CN117778574A - Use of MIR937 genome copy number amplification in diagnosis and/or treatment of ovarian cancer - Google Patents
Use of MIR937 genome copy number amplification in diagnosis and/or treatment of ovarian cancer Download PDFInfo
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- CN117778574A CN117778574A CN202311802494.0A CN202311802494A CN117778574A CN 117778574 A CN117778574 A CN 117778574A CN 202311802494 A CN202311802494 A CN 202311802494A CN 117778574 A CN117778574 A CN 117778574A
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Abstract
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to an application of MIR937 genome copy number amplification in diagnosis and/or treatment of ovarian cancer. According to the invention, through screening miRNA amplified in the 8q24.3 frequently amplified gene locus of the HGSOC patient by the TCGA database, further finding that MIR937 amplification can promote proliferation of HGSOC cells. Mechanistically, MIR937 amplification increased MIR-937-5p expression, reduced FBXO16 and its degradation to ULK1 protein, and thus enhanced autophagy. The research of the invention reveals the regulating shaft of HGSOC progress and provides a new combined therapeutic target for patients, thus having good practical application value.
Description
Technical Field
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to an application of MIR937 genome copy number amplification in diagnosis and/or treatment of ovarian cancer.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Ovarian cancer (OV) is a malignant tumor that severely threatens female health. Among various tissue types of OV, high grade cancer serous carcinoma (HGSOC) is most common, accounting for 60-80% of new cases annually. It is estimated that clinically, over 75% of HGSOC patients are diagnosed with advanced stages, which makes it the most fatal and difficult to interfere with. Thus, five-year survival of HGSOC was less than 30% and was not significantly improved in the last three decades due to the lack of early diagnostic markers and effective therapeutic targets. The need for more rigorous studies has been intensified to enhance our understanding of HGSOC tumor initiation and progression, thereby providing new intervention strategies.
HGSOC patients are characterized by high frequency of somatic copy number Changes (CNAs), including deletions of tumor suppressor genes and amplifications of oncogenes. In most cases, CNA is often associated with changes in gene expression and protein abundance. There is increasing evidence that gene amplification always leads to a proliferative phenotype, due to the increased expression of a particular gene or the cumulative effects of gene collection. In HGSOC, functional gain of gene amplification in tumorigenesis has been widely studied. For example, OV cells with genome amplified URIs rely on enhanced URI proteins to sustain their survival. USP13 amplification and overexpression reprograms cancer cell metabolism and aggravates tumor progression of HGSOC. In addition, amplified RAD21 enhances its interaction with YAP/TEAD4 to inhibit interferon signaling and promote immune evasion of HGSOC cells. Accordingly, researchers have been working to reveal accurate medical targets of clinically relevant genomic alterations. HER2 antibodies (trastuzumab) have evolved in the treatment of HER2 amplified breast cancer. Although HGSOC is not so, this success has led to an encouraging possibility that tumor drivers of targeted expansion have great potential in cancer treatment. Not only for protein-encoding genes, CNAs of small non-coding micrornas (mirnas) represent a type of genomic aberration with cancer driving capability. However, lack of explanation of the underlying mechanism of miRNA amplification in carcinogenesis severely limits the progress of HGSOC treatment.
mirnas consist of 19-24 nucleotides and have profound biological functions in cell survival, proliferation and differentiation, mainly through complementary binding of mrna targets. Functional studies have demonstrated that mirnas can also act as tumor inhibitors or oncogenes, and that genomic aberrations in mirnas can lead to deregulation of mirnas and are continuously associated with cancer progression. This concept has been demonstrated in HGSOC, i.e., increased copy number of miR-569 increases expression and increases tumor survival and proliferation by attenuating TP53INP1 transcription. Intervention of mirnas by mimetics or inhibitors (with anti-miR function) has shown great potential in the clinical treatment of cancer and related diseases. For example, a mimetic of the tumor suppressor miR-16 (MesomiR-1) and an anti-miR (MRG-106) that targets miR-155 have achieved a phase I trial for the treatment of malignant pleural mesothelioma and cutaneous T-cell lymphoma, respectively. Given the popularity of miRNACNAs in cancer, and the feasibility of manipulating miRNA expression in receptors, we have encouraged us to unravel the functional mechanisms of amplifying mirs in HGSOC to provide prospective therapeutic targets.
