitor has the potential application value of reversing PTX chemotherapy resistance in ovarian cancer. However, there is no report on the mechanism of action of targeted knockdown of RBM15 in reversing PTX chemotherapy resistance in tumors as in the present invention. Summary of the invention

[0004] The first objective of the present invention is to provide an application of an inhibitor of RBM15 in the preparation of a drug for treating paclitaxel-resistant ovarian cancer in view of the deficiencies in the prior art.

[0005] The second object of the present invention is to provide a pharmaceutical composition.

[0006] The third object of the present invention is to provide another application of RBM15 inhibitors

[0007] To achieve the above first purpose, the technical solution adopted by the present invention is:

[0008] Application of RBM15 or its gene inhibitor in the preparation of medicine for treating paclitaxel-resistant ovarian cancer.

[0009] As a preferred example, the BM15 inhibitor is antisense nucleic acid, siRNA, miRNA or shRNA.

[0010] More preferably, the sequence of the siRNA is as shown in SEQ ID NO:1.

[0011] More preferably, the shRNA is constructed from siRNA targeting RBM15, and the sequence of the siRNA is shown in SEQ ID NO:1.

[0012] More preferably, the inhibitor can inhibit the spheroidization ability of drug-resistant cells and promote their sensitivity to paclitaxel.

[0013] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0014] A pharmaceutical composition for treating paclitaxel-resistant ovarian cancer, characterized in that the pharmaceutical composition contains:

[0015] a) an inhibitor of RBM15 or its gene, and

[0016] b) a pharmaceutically acceptable carrier;

[0017] The sequence of RBM15 is shown in SEQ ID NO:2 and SEQ ID NO:3.

[0018] The present invention has the advantages that:

[0019] 1) The entry point is close to the clinic. The purpose of the present invention is clear, which is to solve the practical problems that plague the treatment of epithelial ovarian cancer in the clinic. Starting from the shortcomings of current treatment methods, a new potential treatment method has been invented. If it can be put into clinical practice one day, it can improve the current situation in a targeted manner, thereby benefiting a large number of patients.

[0020] 2) The guiding concept is novel. The present invention combines the latest and most advanced basic research results at home and abroad, puts forward a creative hypothesis, and proves the practical feasibility of the hypothesis based on sufficient experiments.

[0021] 3) It has great economic potential. The present invention focuses on the actual clinical problem with broad market demand. Currently, there are many patients with epithelial ovarian cancer, and the treatment method needs to be improved urgently. Therefore, once the present invention is successfully transformed, it will obtain good market benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Attached Figure 1The expression levels of RBM15 in sensitive and resistant cells. (A) Comparison of the mRNA expression of RBM15 in SK3R-PTX and A2780-PTX cells and PTX-sensitive ovarian cancer cells; (B) Comparison of the protein expression of RBM15 in SK3R-PTX and A2780-PTX cells and PTX-sensitive ovarian cancer cells.

[0023] Attached Figure 2 The expression level of RBM15 in PTX-resistant cell lines after transfection with si-RBM15. (A) The knockdown effect of RBM15 mRNA was detected after transfection with si-NC and si-RBM15 in A2780-PTX cells for 24 hours; (B) The knockdown effect of RBM15 protein was detected after transfection with si-NC and si-RBM15 in A2780-PTX cells for 48 hours.

[0024] Attached Figure 3 is the protein expression level of RBM15 in the PTX-resistant cell line after transfection with lentivirus sh-RBM15.

[0025] Attached Figure 4 To detect the IC50 value of cells to PTX after knocking down RBM15 in drug-resistant cells SK3R-PTX and A2780-PTX, as well as the results of the PTX action time gradient. (A, B and D, E) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15. After adding different concentrations of PTX for 48 hours, the OD values ​​of each group of cells were detected by CCK8 method, and the curves were drawn, the IC50 values ​​were calculated, and statistical analysis was performed. (C and F) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15, and then treated with 1μM and 2μM PTX. CCK8 reagent was added at three time points of 24 hours, 48 ​​hours, and 72 hours to measure the OD values ​​and draw the curves.

[0026] Attached Figure 5 The drug-resistant cells were transfected with si-RBM15 and the apoptosis was detected by flow cytometry after drug addition. (A) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15, and then the apoptosis of each group of cells was detected after 24 hours of treatment with or without 1μM and 2μM PTX; (BC) The apoptosis rate (early + late) of each group in A was statistically analyzed.

