Circular RNA circFam169b and its applications

Abstract

This invention belongs to the field of biomedical technology and relates to the circular RNA circFam169b and its applications. This invention discovers a circular RNA circFam169b and, based on triple-negative breast cancer cell line models and nude mouse xenograft tumor models, demonstrates that overexpression of circFam169b can significantly inhibit the proliferation and migration of breast cancer cells and promote tumor cell apoptosis in vivo and in vitro. This invention provides a new approach for the clinical treatment of breast cancer and has broad application prospects in clinical practice.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a circular RNA circFam169b and its applications. Background Technology

[0002] Circular RNA (circRNA) is a class of non-coding RNA molecules with a closed circular structure, formed through back-splicing, and widely distributed in eukaryotes. Clinical research on circRNA has expanded from oncology to multiple disease areas, demonstrating broad diagnostic and therapeutic potential. For example, in neurological diseases, circRNAs (such as circHomer1 and CDR1as) can serve as early diagnostic biomarkers for Alzheimer's disease, and their expression levels are closely related to β-amyloid protein deposition and cognitive decline. In metabolic diseases, circRNA 0001946 is associated with insulin resistance and can predict the progression of diabetes; circMET alleviates diabetic retinopathy by regulating retinal angiogenesis. In autoimmune diseases, the expression of hsa_circ_0004018 in the peripheral blood of rheumatoid arthritis patients is positively correlated with disease activity, and the biomimetic delivery system developed by the Shenzhen Institutes of Advanced Technology significantly improves joint damage by targeting and regulating circRNA.

[0003] Currently, a company has successfully restored salivary gland function damaged by radiotherapy by delivering circular RNA expressing the aquaporin AQP1, becoming the world's first circular RNA drug to enter clinical trials. Despite challenges such as standardized testing and long-term safety, the stability, tissue specificity, and low immunogenicity of circular RNA make it an important candidate for precision medicine, and its clinical application prospects are broad with breakthroughs in gene editing and delivery technologies.

[0004] RNA therapy (such as siRNA and mRNA vaccines) has shown promise in cancer treatment in recent years, but its application still faces multiple challenges, such as RNA stability, targeting, and a single mechanism of action. For example, linear RNA is easily degraded by nucleases, has a short half-life, and requires chemical modification or delivery systems (such as lipid nanoparticles) to maintain its activity. In contrast, circular RNA, due to its closed structure, has the advantage of naturally resisting nuclease degradation, and can achieve protein translation by designing circularization sites or inserting internal ribosome entry sites (IRES), theoretically possessing greater potential for drug development.

[0005] A search revealed no research on the circular RNA circFam169b, or its application in breast cancer drugs. Summary of the Invention

[0006] To address the aforementioned issues, this invention, through large-scale screening and functional verification, discovered a novel circular RNA molecule, circFam169b. This circular RNA molecule exhibits excellent anti-breast cancer properties, providing a new candidate molecule for the development of anti-tumor circular RNAs.

[0007] This invention protects the circular RNA circFam169b, the nucleotide sequence of which is shown in SEQ ID NO.1. This circular RNA is selected from the Fam169b gene on chromosome number scaffold0031 of the giant mouse-eared bat. It is generated by backsplicing of exons 2 and 3 of the Fam169b gene.

[0008] This invention also protects the use of the circular RNA circFam169b expression promoter in the preparation of drugs for the prevention and treatment of breast cancer.

[0009] Furthermore, the breast cancer mentioned is triple-negative breast cancer.

[0010] Furthermore, the efficacy in preventing and treating breast cancer is achieved by upregulating the expression level of the circular RNA. The circular RNA significantly inhibits the proliferation and migration of breast cancer cells and promotes apoptosis.

[0011] The present invention also protects a medicament for treating breast cancer, the medicament comprising an overexpression vector of a circular RNA, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0012] In Example 1 of this invention, the inventors screened a specifically expressed circular RNA, circFam169b, from samples of *Rhizoctonia solani*, the sequence of which is shown in SEQ ID NO.1. After sequence alignment with the *Rhizoctonia solani* genome and annotations, the inventors determined that circFam169b originates from the *Fam169b* gene on chromosome number scaffold0031 of *Rhizoctonia solani*, and is generated by reverse splicing of exons 2 and 3 (a total of 231 nt) of the *Fam169b* gene (SEQ ID NO.2-3). Combined with PCR detection and Sanger sequencing, the inventors demonstrated that it can correctly circularize to form circular RNA. Subsequently, the inventors used the circular RNA overexpression vector pCDH-Mut4 (SEQ ID NO.4) to construct the linear sequence of circFam169b into the pCDH-Mut4 vector, obtaining the pCDH-Mut4-circFam169b overexpression vector (as shown in SEQ ID NO.5). Subsequently, the inventors transfected the overexpression vector into human breast cancer cell lines MDA-MB-231 and HCC1806 via lentiviral transfection. The expression level of circFam169b was detected by RT-qPCR, and the results showed that the above-mentioned circular RNA expression vector could significantly express circFam169b compared with the control group (pCDH-Mut4).