Disclosure of Invention
In view of the above prior art, it is an object of the present invention to provide the use of MIR937 genomic copy number amplification in the diagnosis and/or treatment of ovarian cancer. Specifically, the invention screens miRNA amplified in the 8q24.3 frequently amplified gene locus of the HGSOC patient through a TCGA database, and further discovers that MIR937 amplification can promote proliferation of HGSOC cells. Mechanistically, MIR937 amplification increased MIR-937-5p expression, reduced FBXO16 and its degradation to ULK1 protein, and thus enhanced autophagy. The research of the invention reveals the regulating axis of HGSOC progression and provides a new combined therapeutic target for patients. Based on the above results, the present invention has been completed.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided the use of an agent for detecting the expression of a MIR 937-related biological element in the manufacture of a product for screening, (co) diagnosing, detecting, monitoring or predicting the progression of ovarian cancer.
Wherein the MIR937 related biological element expression conditions include, but are not limited to, changes in MIR937 gene copy number, changes in miR-937-5p expression levels, changes in FBXO16 expression levels, and changes in ULK1 expression levels.
Specifically, the research shows that the MIR937 gene copy number is amplified in HGSOC patients, the expression of miR-937-5p is further increased, and the degradation of FBXO16 and ULK1 protein is reduced, namely MIR937 regulates ovarian cancer progression through miR-937-5p/FBXO16/ULK 1. The concrete steps are as follows: MIR937 amplification mediates MIR-937-5p overexpression, thereby inducing ULK1 overexpression and FBXO16 overexpression, while resulting in malignant progression in HGSOC patients. Thus, the above-described MIR 937-related biological elements can be used as a diagnostic and prognostic marker for ovarian cancer, particularly high-grade serous ovarian cancer.
The product may be a kit, a detection device or a detection apparatus, etc., and is not particularly limited herein.
In a second aspect of the invention, there is provided a system for screening, (aiding) diagnosis, detection, monitoring or prediction of the progression of ovarian cancer, the system comprising:
an acquisition unit configured to: acquiring the expression condition of MIR937 related biological elements of a sample to be tested of a subject;
an evaluation unit configured to: and judging the disease condition of the subject according to the expression condition of the biological element related to the MIR937 of the subject obtained by the obtaining unit.
Wherein the MIR937 related biological element expression conditions include, but are not limited to, changes in MIR937 gene copy number, changes in miR-937-5p expression levels, changes in FBXO16 expression levels, and changes in ULK1 expression levels.
The ovarian cancer may be a high grade serous ovarian cancer.
The sample to be tested may be an ovarian (cancerous) tissue or a cell.
In a third aspect of the invention, the application of MIR937 related biological element as a target in preparing and/or screening ovarian cancer drugs is provided.
The effect of the drug candidate on the MIR 937-related biological element before and after use can be utilized to determine whether the drug candidate can be used to prevent or treat ovarian cancer. Wherein the ovarian cancer is specifically high grade serous ovarian cancer.
Specifically, the method for screening ovarian cancer drugs comprises the following steps:
1) Treating a system expressing and/or containing said MIR 937-related biological element with a candidate substance; setting a parallel control without candidate substance treatment;
2) After step 1) is completed, detecting the expression level of the MIR937 related biological element in the system; if the expression level of the MIR 937-related biological element is significantly reduced or increased in a system treated with a candidate agent, the candidate agent can be a candidate ovarian cancer drug, as compared to a parallel control.
Wherein the MIR937 related biological element comprises at least one of MIR937, miR-937-3p, FBXO16 and ULK 1.
In a fourth aspect of the invention there is provided the use of a substance which inhibits or reduces the expression of MIR937 in any one or more of:
a) Inhibiting miR-937-5p expression or preparing a product for inhibiting miR-937-5p expression;
b) Inhibiting ULK1 expression or preparing a product that inhibits ULK1 expression;
c) Promoting FBXO16 expression or preparing a product that promotes FBXO16 expression;
d) Inhibiting autophagy of ovarian cancer cells or preparing a product for inhibiting autophagy of ovarian cancer cells;
e) Inhibiting the proliferation of ovarian cancer cells or preparing a product for inhibiting the proliferation of ovarian cancer cells;
f) Inhibiting ovarian cancer growth or preparing a product for inhibiting ovarian cancer growth;
g) Products for treating ovarian cancer.
Wherein the agent that inhibits or reduces MIR937 expression may be an agent that includes, but is not limited to, an RNA interfering molecule or antisense oligonucleotide directed against MIR937, a small molecule inhibitor, shRNA, siRNA, performs lentiviral infection or gene knockout (e.g., knocks MIR937 based on CRISPR-Cas9 technology).
The ovarian cancer may be a high grade serous ovarian cancer.
The product may be a drug or an experimental reagent for use in basic research. For example, the product can be used for in vitro regulation and control of proliferation of ovarian cancer cells, so as to prepare an ovarian cancer cell related biological model, and be used for research of an ovarian cancer related mechanism.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredients may be carriers, excipients, diluents and the like which are generally used in pharmacy. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.
