Application of circular RNA circ0120175 in regulating ovarian cancer

Abstract

The present invention belongs to the field of bioengineering technology, and in particular, to the use of circular RNA circ_0120175 in regulating ovarian cancer. The following scheme is proposed, including the use of a circular RNA in regulating ovarian cancer, wherein the circular RNA is circ_0120175, whose nucleic acid sequence is shown in SEQ ID NO. 1. The hsa_circ_012015 is involved as a positive regulator in the FIGO staging and lymph node metastasis of ovarian cancer. The present invention also includes the use of the circular RNA in the preparation of a biological product for ovarian cancer diagnosis, comprising the circ_0120175. The biological product comprises a reagent, a kit, and a chip. The present invention clarifies the effect of hsa_circ_0120175 on the growth of ovarian malignant tumors, and the circular RNA hsa_circ_0120175 is a good biomarker for diagnosing ovarian cancer and a potential target for treating ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to the application of circular RNA circ0120175 in regulating ovarian cancer. Background Art

[0002] Ovarian cancer (OC) is the most lethal malignancy among female reproductive organ cancers. As of 2018, OC was the seventh most common cancer in women worldwide. OC is often asymptomatic in its early stages, lacks effective early diagnostic indicators, and progresses insidiously and rapidly. Over 80% of OC patients are diagnosed in the advanced stages (III or IV). The poor prognosis and high mortality rate pose a serious threat to women's health. Therefore, further exploring the pathogenesis and development of OC and identifying tumor biomarkers for early diagnosis or potential therapeutic targets are of great significance.

[0003] Circular RNA (circRNA) was first discovered in eukaryotes in the 1970s and is considered to be a new type of non-coding RNA molecule with a closed-loop structure, high abundance, high stability and tissue specificity. CircRNA has a variety of different biological functions, such as regulating linear RNA transcription, downstream gene expression and protein production, and plays an important role in many biological processes. More and more studies have shown that circRNA has both oncogenic and tumor suppressor functions, plays a core regulatory role in tumor occurrence and progression, and is suggested as a biomarker for tumor diagnosis or new therapeutic targets; for example, hsa_circ_0005273 promotes breast cancer tumorigenesis by regulating the YAP1-Hippo signaling pathway; circCELSR1 promotes paclitaxel resistance in OC cells by regulating FOXR2 expression through miR-1252; hsa_circ_0077837 is significantly associated with the clinicopathological characteristics and poor prognosis of bladder cancer patients, affecting the biological functions of bladder cancer;

[0004] In order to clarify how it affects the proliferation, migration and invasion of ovarian cancer cells, the application of circular RNA circ0120175 in regulating ovarian cancer is needed. Summary of the Invention

[0005] The application of circular RNA circ0120175 in regulating ovarian cancer proposed in the present invention solves the problems existing in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of a circular RNA in regulating ovarian cancer.

[0008] Preferably, the circular RNA is circ_0120175, and its nucleic acid sequence is shown as SEQ ID NO.1.

[0009] Preferably, the hsa_circ_012015 is involved in FIGO staging and lymph node metastasis of ovarian cancer as a positive regulator.

[0010] A circular RNA biological product for the diagnosis of ovarian cancer is prepared, comprising circ_0120175.

[0011] Preferably, the biological products include: reagents, kits, and chips.

[0012] A drug for treating ovarian cancer, comprising: the circ_0120175, or an inhibitor of the circ_0120175.

[0013] Preferably, the inhibitor includes: (specific name and sequence need to be supplemented).

[0014] Preferably, the ovarian cancer treatment drug has at least the following functions:

[0015] Inhibit the FIGO staging of ovarian cancer and / or inhibit lymph node metastasis.