[0013] In Example 2 of this invention, after confirming that the pCDH-Mut4-circFam169b vector could significantly express circFam169b in human breast cancer cell lines MDA-MB-231 and HCC1806, the inventors performed Transwell migration, cell scratch assays, cell proliferation assays, and apoptosis assays. The results showed that overexpression of circFam169b in this invention can inhibit breast cancer cell migration and proliferation and promote apoptosis at the cellular level, thereby inhibiting tumor progression.

[0014] In Example 3 of this invention, the inventors seeded HCC1806 cells overexpressing circFam169b into the fat pads of the fourth pair of mammary glands in nude mice. After tumor formation, the tumor size was measured periodically. The results showed that the growth of tumor cells overexpressing circFam169b was inhibited, indicating that circFam169b of this invention has a good inhibitory effect on tumor progression at the animal level.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] This invention is the first to discover the circular RNA circFam169b, providing a novel candidate molecule for the development of anti-breast cancer tumor circular RNAs. Both cellular (in vitro) and animal (in vivo) experiments have demonstrated that overexpression of circFam169b significantly inhibits the proliferation and migration of breast cancer cells and promotes tumor cell apoptosis in both in vivo and in vitro. This invention provides a new target for the clinical treatment of breast cancer and has broad application prospects in clinical practice. Attached Figure Description

[0017] Figure 1 The diagram shows the structure of circFam169b and the results of significant expression of circFam169b in MDA-MB-231 and HCC1806 cells; Figure 1 A represents circFam169b, which originates from the Fam169b gene on chromosome number scaffold0031 of the giant mouse-eared bat, and is produced by the back splicing of Exon2 and Exon3 (231nt). Figure 1 B represents the amplification of cDNA and genomic DNA of *Rhizoctonia solani* using Convergent and Divergent primers. The agarose gel electrophoresis results are shown in the figure. Figure 1 C is a schematic diagram of the construction of the insertion sequence of the overexpression vector pCDH-Mut4; Figure 1 DE is a graph showing the overexpression effect of circFam169b transfection in MDA-MB-231 and HCC1806 cells as detected by qRT-PCR.

[0018] Figure 2 The image shows the results of circFam169b overexpression inhibiting tumor cell proliferation and migration; Figure 2 A and Figure 2 Figure B shows the results of the Transwell migration assay detecting the migration ability of MDA-MB-231 and HCC1806 cells overexpressing circFam169b. Figure 2 C represents the overexpression effect of circFam169b in HCC1806 cells; Figure 2 D is the result of the scratch assay to detect the migration ability of HCC1806 cells overexpressing circFam169b; Figure 2 E shows the proliferation capacity of MDA-MB-231 cells overexpressing circFam169b. Figure 2 F shows the apoptosis rate of HCC1806 cells overexpressing circFam169b.

[0019] Figure 3 This is a graph showing the results of circFam169b overexpression inhibiting tumor growth; in which... Figure 3Figure A shows the results of qRT-PCR detection of the overexpression effect of circFam169b in HCC1806 cells used for in situ tumorigenesis in nude mice. Figure 3 B is the result of in situ injection of HCC1806 cells overexpressing circFam169b into the fourth mammary gland of nude mice. After tumor formation, the tumor size was measured and statistically analyzed every two days. Figure 3 C shows the results of tumor size measurement in nude mice using in situ tumor formation. Detailed Implementation

[0020] Exemplary embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. Unless otherwise specified, the reagents and consumables used in the implementation process are commercially available.

[0021] Example 1: Construction of circFam169b expression vector and verification of overexpression effect

[0022] The inventors screened a specifically expressed circRNA, circFam169b, from samples of the giant mouse-eared bat, with its sequence shown in SEQ ID NO.1. To confirm that it is a circular RNA, the inventors verified the circular characteristics of circFam169b. By comparing the sequence with the giant mouse-eared bat genome and annotations, the inventors determined that circFam169b originates from the Fam169b gene on chromosome number scaffold0031 of the giant mouse-eared bat, and is generated by back splicing of exon 2 (SEQ ID NO.2) and exon 3 (SEQ ID NO.3) (a total of 231 nt) of the Fam169b gene.