The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.
The subject to be administered may be human or non-human mammals such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and gorillas, without specific limitation herein.
The beneficial technical effects of one or more of the technical schemes are as follows:
the above technical scheme shows that MIR937 has dependence on gene copy number amplification, and the amplification enhances autophagy and determines proliferation activity of HGSOC. Data mining of TCGA database showed that MIR937 amplification was associated with MIR937 expression and increased cell proliferation of HGSOC. The loss of MIR937 in HGSOC cells resulted in impaired autophagy and retarded cell proliferation, the extent of inhibition being proportional to the extent of MIR937 copy loss. Rescue experiments demonstrated that miR-937-5p, a mature MIR937 product, was sufficient to restore its oncogenic function. Mechanistically, MIR937 amplification increases MIR-937-5p expression and enhances its binding to the 3' utr of FBXO16 transcript, thereby limiting the degradation of ULK1 by FBXO 16. The result of the technical proposal shows that the MIR937 amplification enhances the autophagy and proliferation of cells, and provides a MIR937/FBXO16/ULK1 targeted alternative strategy for HGSOC treatment, thereby having good potential practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows that MIR937 was amplified in HGSOC patients and correlated with increased MIR937 expression in the examples of the invention. Schematic of three mirs in the (a) 8q24.3 locus. (B) Expression data of three mirs were retrieved from the cBioportal tool for TCGA database analysis. (C) Genomic amplification of MIR937 in human cancers retrieved from cBioportal tools for TCGA database analysis. (D and E) correlation between MIR937 copy number and its expression was performed by data from TCGA database (D) or CCLE OV cancer cell line (E). (F) MIR937 expression in HGSOC patients was quantified and MIR937 diploids compared to amplified patients. (G) Pearson correlation between MIR937 copy number and ssGSEA calculated proliferation score. (H) Pearson correlation between MIR937 mRNA expression and proliferation scores calculated by ssGSEA. The gene set named "positive regulation of cell population proliferation" was obtained from the Gene Ontology (GO) knowledge base (https:// www.geneontology.org /). ssGSEA: single sample gene set enrichment analysis. Statistical analysis is carried out by adopting single-factor analysis of variance (B) and double-tail Student t test (F); the Pearson correlation coefficient (R) was used to evaluate the correlation between two consecutive variables (D, E, G and H). * P < 0.001.
FIG. 2 shows that MIR937 lacks in reducing proliferation of ovarian cancer cells in the examples of the invention. (A) MIR937 knockout strategy based on CRISPR-Cas9 in OVCAR3 and a2780 cells. (B) Sanger sequencing was used to determine the genotypes of MIR 937+/-and MIR937-/-OVCAR3 and A2780 cells. (C and D) MIR937+/+, MIR 937+/-and MIR 937-/-cell growth curves for OVCAR3 (C) and A2780 (D). Cells were seeded in an initial number of 2000 96-well plates and growth was continuously monitored for 5 days. (E and F). Xenograft tumors derived from MIR937+/+, MIR 937+/-and MIR937-/-OVCAR3 cells (E) and A2780 cells (F) are shown. 5x106 of each cell was subcutaneously injected to the right of nude mice (n=5). Mice were sacrificed three weeks after inoculation. (G and H) growth curves of xenograft tumors with indicated MIR937 gene-deleted OVCAR3 (G) and a2780 (H) cells. Volume was measured every three days (day 6) after tumor appearance. (I and J) tumor weights were measured after mice were sacrificed. (K) Representative bioluminescence images of mice injected intraperitoneally with luciferase-expressing OVCAR3 and a2780 cells (MIR 937+/+, MIR937+/-, and MIR 937-/-) were collected on day 12 post-inoculation. Data are shown as mean ± SD. Statistical analysis was performed using one-way analysis of variance (C, D and G-J); * P <0.05; * P <0.01; * P < 0.001; * P < 0.0001.