[0016] In the present invention, total RNA from three pairs of OC and adjacent non-cancerous tissue samples was analyzed by RNA sequencing (RNA-seq) to obtain a large number of circRNA transcripts; hsa_circ_0120175 was determined to be highly expressed in OC tissue; subsequent verification in a large number of ovarian cancer specimens found that the expression level of hsa_circ_0120175 in ovarian cancer tissue was significantly higher than that in adjacent ovarian tumor tissue; after knocking out hsa_circ_0120175, the proliferation, migration and invasion rates of ovarian cancer cells were inhibited, and nude mouse tumor formation experiments further verified the effect of hsa_circ_0120175 on the growth of ovarian malignant tumors; circRNA hsa_circ_0120175 is a good biomarker for diagnosing ovarian cancer and a potential target for treating ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The expression of circ_0120175 in OC tissues and cell lines;

[0018] Figure 1 A is the clustering heat map of differentially expressed circRNAs in three normal ovarian tissues and three ovarian cancer tissues;

[0019] Figure 1 B is the Sanger sequencing of the hsa_circ_0120175 product amplified by PCR, showing that hsa_circ_0120175 is a circular structure;

[0020] Figure 1 C is the nuclear-cytoplasmic separation verification that hsa_circ_0120175 is located in the OC cytoplasm;

[0021] Figure 1 D indicates that the expression level of hsa_circ_0120175 is upregulated in ovarian cancer tissues;

[0022] Figure 1 E: Hsa_circ_0120175 is upregulated in human ovarian cancer cell lines (HO8910 and SKOV3).

[0023] Figure 2 Schematic diagram of the relationship between circ_0120175 expression level and overall survival rate in OC;

[0024] Figure 3 The effect of circ_0120175 on the biological function of OC cells;

[0025] Figure 3 A is the interference efficiency of small interfering RNA si-circRNA in ovarian cancer cell lines;

[0026] Figure 3 B is the CCK8 experiment showing that the proliferation ability of HO8910 and SKOV3 cell lines decreased after hsa_circ_0120175 interference;

[0027] Figure 3 C Colony formation showed that the colony formation ability of HO8910 and SKOV3 cell lines decreased after interfering with hsa_circ_0120175;

[0028] Figure 3 DE is the Transwell experiment showing that the invasion and migration ability of HO8910 cell line decreased after interfering with hsa_circ_0120175;

[0029] Figure 3 FG are Transwell experiments showing that the invasion and migration abilities of SKOV3 cell lines were reduced after hsa_circ_0120175 interference;

[0030] Figure 3 HI is the scratch test showing that the proliferation and migration abilities of HO8910 and SKOV3 cell lines decreased after hsa_circ_0120175 interference;

[0031] Figure 4 Effect of hsa_circ_0120175 on the growth of subcutaneous xenograft tumors in nude mice

[0032] Figure 4A is a schematic diagram of the subcutaneous xenograft tumor in nude mice after interference with hsa_circ_0120175;

[0033] Figure 4 B: Compared with the control group, the subcutaneous tumor volume was reduced after interfering with hsa_circ_0120175;

[0034] Figure 4 C is a schematic diagram of the growth of subcutaneous xenograft tumors in nude mice;

[0035] Figure 4 D: Compared with the control group, the subcutaneous tumor weight decreased after interfering with hsa_circ_0120175. DETAILED DESCRIPTION

[0036] The present invention will be further explained below with reference to specific embodiments.

[0037] From September 2017 to June 2018, the applicant selected a total of 80 patients from the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College) for this study.

[0038] All patients did not receive preoperative radiotherapy and signed informed consent before surgery. The samples obtained from these 80 patients included 40 OC tissues and 40 control tissues from normal OC parts (all from patients undergoing surgery for benign ovarian disease).

[0039] All samples were evaluated by two independent pathologists from the Department of Pathology at the First Affiliated Hospital of Wannan Medical College to confirm the diagnosis. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College).

[0040] The detection methods used in the following examples are as follows:

[0041] 1. RNA-seq analysis and circRNA identification

[0042] Total RNA was extracted from cells or tissues using RNAisoPlus (Takara, Japan), and the quality and quantity of the total RNA samples were checked. Libraries were then constructed using the KAPA RNA HyperPrep Kit with Ribozyme (HMR) (Illumina, New England Biolabs, Beverly, MA), and sequenced using Novasek 6000 (Illumina, San Diego, CA).

