Application of reagents for knocking down circAKT3 in the preparation of drugs for treating prostate cancer

Abstract

The present invention discloses the use of a reagent for knocking down circAKT3 in the preparation of a drug for treating prostate cancer, and the sequence of the circAKT3 is shown in SEQ ID NO. 1. The present invention proves for the first time that knocking down circAKT3 can inhibit the proliferation and migration of prostate cancer cells in vitro; knocking down circAKT3 can inhibit tumor growth in vivo, and the weight of tumors with low expression of circAKT3 is significantly lighter than that of epithelial tumors with normal expression of circAKT3; in addition, LNP-encapsulated si circAKT3 can effectively inhibit the growth of prostate cancer cells in the bone environment; and provides new targets and medication strategies for the treatment of prostate cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a reagent for knocking down circAKT3 in the preparation of a drug for treating prostate cancer. Background Art

[0002] The pathogenesis of prostate cancer is complex. Studies have pointed out that the onset of prostate cancer is closely related to certain susceptibility genes, such as PTEN, AR, GOLPH3, PAK1 and other genes. Among them, androgen and androgen receptor signaling pathways play an important role in the development of prostate cancer. Early focal prostate cancer can be cured by radical surgery, radioactive particle implantation or external radiation. However, prostate cancer is prone to bone and brain metastasis, and the process is relatively hidden. Once prostate cancer metastasizes and enters the late stage, the treatment options are relatively limited. The treatment for advanced prostate cancer is still mainly androgen removal therapy, but such patients will eventually turn into castration-resistant prostate cancer, which greatly shortens their survival time.

[0003] Circular RNA (circRNA) is a single-stranded covalently closed RNA molecule that is formed by reverse shearing of pre-mRNA. Compared with linear RNA, circRNA has higher stability and can also be detected in urine, and has the potential to be used as a biomarker for prostate cancer. In many tumors, the expression levels of certain specific circRNAs change abnormally, which is related to tumor proliferation, migration, and invasion. In recent years, mRNA drugs have gained wide attention and flourished due to their significant efficacy and relatively low production costs. Compared with linear mRNA, circRNA lacks a 5' cap structure and a 3' polyA tail, is not easily degraded by RNases, has a relatively stable physical structure, and has a longer action time in the body, so it has an inherent advantage as an RNA drug. Studies have shown that circRNA drugs have shown relatively ideal effects in the treatment of refractory melanoma, breast cancer and other diseases. However, there are currently no reports on the use of circRNA drugs in the treatment of prostate cancer. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of a reagent for knocking down circAKT3 in the preparation of a drug for treating prostate cancer.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides use of a reagent for knocking down circAKT3 in preparing a drug for treating prostate cancer, wherein the sequence of the circAKT3 is shown in SEQ ID NO.1:

[0007] .

[0008] Furthermore, knockdown of circAKT3 inhibited the proliferation, migration and invasion of prostate cancer cells.

[0009] Furthermore, knockdown of circAKT3 inhibited tumor growth in vivo.

[0010] Furthermore, knockdown of circAKT3 inhibited the growth of prostate cancer cells in the bone environment.

[0011] Furthermore, the reagent for knocking down circAKT3 includes one or more of single-stranded small interfering RNA directly synthesized by chemical methods, a plasmid expressing short hairpin RNA, and a viral vector.

[0012] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0013] The present invention demonstrates for the first time that knocking down circAKT3 can inhibit the proliferation and migration of prostate cancer cells in vitro; knocking down circAKT3 can inhibit tumor growth in vivo, and the weight of tumors with low circAKT3 expression is significantly lighter than that of epithelial tumors with normal circAKT3 expression; in addition, LNP-encapsulated si circAKT3 can effectively inhibit the growth of prostate cancer cells in the bone environment; it provides new targets and medication strategies for the treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The experimental results of proliferation detection in Example 1 are shown, wherein (A) is a schematic diagram of two specific siRNAs targeting the reverse splicing site of circAKT3; (B) is RT-qPCR analysis of the knockdown efficiency of siRNAs in PCa cells; the data are expressed as mean±SD, ***p<0.001, **p<0.01, t-test, n=3; (C) is a colony formation experiment to evaluate the effect of circAKT3 knockdown on the proliferation ability of PCa cells; the data are expressed as mean±SD, ***p<0.001, **p<0.01, t-test, n=3; (D) is a CCK-8 experiment to detect the effect of circAKT3 knockdown on the proliferation ability of PCa cells; the data are expressed as mean±SD, ***p<0.001, **p<0.01, t-test, n=3.

[0015] Figure 2 The experimental results of migration detection in Example 2 are shown, wherein (A) is a Transwell experiment to evaluate the effect of circAKT3 knockdown on the migration and invasion ability of PCa cells; scale, 100 mm; data are expressed as mean ± SD, ***p < 0.001, t test, n = 3; (B) is a scratch experiment to evaluate the effect of circAKT3 knockdown on the migration ability of PCa cells; scale, 100 mm; data are expressed as mean ± SD, ***p < 0.001, t test, n = 3.

[0016] Figure 3 The results of the xenograft tumor animal model evaluation in Example 3 show the effect of circAKT3 knockdown on tumor volume.