FIG. 3 is a graph of miR-937-5p in the examples of this invention, illustrating the loss of MIR937 function in ovarian cancer cells. (A and B) growth curves of OVCAR3 (A) and A2780 (B) cells were monitored by CCK8 analysis of miR-937-5p or 3p mimetic transfection. (C and D) after transfection of miR-937-5p or 3p inhibitors, the growth curves of OVCAR3 and A2780 cells were examined. Comparison of growth curves (E) and A2780 (F) MIR937+/+ cells of OVCAR3 with MIR 937-/-cells transfected with miR-937-5p or 3p mimics. Schematic summary of the injection of agoniR into xenograft tumors by mock-transfected MIR937+/+ cells, and MIR 937-/-cells (G) transfected with miR-937-5p or 3p mimics. (H and I) shows xenograft tumors derived from OVCAR3 (H) and A2780 (I) cells. MIR937+/+ tumors were mock-treated with agoniR NC, and for MIR 937-/-tumors, use of agoniR-937-5 p and-937-3 p interfered with tumor growth. (J and K) growth curves of OVCAR3 (J) and A2780 cells (K) xenograft tumors affected by agoniR-937-5 p and-937-3 p. Tumor weights of (L and M) OVCAR3 (L) and a2780 (M) cells were measured on day 25, at which time mice were sacrificed after five rounds of agonir injection. Data are shown as mean ± SD. Statistical analysis was performed using one-way analysis of variance (A-F and J-M); * P <0.01; * P < 0.001; * P < 0.0001.
FIG. 4 is a diagram of a tumor suppressor gene directly targeting FBXO16 by miR-937-5p in an embodiment of the invention. (A) The Venn diagram shows 11 potential targets overlaid by a list of TSG and miR-937-5p targets obtained from the TargetScan database. (B) After transfection of miR-937-5p mimics in OVCAR3 cells, expression of 11 predicted genes was detected by qPCR analysis. (C) The TargetScan predicts potential miR-937-5p binding sequences in the 3' UTR of FBXO16 mRNA. The sequences of the mutant constructs used for luciferase reporter gene assays are also indicated. (D) Following co-transfection of the previously described reporter plasmid with the miR-937-5p mimetic in HEK293T cells, relative luciferase activity was monitored. Firefly luciferase activity was normalized to renilla activity. (E) FBXO16 protein levels were detected following transfection of miR-937-5p mimics or inhibitors in OVCAR3 and a2780 cells. GAPDH is used as load control. (F) Xenograft tumors derived from OVCAR3 cells are shown. Two OVCAR3 cell lines with stable FBXO16 knockdown and one control cell line (vector) were injected subcutaneously into nude mice. (G) tumor weight was measured on day 21 after mice were sacrificed. Data are shown as mean ± SD. Statistical analysis was performed using the two-tailed Student's t test (B and D) and single-factor analysis of variance (G); * P <0.05; * P < 0.001; * P < 0.0001.
Figure 5 is a graph showing that FBXO16 in the examples of the present invention down regulates autophagy in ovarian cancer. (A) Gene ontology enrichment analysis of the up-regulated (up-regulated) and down-regulated (down-regulated) gene sets in OVCAR3 cells stably knocked down with FBXO 16. (B and C) IB analysis of autophagy-related proteins affected by FBXO16 stable knockdown (B) and overexpression (C) in OVCAR3 cells under normal and hypoxic conditions. Cells were treated with 1% oxygen at 37 ℃ for 12 hours under hypoxic conditions.
Fig. 6 is a diagram of how FBXO16 interacts with ULK1 and facilitates K48 ligation thereof in an embodiment of the present invention. (A) Schematic of predicted interactions between ULK1 and CUL1-SKP1-FBXO16 complex. (B and C) Co-IP analysis was performed with anti-Flag antibodies to detect interactions between Flag-FBXO16 and Myc-ULK1 (B) and Myc-FBXO16 and Flag-ULK1 (C) in HEK293T cells Co-transfected with the plasmids shown. (D and E) the schematic structures of Wild Type (WT) and truncation mutants of ULK1 (D) and FBXO16 (E) are shown (up) and co-IP with Flag antibodies was performed to visualize interactions between FBXO16 and ULK1 truncations (down) and ULK1 and FBXO16 truncations mutants (down). (F and G) Co-IP with anti-Flag antibody followed by immunoblot analysis (IB) to detect ULK1 ubiquitination in HEK293T cells affected by FBXO 16. WT, K48 and K63 mutant forms of HA-Ubs were used in (F), while wild-type and K48R mutants of HA-Ubs were used in (G).
FIG. 7 shows that clinical MIR937 amplification was associated with FBXO16 and ULK1 expression in examples of the invention. (A) IB analysis of protein levels of ULK1, P62, LC3II/I in MIR937+/+, MIR937 +/-and MIR937-/-OVCAR3 (upper) and a2780 (lower) cells. GAPDH is used as load control. (B) IB analysis was used to compare FBXO16, ULK1 protein abundance in clinical OV and control samples. Ctrl: normal ovaries, MIR937 WT, OV patient samples, non-amplified MIR937.MIR937 AMP OV patient samples were amplified with MIR937. (C) Representative image of ULK1 protein IHC analysis in OV patient samples or normal controls, scale bar: the marker information is identical to that in the (D and E) Kaplan-Meier survival analysis of OV patients based on the GSE26193 dataset of FBXO16 (D) and ULK1 (E) expression packets. (F) Kaplan-Meier survival analysis of OV patients grouped according to FBXO16 and ULK1 expression in GSE 26193.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.