[0043] Finally, the valid reads were aligned to the human reference genome (GRCh37 / hg19) to identify circRNAs by bioinformatics methods.

[0044] 2. Cell culture

[0045] Human ovarian cancer cell lines, including SKOV3 and HO8910 cells, and normal ovarian cells, IOSE-80, were purchased from the Shanghai Institute of Materia Medica. The SKOV3 cell line was cultured in McCoy's 5A medium (Gibco, USA), and the HO8910 and IOSE-80 cell lines were cultured in RPMI1640 medium (Gibco, USA). Both culture media were supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) and 1% penicillin-streptomycin antibiotics. Cells were routinely cultured in an incubator at 37°C and 5% CO2 and observed under an inverted light microscope. When the cell growth density reached 80% confluence, the cells were passaged by digestion with EDTA / trypsin (Cat. No. 252000-056, Gibco, USA).

[0046] 3. Cell transfection

[0047] siRNA negative control (si-NC) and small interfering RNA (siRNA) targeting hsa_circ_0120175 were purchased from GENESEED (Guangzhou, China); SKOV3 and HO8910 cells in the logarithmic growth phase were seeded in 6-well plates at 1.5×105 cells per well and incubated in a 37°C incubator; when the cell density reached approximately 50%, ovarian cancer cells were transfected according to the instructions of Lipofectamine 3000 transfection reagent (Invitrogen, USA).

[0048] 4. RNA Extraction and RT-qPCR

[0049] Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, USA) and RNAisoPlus (Cat. No. 9010, Takara, Japan), and the purity and concentration of the RNA samples were determined using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, USA). Total RNA was reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit (GS0201-2, Geneseed, China). Finally, the cDNA was subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) using SYBR Green qPCR Mix (GS0201-3, Geneseed, China) on an ABI 7900HT sequencer (Thermo Fisher Scientific, Waltham, MA). The experimental results were analyzed using 2 -ΔΔCtMethods: Calculation; Experiments were repeated three times; The following primers were used:

[0050] Forward primer (hsa_circ_0120175-F1): 5′-ATGATAGTAAAAGTTTGCGGACAAT-3′;

[0051] Reverse primer (hsa_circ_0120175-R1): 5′-GTGCACCAACTGAAGTACAC-3′.

[0052] 5. Nuclear and cytoplasmic separation

[0053] Cellular components were separated using a Nuclear / Cytoplasmic Fractionation Kit (Catalog No. AM1921; Life Technologies; Carlsbad, CA, USA) according to the manufacturer's protocol; cells were trypsinized, washed in PBS, and incubated in cell fractionation buffer; after centrifugation, the supernatant was the cytoplasmic fraction, and the nuclear fraction was extracted using nuclear extraction buffer.

[0054] 6. Cell Viability Assay

[0055] SKOV3 and HO8910 cells were seeded in 96-well plates at 4 × 104 cells per well and incubated in a cell culture incubator. 10 μl of Cell Counting Kit-8 (CCK-8, WST-8, Dojindo, Japan) was added to each well on days 1, 2, 3, and 4, respectively. After culturing in the incubator for 2 hours, the absorbance (OD) at 450 nm was measured on a spectrophotometric microplate reader (BioTek, VT, USA).

[0056] 7. Colony formation test

[0057] Cells (200 cells per well) were seeded in 6-well plates; the cells were then transfected and cultured in a 37°C, 5% CO2 incubator for 2 weeks before use; the culture medium was replaced every 3 days, the cells were washed with PBS (Solarbio, Beyotime Technology, China), and the colonies were fixed with paraformaldehyde (Beyotime Technology, China) for 30 minutes and then stained with crystal violet (Millipore, USA) for 15 minutes; the colony formation rate was calculated using the following formula: (number of clones / number of seeded cells) × 100%.