[0027] Attached Figure 6Knockdown of RBM15 in drug-resistant cells down-regulates cell stemness. (A) A2780-PTX / SK3R-PTX-shNC / sh-RBM15 cells were cultured into stem cell spheres; cells were cultured in serum-free DMEM / F12 medium supplemented with EGF, bFGF, heparin, and B27 for 11 days, and photos were taken every two days; (BC) The average diameter of each sphere in Figure A (n = 3 random spheres) was measured and calculated, and the growth curve was drawn. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0029] It should be noted that, due to formatting issues, the siRNA involved in the examples in the sequence listing needs to replace U with T in SEQ ID NO: 1. The original sequence is: GAGACUACCCGUUCUAUGA. See the sequence listing for the replaced sequence.

[0030] Example 1

[0031] 1. Experimental methods

[0032] 1.1 siRNA and shRNA design and synthesis

[0033] (1) The mRNA sequence of RBM15 protein was downloaded from the GENBNAK website (https: / / www.ncbi.nlm.nih.gov / nucleotide / ), with the sequence number NM_022768.5. siRNA targeting RBM15 was designed by ourselves, and the sequence is as follows:

[0034]

[0035] # refers to the targeting sequence in NM_022768.5

[0036] (2) siRNA was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. and prepared into a working concentration of 20 uM using sterile ultrapure water free of DNA and RNase.

[0037] (3) The siRNA sequence was inserted into pLKO.1puro by gene cloning to construct an shRNA targeting RBM15, named sh-RBM15. At the same time, Shanghai Wenbei Biotechnology Co., Ltd. was commissioned to package it into lentivirus and control virus for use (virus titer 10^8 virus / ml).

[0038] 1.2 Cell culture.

[0039] Ovarian cancer cells A2780 (Keygen Biotech, Nanjing, Jiangsu, China) and PTX-resistant A2780-PTX (Keygen Biotech) were cultured in DMEM medium (Gibco, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum. SKOV-3 and PTX-resistant cell lines SK3R-PTX were cultured in McCoy's 5A medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum. Paclitaxel-resistant cells SK3R-PTX (derived from PTX-sensitive ovarian cancer cells SK-OV-3, catalog number HTB-77, ATCC) were constructed by our research group. All cells used without special emphasis were cultured in culture medium supplemented with 10% fetal bovine serum in culture plates or culture flasks.

[0040] 1.3siRNA or plasmid transfection experiment.

[0041] The cells to be transfected were plated in a 6-well plate. After culturing for 24 hours, the transfection reagent X-tremeGENE siRNA Transfection Reagent (Cat. No. 4476093001, Sigma) and 5 μL of the corresponding siRNA were added. After standing in the incubator for 6-8 hours, the old culture medium was removed, and new culture medium was added to culture for another 48 hours before conducting subsequent experiments.

[0042] 1.4 Lentivirus infection

[0043] (1) One day before infection, A2780-PTX and SK3R-PTX cells were transferred into T25 flasks at a density of 8×10^5 cells.

[0044] (2) After the cells have grown to 50%, 10 μL of the control virus and 30 μL of the sh-RBM15 lentivirus are added to 2 mL of freshly replaced complete medium. After culturing in a cell culture incubator for 6 hours, the complete medium is replenished to 4 ml.

[0045] (3) After the cells were cultured for 48 hours, the culture medium was replaced with complete medium containing 40 ng / mL (for A2780-PTX cells) or 10 ng / mL (for SK3R-PTX cells) puromycin and the cells were cultured for another 48 hours.

[0046] (4) According to the cell culture method of A2780-PTX and SK3R-PTX, the medium was changed and the cells were subcultured. The culture medium was complete medium containing corresponding concentrations of puromycin.

[0047] (5) After the cells can stably grow in complete medium containing puromycin for more than two generations, they are expanded and cryopreserved for future use.

[0048] 1.5 Western blot

[0049] Prepare the cell protein sample that has been extracted with SDS in advance, perform electrophoresis on a 10% SDS-PAGE gel, transfer to a PVDF membrane, and incubate with the corresponding protein antibody at 4°C overnight. The next day, incubate with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour and expose to light for development.

[0050] 1.6qRT-PCR (real-time quantitative PCR)

[0051] First, extract the total RNA of the cells to be treated, then reverse transcribe it into cDNA, and finally design corresponding primers according to different genes for qRT-PCR amplification reaction, and finally perform statistical analysis of the CT value results. The primer sequences are as follows:

[0052]

[0053] 1.7 Knockdown of RBM15 to verify the half maximal inhibitory concentration (IC50) of paclitaxel chemotherapy drugs in cells (concentration gradient)

[0054] (1) One day before drug addition, A2780-PTX and SK3R-PTX cells transfected with si-NC and si-RBM15 were digested and counted, and plated into 96-well plates at a density of 7,000 to 10,000 cells per well.

[0055] (2) On the second day, a gradient dilution method was used to prepare a drug concentration gradient of PTX using a culture medium containing 10% FBS. Three cell replicates were set up for each drug concentration. The PTX concentrations used were: A2780-PTX: 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0.0001 μM, 0 μM; SK3R-PTX: 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0 μM.