Examples
1. Materials and methods
1.1 cell culture and tissue samples
HEK293T, OVCAR3, a2780, TOV21G and SKOV3 cells were obtained from Procell life sciences technology limited (chinese martial arts). HEK293T cells were maintained in low glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum at 37℃with 5% CO 2 . OVCAR3, a2780, TOV21G and SKOV3 cell lines in RPMI medium containing 10% fetal bovine serum at 37 ℃ and 5% co 2 And (3) growing in the middle. All cell lines were confirmed to be free of mycoplasma contamination. Frozen tissue specimens from the Shandong university of first medical science affiliated cancer Hospital for normal ovarian and ovarian epithelial malignancy.
1.2 plasmids, antibodies and reagents
Human ULK1, FBXO16, CUL1, SKP1 and RBX1 genes were amplified and subcloned into pcdna3.1 vector with Flag or Myc tag. HA-tagged WT, K48-and K63-Ub encoding plasmids were subcloned into the pcDNA3.1 vector. Antibodies against ULK1 (catalog 8054S), p62 (catalog 23214S), LC3A/B (catalog 4108S), CUL1 (catalog 4995S), ATG7 (catalog 8558S), ATG13 (catalog 13273S), beclin-1 (catalog 3495S), flag (catalog 14793S), myc (catalog 2278S) and HA (catalog 3724S) were purchased from Cell Signaling Technology. Antibodies against GAPDH (catalog ab 181602) were obtained from Abcam. anti-FBXO 16 antibody (catalogue LS-C82721) was purchased from LSBio. Cell counting kit-8 (CCK-8) (catalog C0038), D-fluorescein potassium salt (catalog ST 196) and crystal violet staining solution (catalog C0121) were purchased from Beyotime Biotechnology. Cyclohexanamide was purchased from MedChemexpress (catalog HY-12320). Cell Light EdU Apolo567 in vitro kit (catalog C10310-1) was purchased from Ribobio.
1.3 production of MIR937 KO cell line
For MIR937 knockdown, slenticrispr V2 (adedge, catalog 52961) was digested with kpnl (NEB, catalog R3142V) and EcoRI (NEB, catalog R0101V). Two sgrnas flanking the MIR937 locus were designed to delete the MIR937 coding sequence. The sequence of the first sgRNA is: TGCCCCCGGTGAGTCAGGGT (SEQ ID NO. 1), the second sgRNA sequence is: GTTCCCGAGCTCCTGCAGGT (SEQ ID NO. 2) driven by the U6 or H1 promoters, respectively. The cassette for the U6 promoter sgRNA 1-H1-promoter sgRNA2 was synthesized and ligated into pLentiCRISPR v 2. The constructed plasmid pMD2.G (adedge, catalog 12259) and psPAX2 (adedge, catalog 12260) were co-transfected into HEK293T cells for viral packaging. OVCAR3 and a2780 cells were infected with puromycin (MCE, catalog HY-B1743A) and single cells were screened for MIR937 knockout verification.
1.4 genomic DNA isolation, PCR and copy number verification
Total genomic DNA of ovarian cancer cell lines and human ovarian tissue cultured in vitro was extracted by a DNA isolation kit (Vazyme, DC 112-01). For MIR937 knockout analysis, PCR amplification and sequencing was performed using genomic DNA of the cultured ovarian cancer cell line as template, using the following pair of primers: f GTGGGGGCGTATAGTCTCTTG (SEQ ID NO. 3), R GCATCGGTTAGTGCCTG (SEQ ID NO. 4). For clinical samples, FAM-labeled probes and amplification primers for genomic MIR937 sequences were designed, VIC-labeled Rnase-P probes and primers were used to quantify the copy number of the reference gene, and genomic DNA from TOV21G cells was used as a diploid control. TaqMan copy number assays were performed with TaqManFast Advanced Master Mix (ThermoTM Fisher Scientific, catalog 4444554). Results were collected on Quantum 1Plus and analyzed by CopyCaller software (ThermoTM Fisher Scientific).