[0058] 8. Transwell assay

[0059] Transwell chambers (Corning, 24-well inserts, 8 μm pore size) were used to detect cell migration and invasion, respectively. Each group of cells to be tested was subcultured in the Transwell chamber at a density of 1×10 5 cells / ml in serum-free medium, and normal culture medium was added to the lower layer; 24 hours later, the cells on the bottom surface of the membrane were stained with 0.1% crystal violet (Millipore, USA) and counted under a microscope in five high-power random fields.

[0060] 9. Scratch test

[0061] The cell groups to be examined were resuspended and seeded on six-well plates (5×105 cells per well). When the cells reached 90–100% confluence, a vertical scratch was made in each well using a 200 μl pipette tip. Excess suspended cells were rinsed with PBS, and then 2 ml of serum-free medium was added to each well and cultured in a 5% CO2, 37°C incubator. Cells were photographed at 0, 12, and 24 h after the scratch, and the scratch area was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0062] 10. Subcutaneous xenograft tumor formation model

[0063] Five-week-old female BALB / c nude mice (Nanjing Institute of Life Sciences, Nanjing, China) were housed in a pathogen-free environment. SKOV3 cells (1 × 107) expressing or not expressing hsa_circ_0120175 were subcutaneously injected into the right abdominal wall of each nude mouse. The tumor volume of each nude mouse was then measured and calculated weekly. After 6 weeks, the nude mice were photographed, cervical dislocation was performed, and the tumors were removed. The tumors were then resected and weighed. The volume was calculated according to the formula: volume (mm 3 ) = width × length × height; This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Wannan Medical College (Yijishan Hospital).

[0064] 11. Statistical analysis

[0065] SPSS 24.0 software was used for statistical analysis, and GraphPad 5.0 was used for graphing. Statistical comparisons were performed using the t-test or one-way analysis of variance (ANOVA). Survival curves were calculated using the Kaplan-Meier method and the log-rank test. Differences were considered statistically significant when P < 0.05.

[0066] Example 1:

[0067] RNA-seq was used to analyze the total RNA after ribosomal RNA removal in three ovarian cancer tissues and three normal ovarian tissues, and a significantly upregulated circRNA, named hsa_circ_0120175, was found. Figure 1 As shown in A;

[0068] To verify the formation of the ring and determine the circularization site of hsa_circ_0120175, the PCR product of hsa_circ_0120175 was subjected to Sanger sequencing, and Sanger sequencing confirmed the circularization site of hsa_circ_0120175, as shown in Figure 1 As shown in B;

[0069] Since the subcellular localization of hsa_circ_0120175 is still unclear, nuclear-cytoplasmic fractionation experiments were performed, and the data showed that hsa_circ_0120175 is mainly located in the cytoplasm, such as Figure 1 As shown in C

[0070] At the same time, RNA-seq revealed that hsa_circ_0120175 was upregulated in OC tissues. We then used qRT-PCR to verify the expression of hsa_circ_0120175 in 40 OC tissues, normal ovarian tissues, and OC cell lines. The results showed that the expression level of hsa_circ_0120175 in OC tissues was significantly higher than that in normal ovarian tissues. Figure 1 As shown in D;

[0071] Compared with the normal ovarian cell line IOSE80, hsa_circ_0120175 was significantly overexpressed in OC cell lines (SK0V3 and HO8910), as shown in Figure 3. Figure 1 As shown in E.

[0072] Example 2:

[0073] To explore the relationship between hsa_circ_0120175 expression levels and clinicopathological characteristics of OC patients;

[0074] Patients were divided into two groups according to the median level of hsa_circ_0120175 expression in OC tissues (low expression < median, high expression ≥ median), as shown in Table 1 ;

[0075] Table 1 shows the expression of Hsa_circ_0120175 and clinicopathological characteristics of OC patients.

[0076]

[0077]

[0078] The expression level of hsa_circ_0120175 was significantly correlated with FIGO stage and lymph node invasion, but not with tumor size or tumor grade;

[0079] The Kaplan-Meier survival curve showed that the survival rate of OC patients with high expression of hsa_circ_0120175 was relatively poor, e.g. Figure 2 shown.