[0056] (3) The culture medium in the 96-well plate was discarded, and the culture medium containing PTX drug was added to each well and the culture was continued.

[0057] (4) After 48 h of PTX treatment, 100 μL of culture medium containing CCK8 solution was added to each well. After 2 h of reaction, the absorbance OD value was measured at a wavelength of 450 nm.

[0058] (5) Based on the OD values ​​of different concentrations, draw the PTX drug concentration inhibition curve and calculate the IC50 value of each experimental group.

[0059] 1.8 Determination of the time effect of half maximal inhibitory concentration (IC50) of paclitaxel chemotherapy drug by knocking down RBM15

[0060] Time gradient:

[0061] (1) PTX-resistant cells were transfected with si-NC and si-RBM15, and the next step was performed 24 h after transfection.

[0062] (2) Rinse with conventional PBS, digest and centrifuge the cells to be treated, and then resuspend the cell pellet and count the cells. 7000-10000 cells per well, inoculate the cells into a 96-well plate while blowing the cell suspension, observe under a microscope and place in a cell culture incubator for growth.

[0063] (3) Complete culture medium containing 2 μM PTX (for A2780-PTX cells) and 1 μM PTX (for SK3R-PTX cells) was added every other day. The old culture medium was removed at 24, 48, and 72 hours, and 100 μL of the prepared culture medium containing CCK8 was added. After incubation in an incubator for 2 hours, the absorbance was measured with a multifunctional microplate reader.

[0064] (4) Calculate the cell viability ratio (%) at each time point = [PTX intervention group - blank group] / [0 μM PTX group - blank group] × 100, where the blank group is the absorbance value of the well without cells.

[0065] 1.9 Knockdown of RBM15 or addition of PTX drug treatment, flow cytometry detection of cell apoptosis

[0066] (1) A2780-PTX or SK3R-PTX cells were plated in 6-well plates according to experimental requirements, and si-NC and si-RBM15 were transfected when the cell confluence reached about 80%, and the cells were then cultured.

[0067] (2) After transfection for more than 24 hours, a culture medium containing 10% FBS with corresponding PTX concentration was prepared. Four groups were set up, namely A2780-PTX / SK3R-PTX+si-NC group, A2780-PTX / SK3R-PTX+si-RBM15 group, A2780-PTX / SK3R-PTX+si-NC+PTX group, and A2780-PTX / SK3R-PTX+si-RBM15+PTX group. The first two groups replaced the culture medium containing 10% FBS (without PTX) in the corresponding cell wells, and the latter two groups replaced the culture medium containing 10% FBS with 2μM (A2780-PTX cells) and 1μM PTX (SK3R-PTX cells) in the corresponding cell wells.

[0068] (3) After culturing for 24 hours, the cells were digested with EDTA-free trypsin, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0069] (4) Add 2 mL of pre-cooled PBS and wash twice, centrifuging at 1000 rpm for 5 minutes each time, and discard the supernatant.

[0070] (5) Resuspend the cells in 100 μL of binding buffer from FITC Annexin V Poptosis Detection Kit I (Cat. No. 556547, BD Biosciences), add 1 μL of FITC-labeled Annexin V and 2 μL of PI dye, and stain for 15 min in the dark.

[0071] (6) Add 400 μL of binding buffer to each tube, perform detection on a flow cytometer, and analyze the proportion of apoptotic cells using software.

[0072] 1.10 Tumor stem cell sphere formation experiment:

[0073] (1) Preparation of medium for tumor stem cell sphere culture: Add appropriate amount of heparin (4 μg / mL), basic fibroblast growth factor (20 ng / mL), epidermal growth factor (20 ng / mL) and B27 (0.4 μg / mL) to DMEM / F12 medium and mix well for later use;

[0074] (2) Stably transfected cells A2780-PTX-sh-NC / sh-RBM15 and A2780-PTX-sh-NC / sh-RBM15 with good growth status were taken, digested and centrifuged, resuspended in the prepared DMEM / F12 medium, and the cells were counted under a microscope;

[0075] (3) Prepare the culture medium with the required number of cells according to the counting results, about 1000 cells per well of an ultra-low adhesion 6-well plate, add and mix, and then culture in an incubator. Then, add 500 μL of culture medium to each well every 3 days;

[0076] (4) Starting from the second day after plating, the cell growth was observed under a microscope and photographed and measured every other day until the 11th day. The tumor spheres were measured by measuring two mutually perpendicular diameters (long diameter d1 and short diameter d2), and then the average radius of the tumor spheres was calculated according to the formula r = 1 / 2 × √ (d1 × d2), and finally the growth curve of the tumor spheres was drawn.

[0077] 2 Experimental results

[0078] 2.1 Expression level of RBM15 in sensitive and resistant cells.