[0023] Based on the sequence of circFam169b, the inventors designed Convergent primers (SEQ ID NO. 6-7) and Divergent primers (SEQ ID NO. 8-9) to perform PCR detection of cDNA and genomic DNA (gDNA) of the greater mouse ear falcon, and performed Sanger sequencing on the PCR products. Figure 1 A). The results showed that the Convergent primer amplified a band of the correct size (77 nt) in both cDNA and genomic DNA, while the Divergent primer amplified a band of the correct size only in cDNA (90 nt). Figure 1 B). This indicates that circFam169b is indeed a circular RNA.

[0024] Subsequently, the inventors constructed the circFam169b overexpression vector (SEQ ID NO.5) based on the circRNA overexpression vector pCDH-Mut4 (SEQ ID NO.4). The linear sequence of circFam169b was synthesized and incorporated into the pCDH-Mut4 vector to obtain the pCDH-Mut4-circFam169b overexpression vector. Figure 1 C). The overexpression vector is packaged into a lentivirus to obtain viral fluid. The lentivirus packaging steps are as follows:

[0025] (1) Seed healthy HEK293T cells into culture dishes and adjust the culture density to approximately 70-80%;

[0026] (2) Before virus encapsulation, the HEK293T cell culture medium was replaced with serum-free DMEM medium for cell starvation;

[0027] (3) After waiting for two hours, prepare the infection system. Calculate the required volume of plasmid based on the plasmid concentration and molecular weight. Add the required packaging plasmid to 500 μL of serum-free medium in Opti-MEM (Gibco) according to the molar ratio of the required packaging plasmid to the target plasmid (pMD2.G: psPAx2: target vector) in 1:1:1. Mix well and let stand for 5 minutes. Then mix the transfection reagent PEI to plasmid in a ratio of 2:1. After standing for 20 minutes, add it to the starved HEK293T cells and gently shake to mix evenly.

[0028] (4) After 4-6 hours, replace the culture medium with DMEM (Gibco) medium + 5% fetal bovine serum (FBS, Gibco) + 1% penicillin and streptomycin (PS) medium for further culture;

[0029] (5) Collect the virus solution twice at 48 hours and 72 hours after transfection, mix them together, centrifuge at 4°C for 500g, and after centrifugation for 5 minutes, filter the virus solution through a 0.45 μm filter membrane and store it in a -80°C refrigerator.

[0030] Subsequently, the pCDH-Mut4-circFam169b overexpression vector was transfected into human breast cancer cell lines MDA-MB-231 and HCC1806 via lentiviral transfection. The viral infection experimental procedures are as follows:

[0031] (1) Take out the required volume of virus solution stored in a -80℃ freezer in advance and thaw it at room temperature or 4℃.

[0032] (2) Use trypsin to digest and count the cells, and adjust the cell suspension concentration to about 1×106 cells per milliliter;

[0033] (3) Taking a 10 cm dish as an example, pour in 1×10 6 After adding 100 cells / plate, add 200 μL of virus solution and 20 μL of polybrene, then add 10 mL of complete culture medium, mix well, and incubate in a cell culture incubator for 24 hours.

[0034] (4) Remove the virus-containing mixture from the petri dish and replace it with complete culture medium, and incubate for 24 hours;

[0035] (5) 48 hours after infection, the cell culture medium was replaced with a complete culture medium containing puromycin and cultured for 24 hours to screen for successfully infected cells.

[0036] Subsequently, the inventors detected the expression level of circFam169b by qRT-PCR, amplified circFam169b using Divergent primers (SEQ ID NO. 8-9), and amplified Gapdh using Gapdh primers (SEQ ID NO. 10-11) as an internal reference.

[0037] The RT-qPCR reaction system is as follows:

[0038]

[0039] The RT-qPCR reaction conditions are as follows:

[0040]

[0041] The results showed that the pCDH-Mut4-circFam169b vector could achieve significant overexpression of circFam169b in both cell lines. Figure 1 CD).

[0042] Example 2: Overexpression of circFam169b inhibits tumor cell proliferation and migration.