1.5 production of stable cell lines
The FBXO16 coding sequence was subcloned into the Flag tagged pCDH-GFP vector. The ShRNA oligonucleotide targeting FBXO16 was annealed to the pLKO.1-EGFP vector. The shRNA sequence is as follows: FBXO16-shRNA 1: 5'-GCTATTGAATGACCGGGTA-3' (SEQ ID NO. 5); FBXO16-shRNA2:5'-CAAGCTTCCAAGGTGTTA-3' (SEQ ID NO. 6). Phase Guan Zhili was packaged into lentiviruses with psPAX and pMD2.G plasmids for OVCAR3 infection, and infected OVCAR3 cells were screened with 1. Mu.G/ml puromycin.
1.6MiRNA, siRNA transfection
miR-937-5p/3p mimics, mimic Negative Controls (NC), inhibitors or inhibitor NC oligonucleotides were transfected into OVCAR3, A2780, TOV21G and SKOV3 cells by HiperFect transfection reagents (Qiagen, catalog 301704) according to the manufacturer's instructions. For transient silencing of FBXO16, the replicaexs of siRNA were transfected into ovarian cancer cells with LipofectamineTM RNAiMAX agent (Thermo Fisher Scientific, catalog 13778075) according to protocol. Target sequences for FBXO16 knockdown were purchased from GenePharma. All sequences used are provided in table 1.
TABLE 1 sequence information
1.7RNA isolation and quantitative RT-PCR
Total RNA from tissue or cultured cells was isolated by the FastPure cell/tissue Total RNA isolation kit (Vazyme, catalog RC 101-01). RNA was reverse transcribed using PrimeScript RT kit for mRNA (Toyobo, catalog RR 037A) or using the miRNA first strand cDNA synthesis kit for microRNA (Vazyme catalog MR 101) as per the manufacturer's instructions. qRT-PCR assays were performed on Quantum studio 1Plus instrument by using SYBR Green (CWBIO, catalog CW 2601H). Relative RNA expression was quantified by the comparative 2- ΔΔCt method. The RT-qPCR primer sequences of the relevant genes are listed in Table S2. Primers for microRNA detection were purchased from GenePharma, U6 was used as a control for normalization of microRNA expression.
1.8 cell proliferation and colony formation assay
OVCAR3, a2780 and TOV21G cells were seeded at 2000 cells per well in 96-well dishes, SKOV3 cells were seeded at 1500 cells per well. Cell count kit-8 (CCK-8) kit (Beyotime, catalog C0038) was used to determine cell count and viability and absorbance was measured at 450nm by a spectrophotometric reader (MD, spectramax 190). For colony formation assays, MIR937 knockout OVCAR3 and a2780 cells were seeded into 6-well plates (600 cells/well) and cultured for 10 days. Colonies were fixed with 4% Paraformaldehyde (PFA) and stained with 0.5% crystal violet. Colony numbers were counted by ImageJ software and data are expressed as mean ± SD. Each experiment was repeated three times.
1.9 5-ethynyl-2' -deoxyuridine (EdU) incorporation assay
Transiently transfected or MIR937 knocked out OVCAR3 and a2780 cells were seeded in 96-well plates with 1 x 104 cells per well. The EdU assay was performed using a Cell Light EdU Apollo 567 in vitro imaging kit (RiboBio, catalog C10310-1) according to the manufacturer's instructions. Briefly, after 2 hours incubation with 10mM EdU, cells were fixed with 4% PFA, permeabilized in 0.5% Triton X-100, and labeled with a fluorescent dye of Apollo 567. Nuclei were stained with DAPI (Solarbio, catalog C0065) for 15 min. Images were captured with a fluorescence microscope (Nikon, TS 2R). EdU positive cells were quantified using imageJ software and summarized as mean.+ -. SD using at least three random fields.
1.10 Dual luciferase reporter assay
pGL3-3M-Luc vectors are used to insert the 3'UTR of the FBXO16 transcript or its mutant 3' UTR without the miR-937-5p binding site. HEK293T cells were seeded in 24-well dishes one day prior to transfection. WT or mutant plasmids (with FBXO 16' UTR) were co-transfected with NC or miR-937-5p mimics. After the time period of 48 hours, the mixture was stirred,harvesting cells and lysis using bifluorescenceThe reporter assay system (Promega, catalog E1910) measures luciferase activity by a GloMax 20/20 photometer. Relative luciferase activity was normalized to ranila activity. Three independent experiments were performed.
1.11 immunoblotting and immunoprecipitation
Immunoblotting was performed. Briefly, cells were lysed with RIPA lysis buffer supplemented with PMSF (Solarbio, catalogue P0100) and protease inhibitor cocktail (Solarbio, catalogue P1260). After centrifugation, cell lysates were collected, boiled, and subjected to SDS-PAGE, and transferred to Nitrocellulose (NC) filters for immunoblotting. For immunoprecipitation analysis, cells were split on ice for 15 min using IP buffer (50 mM Tris-HCl,150mM NaCl,1.0%NP-40) containing a protease inhibitor cocktail. The supernatant was collected and immunoprecipitated overnight at 4℃with agarose conjugated anti-Flag-Tag antibody (abmart, catalogue M20018). Immunoprecipitates were washed three times with IP buffer, boiled in SDS sample buffer, and analyzed by immunoblotting with the indicated antibodies.