[0079] The mRNA and protein expression levels of RBM15 were increased in PTX-resistant cells. Figure 1 , among which the mRNA expression level of RBM15 in SK3R-PTX and A2780-PTX cells was higher than that in PTX-sensitive ovarian cancer cells (n=3, **: p value < 0.01; ***: p value < 0.001); the protein expression level of RBM15 in SK3R-PTX and A2780-PTX cells was higher than that in PTX-sensitive ovarian cancer cells (n=3, *: p value < 0.05).

[0080] 2.2 Detection of RBM15 expression in PTX-resistant cell lines after transfection of si-RBM15 by qPCR

[0081] A2780-PTX-resistant cells were transfected with si-RBM15, and the knockdown efficiency of the designed si-RNA was verified at the mRNA and protein levels. The results are shown in the attached Figure 2 , where (A) A2780-PTX cells were transfected with si-NC and si-RBM15 to detect the knockdown effect of RBM15 mRNA at 24 hours (n=3, ***: p value <0.001); (B) A2780-PTX cells were transfected with si-NC and si-RBM15 to detect the knockdown effect of RBM15 protein at 48 hours, indicating that si-RBM15 small interfering RNA successfully targeted and knocked down RBM15 mRNA, and then RBM15 protein expression was also reduced.

[0082] 2.3 Protein expression of RBM15 in PTX-resistant cell lines transfected with lentivirus sh-RBM15

[0083] See attached for the results Figure 3After SK3R-PTX and A2780-PTX cells were infected with lentivirus sh-NC and sh-RBM15, the protein expression of RBM15 was significantly reduced, verifying the knockdown effect of lentivirus transfection.

[0084] 2.4 After knocking down RBM15 in drug-resistant cells SK3R-PTX and A2780-PTX, the IC50 values ​​of cells to PTX and the results of the PTX action time gradient were detected

[0085] Knockdown of RBM15 in A2780-PTX and SK3R-PTX paclitaxel-resistant cells can downregulate the expression of MDR1, thereby increasing the sensitivity of resistant cells to PTX. The results are shown in the attached Figure 4 , (A, B and D, E) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15, and after adding different concentrations of PTX for 48 hours, the OD values ​​of each group of cells were detected by CCK8 method, and then the curves were drawn, the IC50 values ​​were calculated, and statistical analysis was performed. That is: after knocking down RBM15, the IC50 value of PTX-resistant cells decreased, and the sensitivity to PTX increased (n=3, **: p value <0.01; ***: p value <0.001); (C and F) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15, and then 1μM and 2μM PTX were added. After adding CCK8 reagent at three time points of 24 hours, 48 ​​hours and 72 hours, the OD values ​​were measured and the curves were drawn. That is: the time gradient also proved that after knocking down RBM15, the sensitivity of PTX-resistant cells to PTX increased with the increase of time (n=3, ****: p value <0.0001).

[0086] 2.5 The drug-resistant cells were transfected with si-RBM15 and the cell apoptosis was detected by flow cytometry after drug addition.

[0087] See attached for the results Figure 5 , (A) SK3R-PTX and A2780-PTX cells were transfected with si-RBM15, and then treated with 1μM and 2μM PTX for 24 hours, and the apoptosis of each group of cells was detected; (BC) The apoptosis rate (early + late) of each group of cells in A was statistically analyzed (n=3, *: p value <0.05; ****: p value <0.0001). That is, after knocking down RBM15, the apoptosis-promoting effect of PTX on drug-resistant cells increased, the apoptosis rate increased, and the killing effect of PTX on drug-resistant cells was enhanced.

[0088] 2.6 Knockdown of RBM15 in drug-resistant cells downregulates cell stemness

[0089] See attached for the results Figure 6, where (A) A2780-PTX / SK3R-PTX-shNC / sh-RBM15 cells were cultured into stem cell spheres; cells were cultured in serum-free DMEM / F12 medium supplemented with EGF, bFGF, heparin and B27 for 11 days, and photos were taken every two days; image magnification ×100, scale bar 100μm; (BC) The average diameter of each sphere in Figure A (n=3 random spheres) was measured and calculated, and the growth curve was drawn (*p﹤0.05, ****p﹤0.0001), indicating that knocking down RBM15 in paclitaxel-resistant cells can downregulate the cell sphere-forming ability, that is, reduce the cell stemness.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. The use of an inhibitor of RBM15 in the preparation of a drug for treating paclitaxel-resistant ovarian cancer, characterized in that: The ovarian cancer is epithelial ovarian cancer, the RBM15 inhibitor is siRNA or shRNA, the sequence of the siRNA is shown in SEQ ID NO: 1, and the shRNA is constructed by siRNA targeting RBM15, and the sequence of the siRNA is shown in SEQ ID NO: 1.

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