[0043] The inventors conducted Transwell migration experiments on MDA-MB-231 and HCC1806 cells overexpressing circFam169b. The control group was transfected with the overexpression vector pCDH-Mut4 (without circFam169b), while the experimental group was transfected with the overexpression vector pCDH-Mut4-circFam169b. The study examined whether tumor cell migration phenotype was inhibited. The Transwell migration experiment steps are as follows:

[0044] (1) After digesting MDA-MB-231 and HCC1806 cells with trypsin, the digestion was terminated with a stop solution, and the cells were collected into centrifuge tubes. The cells were washed once with PBS, resuspended in serum-free medium, and counted. The cell suspension concentration was adjusted to 1×10⁻⁶ cells / mL. 6 Approximately 100 cells, typically 2 × 10⁶ cells per compartment in a Transwell plate (COSTAR, 3422). 5 One cell;

[0045] (2) Cell seeding: Take 100 μL of cell suspension and slowly add it to the upper chamber vertically above the center of the chamber;

[0046] (3) Add 500 μL of serum-containing complete culture medium to the lower chamber of the Transwell plate, and then incubate at 37 °C in a 5% CO2 cell culture incubator for 18-30 hours;

[0047] (4) Remove the upper chamber of the Transwell, discard the culture medium in the well, and carefully wipe the cells on the upper side of the upper chamber membrane with a cotton swab;

[0048] (5) Fix the cells with 4% paraformaldehyde for 20 minutes, then stain with 0.1% crystal violet for 15-20 minutes, rinse twice with PBS, and after air drying, observe and take pictures of different fields of view using a stereomicroscope and an inverted microscope.

[0049] The results showed that, compared with the control group, the migration ability of MDA-MB-231 and HCC1806 cells overexpressing circFam169b was significantly inhibited. Figure 2 AB).

[0050] Subsequently, the inventors conducted parallel verification of circFam169b's inhibitory effect on HCC1806 cell migration using a cell scratch assay. The cell scratch assay procedure is as follows:

[0051] (1) Use a 6-well plate to seed cells. Before seeding cells, draw some horizontal lines on the back of the 6-well plate with a marker.

[0052] (2) Seed an appropriate amount of cells. Taking HCC1806 cells as an example, seed 8 × 10⁶ cells per well. 5 Up to 1×10 6 Approximately 100 cells, with an optimal seeding density to cover the bottom of the plate after adhesion;

[0053] (3) After the cells adhere to the plate, if the density is appropriate, use a 10 μL pipette tip to draw a vertical line in the middle of each well of the 6-well plate;

[0054] (4) If the density does not reach 100%, continue to cultivate until the appropriate density is reached before conducting the experiment;

[0055] (5) Wash the well plate three times with PBS to remove cells and cell debris caused by the scratches;

[0056] (6) Remove the waste liquid, add serum-free culture medium for culture, and take pictures at appropriate time points according to different cell types. The results are statistically analyzed using ImageJ software to calculate the healing area and the healing ratio.

[0057] The results showed that overexpression of circFam169b significantly inhibited the migration ability of HCC1806 cells. Figure 2 (CE). Subsequently, the inventors performed cell proliferation assays on breast cancer cells overexpressing circFam169b. The cell proliferation experiment steps are as follows:

[0058] The MTS (Promega) method was used to detect cell proliferation in the experiment.

[0059] (1) Cell preparation: Take out the cells that have been overexpressed with circFam169b, wash them once with PBS, digest the cells with trypsin, collect them into centrifuge tubes after the cells are suspended, centrifuge at 500 g for 5 minutes to remove the supernatant, suspend the cell pellet in culture medium and count them with a cell counter.

[0060] (2) Cell seeding: Generally, 2000 cells / well are seeded in a 96-well plate, with 6-8 replicates per group, and 3 96-well plates are seeded;

[0061] (3) Culture: Place the 96-well plate with the cells in a 37°C, 5% CO2 cell culture incubator;

[0062] (4) On days 0, 3 and 6, take out the MTS (Promega) from the -20℃ freezer, thaw it in the dark, and prepare a mixture for detecting cell proliferation (MTS: empty medium = 1:5).

[0063] (5) Take out the 96-well plate from the incubator, remove the culture medium, add 100 μL of MTS working solution per well to be measured, and react at 37 °C in the dark for 2 hours.

[0064] (6) After the reaction is complete, use an ELISA reader to set OD 490 to measure the specific values ​​of the wells to be tested, and then perform data processing and calculation.

[0065] The results showed that overexpression of circFam169b significantly inhibited the proliferation of MDA-MB-231 cells. Figure 2 E). Therefore, circFam169b also has an inhibitory effect on cell proliferation.