[0017] Figure 4The PC-3-luc cell bone metastasis model in Example 3 was used to evaluate the therapeutic effect of LNP-si-circ. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0019] The cells used in the experiments of the present invention were purchased from Shanghai Fuheng Biotechnology Co., Ltd., where DU145 was cultured in DMEM, PC-3 was cultured in DMEM / F12, and 22Rv1 was cultured in RPMI1640 medium with a fetal bovine serum concentration of 10%.

[0020] Two small interfering RNAs (si-circAKT3-1 and si-circAKT3-2) designed for circAKT3 were purchased from Genema. Their sequences ( Figure 1 A) is as follows:

[0021] si-circAKT3-1:

[0022] The antisense strand sequence is shown in SEQ ID NO.2: 5'-UGACUCAGCCUCGCCCCCA-3'

[0023] si-circAKT3-2

[0024] The antisense strand sequence is shown in SEQ ID NO.3: 5'-GAUGACUCAGCCUCGCCCC-3'

[0025] Example 1 Proliferation Detection

[0026] After successfully constructing stable prostate cancer DU145, PC-3, and 22Rv1 cell lines with knockdown of circAKT3, the following experiments were performed:

[0027] 1) Colony formation assay: After resuspending the cells, 1000 cells were plated in each well of a six-well plate. The medium was changed every three days and the cells were cultured until colonies were formed. The cells were fixed with formaldehyde and stained with crystal violet.

[0028] 2) CCK-8 experiment: resuspend the cells and inoculate an appropriate amount of cells in each well. After the cells adhere, add 10 μL of CCK-8 reagent to each well. After incubation for 2 hours, detect the OD value at 450 nm. Repeat the above detection steps at 24 hours, 48 ​​hours, 72 hours, and 96 hours.

[0029] The experimental results are as follows Figure 1 As shown:

[0030] Figure 1B shows the knockdown efficiency of siRNAs in PCa cells analyzed by RT-qPCR; Figure 1 Colony formation assay of C. Figure 1 D's CCK-8 experiment showed that knocking down circAKT3 could significantly inhibit the proliferation of prostate cancer cells.

[0031] Example 2 Migration Detection

[0032] After successfully constructing stable prostate cancer DU145 and PC-3 cell lines with knockdown of circAKT3, the following experiments were performed:

[0033] 1) Scratch test: Inoculate an appropriate amount of cells in a 6-well plate so that the cell confluence on the first day reaches more than 90%; on the first day, use a 200μL pipette tip to scratch the lid of the 6-well plate. After washing the scratched cells with PBS, add serum-free culture medium. Take pictures under white light using a 4x microscope. After continuing to incubate in the incubator for a certain period of time, take pictures of the same position again.

[0034] 2) Transwell invasion assay: Dilute Matrigel to 250 μg / mL with serum-free medium; take a 24-well plate, place the Transwell chamber in it, and add 100 μL of Matrigel into the chamber; after incubating in a cell culture incubator overnight, aspirate the liquid gel in the chamber; subsequent operations are the same as the Transwell cell migration assay.

[0035] The experimental results are as follows Figure 2 As shown: Transwell assay and scratch assay showed that knocking down circAKT3 could significantly inhibit the migration and invasion of prostate cancer cells.

[0036] Example 3 Animal Experiment

[0037] 1) Epithelial transplant tumor experiment: 5×10 6 DU145 sh ctrl / shcirc cells were cultured and the length and width of the subcutaneous tumors of nude mice were measured once a week, and the tumor volume was calculated using the formula: volume = (length × width 2 )÷2; 4 weeks after cell inoculation, the mice were sacrificed, and the tumors were removed and weighed.

[0038] The experimental results are as follows Figure 3 As shown: Knockdown of circAKT3 can inhibit tumor growth in vivo, and the weight of tumors with low circAKT3 expression is significantly lighter than that of epithelial tumors with normal circAKT3 expression.

[0039] 2) Mouse bone metastasis model: 5×10 6Luc PC-3 cells were used for in vivo imaging of mice two weeks later. LNP-si-ctr or LNP-si-circ drugs were then injected into the tibia of mice for treatment, once every three days for a total of five injections; in vivo imaging was performed again on the 32nd day.

[0040] The experimental results are as follows Figure 4 As shown: LNP-encapsulated si circAKT3 (LNP-si-circAKT3) can effectively inhibit the growth of prostate cancer cells in the bone environment.

[0041] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent for knocking down circAKT3 in the preparation of a drug for treating prostate cancer, characterized in that: The sequence of circAKT3 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Knockdown of circAKT3 inhibits the proliferation, migration and invasion of prostate cancer cells.

3. The use according to claim 1, characterized in that: Knockdown of circAKT3 inhibited tumor growth in vivo.

4. The use according to claim 1, characterized in that: Knockdown of circAKT3 inhibits the growth of prostate cancer cells in the bone environment.

5. The use according to claim 1, characterized in that: The reagent for knocking down circAKT3 includes one or more of directly chemically synthesized single-stranded small interfering RNA, a plasmid expressing short hairpin RNA, and a viral vector.

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