1.12 immunohistochemistry
Ovarian tissue collected from control donors or OV patients was Formalin Fixed and Paraffin Embedded (FFPE). FFPE ovarian samples were sectioned and mounted on polylysine coated slides. The slices were alkylated and rehydrated with xylene and graded concentrations of ethanol, respectively. Antigen recovery was then accomplished by boiling the sections in 10mM citrate buffer (Solarbio, catalog C1010) for 20 minutes at 100deg.C. Then, a universal two-step assay kit (ZSGB bio, catalog PV-9000) was used according to the IHC analysis instructions of the manufacturer. In detail, endogenous peroxidase activity was blocked and diluted ULK1 antibody (Boster, catalog A00584-1) (1:250) was incubated with the sections overnight at 4 ℃. The reaction enhancer was used for 20 minutes before the Diaminobenzidine (DAB) (ZSBB bio, ZLI-9018) was detected.
1.13 ovarian cancer xenograft mouse model
Female BALB/c athymic nude mice of 6 weeks old were obtained from Hua Fukang biosciences and bred under pathogen-free conditions. To establish a subcutaneous xenograft model, OVCAR3 and a2780 cells (5×106/mouse) were subcutaneously injected to the right side of nude mice in 100ul phosphate buffered saline. The size of xenograft tumors produced by MIR937 wild-type or genome-deleted OV cells was monitored every three days after the first appearance. For rescue experiments, 4X 106OVCAR3 or A2780 cells in PBS were subcutaneously injected and agoniR NC, agoniR-937-5 p and agoniR-9 37-3p (riboBio) were directly injected into the implanted tumors 5 times per 4 days in 20 μl PBS at a dose of 1 nmol. Tumor volume (V) was monitored by measuring length (L) and width (W) with vernier calipers and calculated with the formula V (cm 3) =w2×l/2. Finally, mice were euthanized, all tumors excised and weighed. To establish an intraperitoneal xenograft model, MIR937+/+, MIR937+/-, MIR937-/-OV cells were transfected with pCDNA3.1-Luc plasmid and screened with G418 for three days, single cell proliferation with luciferase activity. 5X 106 luciferase labelled OVCAR3 or A2780 cells were injected intraperitoneally and the in vivo bioluminescence signal was collected by intraperitoneal injection of D-luciferin (150 mg/kg) (Beyotime, catalog ST 196) by an animal imager (PerkinElmer, IVIS Lumina XRMS III) on days 7, 14 and 21 post inoculation. All animal experiments were performed according to the guidelines for care and utilization of laboratory animals (university of Shandong traditional Chinese medicine) and were approved by the institutional animal care and use Committee of Shandong traditional Chinese medicine.
1.14 quantitative proteomic analysis
Proteomic analysis was performed by Jingjie PTM BioLabs (Hangzhou, china). Briefly, 4D label-free proteomic analysis was performed following standard procedures: protein preparation, trypsin digestion, HPLC fractionation, LC-MS/MS analysis and bioinformatic analysis.
1.15 statistics
All experiments were repeated at least three times unless otherwise indicated. Values are expressed as mean ± standard deviation. Statistical differences between the two groups were assessed by the two-tailed Student t test. For the multiple group comparison, statistical significance was assessed by one-way analysis of variance (ANOVA) using GraphPad Prism 9.0 software. For correlation analysis, pearson correlation is used to calculate the r-value and p-value. P.ltoreq.0.05 is considered statistically significant.
1.16 approval of study
The collected human ovarian samples are approved by the ethical committee of the affiliated cancer hospital of the first medical university in Shandong, and conform to all relevant ethical specifications (2019-2005). All animal procedures were performed according to the protocol approved by IACUC, university of traditional Chinese medicine, shandong.