[0080] Example 3:

[0081] To clarify the biological function of hsa_circ_0120175 in OC, HO8910 and SKOV3 cells were transfected with siRNA; its expression in OC was then detected by qRT-PCR to confirm silencing; the results showed that the expression level of hsa_circ_0120175 in the si-circRNA group was lower than that in the blank control group and si-NC group, as shown in Figure 5. Figure 3 As shown in A;

[0082] Among them, there was no significant difference between the blank control group and the si-NC group, e.g. Figure 3 As shown in A; Then, the proliferation, migration and invasion of OC cells were evaluated by colony formation assay, CCK8 assay, Transwell assay and scratch assay; In the CCK8 assay and scratch assay, the cell proliferation ability of the hsa_circ_0120175 interference group was significantly inhibited, as shown in Figure 3 As shown in BC;

[0083] Transwell and Matrigel Transwell assays showed that interference with hsa_circ_0120175 significantly inhibited the migration and invasion abilities of H08910 and SKOV3. Figure 3 As shown in DG;

[0084] In the scratch test, the healing ability of cells after hsa_circ_0120175 interference was significantly lower than that of si-NC control cells. Figure 3 HI; thus, it was shown that hsa_circ_0120175 promoted the proliferation, invasion and migration of OC in vitro.

[0085] Example 4:

[0086] The effect of hsa_circ_0120175 on OC progression was detected by subcutaneous xenograft tumor formation in nude mice; compared with the si-NC group, the tumor growth in the si-circRNA group was significantly inhibited, and the volume and weight of the tumor xenografts were reduced compared with those in the control group. Figure 4AD shows P < 0.05, indicating that hsa_circ_0120175 can promote the growth of OC in vivo.

[0087] In summary, RNA sequencing (RNA-seq) was used to analyze total RNA from three pairs of OC and adjacent non-cancerous tissue samples to obtain a large number of circRNA transcripts; it was determined that hsa_circ_0120175 was highly expressed in OC tissues; then it was verified that in a large group of samples, the expression level of hsa_circ_0120175 in ovarian cancer tissues was significantly higher than that in adjacent ovarian tumor tissues; after knocking out hsa_circ_0120175, the proliferation, migration and invasion rates of ovarian cancer cells were inhibited, and nude mouse tumor formation experiments further verified the effect of hsa_circ_0120175 on the growth of ovarian malignant tumors; thus, it was shown that circRNA hsa_circ_0120175 is a good biomarker for diagnosing ovarian cancer and a potential target for treating ovarian cancer.

[0088] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0089] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature identified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0090] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention. Sequence Listing <110> The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College) <120> Application of circular RNA circ0120175 in regulating ovarian cancer <130> 2010 <141> 2022-05-06 <160> 1 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 415 <212> DNA <213> Artificial sequence <400> 1 aatgtgtctg tgaaaactac aagctggccg taaactgctt tgtgaataat aatcgtcaat 60 gccagtgtac ttcagttggt gcacaaaata ctgtcatttg ctcaaagctg gctgccaaat 120 gtttggtgat gaaggcagaa atgaatggct caaaacttgg gagaagagca aaacctgaag 180 gggccctcca gaacaatgat gggctttatg atcctgactg cgatgagagc gggctcttta 240 aggccaagca gtgcaacggc acctccatgt gctggtgtgt gaacactgct ggggtcagaa 300 gaacagacaa ggacactgaa ataacctgct ctgagcgagt gagaacctac tggatcatca 360 ttgaactaaa acacaaagca agagaaaaac cttatgatag taaaagtttg cggac 415

Claims

1. Use of a primer pair for detecting circular RNA expression levels in the preparation of an ovarian cancer diagnostic reagent, characterized in that: The circular RNA is circ_0120175, and its nucleic acid sequence is shown in SEQ ID NO.1; The forward primer of the primer pair is hsa_circ_0120175-F1, and its nucleic acid sequence is: 5′-ATGATAGTAAAAGTTTGCGGACAAT-3′; The reverse primer of the primer pair is hsa_circ_0120175-R1, and its nucleic acid sequence is: 5'-GTGCACCAACTGAAGTACAC-3'.

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