[0066] In addition, the inventors conducted apoptosis experiments on HCC1806 cells overexpressing circFam169b. The apoptosis experiments are as follows:

[0067] The experiment used the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™, 556547) for detection.

[0068] (1) 72 hours after cell infection, the cells in the supernatant and adherent cells were digested and mixed together, and centrifuged at 500 g for 5 minutes;

[0069] (2) Resuspend and count in PBS, take 2×10 5 Transfer each cell to a 2 mL centrifuge tube (in addition to the experimental groups, control group cells should also be taken as blank control, FITC-Annexin V single label, and PI single label, one tube each).

[0070] (3) Centrifuge at 300 g for 5 minutes, discard the supernatant PBS, and resuspend in 200 μL 1×Binding Buffer;

[0071] (4) Protect from light and add FITC-Annexin V and PI. Note the blank control and two sets of single standards, each 1×10 5 Add 5 μL of antibody to each cell, 2 × 10⁶ cells. 5 Add 10 μL of antibody to each cell, gently pipette to mix, and incubate at room temperature in the dark for 15 minutes.

[0072] (5) Add 300 μL of 1×Binding Buffer to each sample, transfer to a flow cytometer tube, and perform flow cytometer analysis within 1 hour.

[0073] The results showed that, in addition to inhibiting the migration and proliferation of human breast cancer cells, overexpression of circFam169b also promoted apoptosis in HCC1806 cells. Flow cytometry analysis of apoptotic cells showed that the proportion of apoptotic HCC1806 cells overexpressing circFam169b increased, indicating that overexpression of circFam169b promotes tumor cell apoptosis. Figure 2 F). Therefore, circFam169b has a good ability to inhibit tumor cell migration and proliferation and promote tumor cell apoptosis.

[0074] Example 3: Overexpression of circFam169b can inhibit tumor growth (in vivo experiment)

[0075] After confirming that circFam169b inhibits tumor progression by suppressing breast cancer cell migration and proliferation in in vitro cell experiments, the inventors conducted xenograft experiments in nude mice to test the in vivo antitumor activity of circFam169b. The nude mouse xenograft experiment steps are as follows:

[0076] (1) Cell preparation: circFam169b was overexpressed in HCC1806 cells. After the cells grew to a certain number, they were digested with trypsin and collected. After washing twice with PBS, the cells were counted. The cell densities of the control group (overexpressing the vector, without circFam169b on the vector) and the overexpression group (transfected with pCDH-Mut4-circFam169b, overexpressing circFam169b) were adjusted to be consistent. 30% Matrigel was added, and the cell injection concentration was 6×10⁻⁶. 5 One breast tissue per 100 μL / side;

[0077] (2) Cell injection: The abdomen of nude mice was disinfected and cut open to form an inverted Y shape. The fourth pair of mammary glands on both sides were carefully exposed. 100 μL of cell suspension was carefully injected into each side. The skin was then sutured with a suture device. The control group was injected with HCC1806 cells overexpressing the vector without circFam169b. The experimental group was injected with HCC1806 cells transfected with pCDH-Mut4-circFam169b overexpressing circFam169b.

[0078] (3) The sutures were removed one week later, and the tumor size was measured at different time points thereafter;

[0079] (4) The mice were sacrificed about 8 weeks later and the tumor mass was removed for analysis.

[0080] The results showed that overexpression of circFam169b in HCC1806 ( Figure 3 A) When HCC1806 was injected into nude mice, measurements were taken at different time points. It was found that HCC1806 overexpressing circFam169b had significantly smaller tumor volume compared to the control group. Figure 3 B); After sacrificing nude mice, tumor fragments were collected for comparison, and the tumor volume of the experimental group mice was significantly inhibited (B); Figure 3 C). Therefore, overexpression of circFam169b can significantly inhibit the growth of breast tumors in animals, demonstrating a good anti-tumor effect.

Claims

1. Circular RNA circFam169b, characterized in that, Its nucleotide sequence is shown in SEQ ID NO: 1, and it is generated by reverse splicing of exon 2 and exon 3 of the Fam169b gene.

2. The application of the circular RNA circFam169b expression promoter in the preparation of drugs for the prevention and treatment of breast cancer, characterized in that: The circular RNA circFam169b expression promoter is a vector that overexpresses the circular RNA circFam169b. The nucleotide sequence of the circular RNA circFam169b is shown in SEQ ID NO:

1. The breast cancer is triple-negative breast cancer.

3. A drug for treating breast cancer, characterized in that: The drug contains an overexpression vector of circular RNA, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the breast cancer is triple-negative breast cancer.

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