Application of circRNA as osteosarcoma treatment target

Abstract

The invention relates to the field of molecular biology, in particular to application of circular RNA in osteosarcoma targeted therapy. The circular RNA is hsacirc0029634 (circZMYM2), the research finds that after expression of circZMYM2 is knocked down in osteosarcoma cells by using specific shRNA, proliferation and migration of the osteosarcoma cells and growth of nude mouse tumors can be inhibited, and the circular RNA can be used as a target spot for targeted therapy of osteosarcoma.

Description

[0001] Technology Neighborhood

[0002] The present invention belongs to the field of biotechnology and biomedicine, and specifically relates to the application of hsa_circ_0029634 (circZMYM2) as a therapeutic target for osteosarcoma. Background Art

[0003] Osteosarcoma is a malignant tumor that originates from the bone. It is the most common primary bone malignancy, especially common in children and adolescents. It usually occurs in rapidly growing bone areas, such as the distal or proximal end of the femur, tibia, and humerus. The incidence is higher in adolescents (especially between 10 and 20 years old). The 5-year survival rate of osteosarcoma is about 60-70%. The 5-year survival rate of patients with localized osteosarcoma is about 70-75%. The 5-year survival rate of patients with metastatic osteosarcoma (especially lung metastasis) is significantly reduced, at about 20-30%. In recent years, with the advancement of chemotherapy, surgical techniques, and radiotherapy, the prognosis of osteosarcoma patients has improved. However, early diagnosis and comprehensive treatment are crucial to improving survival rates.

[0004] Circular RNA (circRNA) is a unique class of non-coding RNA molecules with a covalently closed circular structure. Unlike linear RNA, circular RNA does not have a 5' end cap and a 3' end tail, which gives them higher stability and resistance to nuclease degradation. Circular RNA is widely present in various organisms, including plants, animals and humans. Many circular RNAs have tissue-specific and developmental stage-specific expression. In recent years, the research on circular RNA has made significant progress, revealing its important role in various biological processes. In particular, its potential applications in cancer, neurodegenerative diseases and cardiovascular diseases have attracted widespread attention. As an emerging RNA molecule, circular RNA has broad prospects in biomedical research and may become a new target for disease diagnosis and treatment in the future. Summary of the invention

[0005] The present invention provides a method for targeted treatment of osteosarcoma, comprising a group of sequences for knocking down the expression of circular RNA.

[0006] The present invention provides an application of a circular RNA-hsa_circ_0029634 (circZMYM2 for short) as a target for targeted treatment of osteosarcoma.

[0007] The first object of the present invention is to provide a sequence for specifically knocking down the expression of circZMYM2.

[0008] The second objective of the present invention is to verify the inhibitory effect of specific knockdown of circZMYM2 on the growth of osteosarcoma cells.

[0009] To achieve the first objective, the technical solution adopted by the present invention is: providing two shRNA sequences that significantly reduce the expression of circZMYM2 in osteosarcoma cell lines without affecting the expression of the circZMYM2 source gene ZMYM2.

[0010] To achieve the second purpose, the technical solution adopted by the present invention is: and using osteosarcoma cell lines to verify that shRNA inhibits the proliferation and migration of osteosarcoma cells by reducing the expression of circZMYM2.

[0011] The circZMYM2-specific shRNA sequence is as follows

[0012] F:5'-ACAGUUCCUGUUCCUGGUUCA-3'

[0013] R:5'-GUUCCUGUUCCUGGUUCAGCA-3' BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Analysis of shRNA knockdown efficiency and specificity.

[0015] Figure 2 To reduce the expression of circZMYM2 and inhibit the proliferation and migration of osteosarcoma cells. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0017] Unless otherwise specified, the following experimental methods are conventional methods, and the experimental materials used are easily available from commercial companies unless otherwise specified.

[0018] Example 1

[0019] 2.1 Selection of circZMYM2-specific shRNA

[0020] Based on the sequence of circZMYM2, two pairs of shRNAs across the circZMYM2 cleavage site were designed (sequences were synthesized by Qingke Company, vectors were constructed, and lentivirus was packaged). The following shRNAs were selected by testing their specificity and knockdown efficiency, and their sequences are as follows:

[0021] F:5'-ACAGUUCCUGUUCCUGGUUCA-3'

[0022] R:5'-GUUCCUGUUCCUGGUUCAGCA-3'

[0023] 2.2 Establishment of shRNA knockdown cell lines

[0024] U-2OS cell line and 143B cell line were plated in 6-well plates. When the cell confluence reached 80%, the cells were infected with shRNA lentivirus (infection multiplicity was 5). After 48 hours, antibiotic selection was performed with puromycin at a concentration of 1.5 μg / mL to remove uninfected cells. RNA was then extracted and reverse transcription and quantitative PCR were performed to detect the expression of circZMYM2 and ZMYM2.

[0025] Example 3

[0026] 3.1 CCK-8 detection of cell proliferation

[0027] The cells in the shRNA knockdown group and the control group were digested and counted, and about 2000 cells were plated in each well of a 96-well plate, with a total volume of 100 μL per well, and 5 time points were measured, with 3 replicates for each time point. After the cells adhered to the wall, 10 μL of CCK-8 reagent (purchased from Beyotime) was added to each well, and cultured at 37°C in the dark for 1 hour, and the absorbance at 450 nm was detected using an enzyme reader.

[0028] 3.2 Scratch assay to detect cell migration

[0029] The cells in the shRNA knockdown group and the control group were digested and passaged into 6-well plates. After the cells were fully grown, the medium was changed to serum-free medium. A 10 μL pipette tip was used to draw a straight line in the well plate. A bright-field photo was taken with a microscope and recorded as the zero point. Photos were taken at the same position 48 hours later to compare the gap width.

[0030] 3.3 Nude mouse tumor formation experiment to detect tumor growth rate in vivo

[0031] U-2OS cells in the shRNA knockdown group and the control group were plated on a 10 cm dish. When the cell confluence reached 90%, the cells were digested and counted. Two million cells were injected subcutaneously into each BALB / c nude mouse. The tumor size was measured every 5 days. The tumor was removed on day 25 and its weight was calculated.

Claims

1. A therapeutic target for osteosarcoma, characterized in that: A shRNA that can specifically reduce the expression of hsa_circ_0029634(circZMYM2).

2. The use according to claim 1, characterized in that The nucleic acid sequence of the shRNA.

3. The use according to claim 1, characterized in that The application of the shRNA in osteosarcoma cell lines is to reduce the cell proliferation ability of osteosarcoma cells.

4. The use according to claim 1, characterized in that The application of the shRNA in osteosarcoma cell lines is to reduce the cell migration ability of osteosarcoma cells.

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