2. Experimental results
2.1MIR937 amplified in HGSOC patients and correlated with increased MIR937 expression
Changes in somatic gene Copy Number (CNA) frequently occur in the whole genome of HGSOC patients, suggesting that the CNA gene may be involved in pathogenesis of HGSOC. Full genome association studies (GWAS) have been performed and revealed that the 8q24 genomic fragment is the susceptibility locus for HGSOC patients. Furthermore, 8q24.3 is one of the most significant fragments amplified in the entire genome, including seventy five typical genes, in this locus. However, most genes located in HGSOC have not been functionally clarified. Micrornas (mirs) are small and short in genomic sequence, and we are most interested in how genomic changes in these small fragments affect the progression of HGSOC. Impressive was that there were three MIRs located at 8q24.3 loci, miR937, miR939 and miR661 (fig. 1A). First, we examined the absolute expression levels of three MIRs in HGSOC patients from the cancer genomic map (TCGA) database, and found that miR937 expression was relatively high, while miR939 and miR661 expression was relatively low (fig. 1B). We also examined the expression of these mirs in all ovarian cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) database and obtained consistent results. Therefore, we selected MIR937 for further study. We screened the amplification frequency of MIR937 in different cancer types by the TCGA database system, and found the MIR937 with the highest amplification frequency in HGSOC patients (FIG. 1C).
To further demonstrate the clinical significance of MIR937, we first performed a correlation analysis between DNA copy number and MIR937 expression in HGSOC patients and cancer cell lines, finding that MIR937 RNA levels correlated positively with their DNA copy numbers (fig. 1, D and E). Meanwhile, if patients or OV cell lines are grouped according to their CNA status of MIR937, it can also be found that the abundance of MIR937 transcripts increases significantly upon their expansion. To verify the correlation, we collected HGSOC patient samples and verified 7 of 45 samples amplified with MIR937 by Taqman qPCR analysis, which showed significantly higher expression of MIR937 (fig. 1F). The data indicate that MIR937 upregulation is the primary outcome of MIR937 amplification. Furthermore, we performed a Gene Ontology (GO) enrichment analysis via TCGA database, and found that the copy number and expression of MIR937 correlated positively with the GO terms "positive regulation of cell population proliferation" (fig. 1, g and H) and "negative regulation of programmed cell death". Taken together, our findings indicate that miR937 genomic amplification is the primary cause of its upregulation and thus may exacerbate its pathological impact in HGSOC development.
2.2MIR937 gene deletion inhibits ovarian cancer cell proliferation
To investigate the potential effect of MIR937 on OV progression, we designed a CRISPR-Cas9 based MIR937 knockout strategy in OVCAR3 and a2780 cells (fig. 2A). In detail, two sgrnas were used on both sides of the MIR937 genomic locus without affecting adjacent exons encoded by the SCRIB. Single cells with MIR937 knockouts were screened and verified by genomic DNA PCR amplification and sanger sequencing. Homozygous and heterozygous deletions of MIR937 cells were selected (MIR 937-/-, MIR 937+/-) (FIG. 2B). We first monitored the genomic loss of MIR937 to OV proliferation by an in vitro CCK8 assay and realized that MIR937 +/-grew slower than MIR937+/+ cells, whereas MIR 937-/-prolonged the inhibitory effect on OVCAR3 and A2780 cells (FIGS. 2, C and D). Furthermore, colony formation assays showed that the ability of the cells to form colonies gradually decreased with loss of the MIR937 gene. Statistical results also demonstrate a significant loss of colony forming ability from MIR937 knockout. An EdU incorporation assay was also performed, which further demonstrated that MIR937 lacks proliferation characteristics that inhibit OVCAR3 and a2780 cells. Statistics show that for OVCAR3 and a2780 cells, edU positive cells were reduced to 78% or 70% by heterozygous deletion of MIR937, but to 42% or 44% by homozygous deletion of MIR937, respectively. The in vitro data highly indicate that the extent of MIR937 deficiency correlates with the extent of inhibition of OV cell proliferation.
To further verify the function of MIR937, we first injected MIR937+/+, MIR937+/-, MIR 937-/-cells subcutaneously into the right flank of nude mice to establish xenograft models. When the tumors grew to the appropriate size, mice were sacrificed to observe the tumor growth differences for the different groups (fig. 2, e and F). By monitoring the growth rate and weight of xenograft tumors, we consistently found that MIR 937-/-cells exhibited more restricted cell growth than MIR937 +/-cells (fig. 2, g-J). Next, we generated luciferase-labeled OVCAR3 and a2780 cells with MIR937 deficiency and injected these cells intraperitoneally to mimic the natural environment of OV tumor growth. The consistently captured in vivo luciferase signal indicated that MIR937 deficiency could severely inhibit proliferation of OV cells (figure 2K). Overall, these data suggest that MIR937 may play a carcinogenic role in the development of OV.
miR-937-5p is responsible for MIR937 function deficiency in ovarian cancer cells
MIR937 was transcribed as a precursor, eventually forming two mature products, miR-937-5p and miR-937-3p. Whether miR-937-5p or miR-937-3p is responsible for the carcinogenesis of MIR937 remains to be elucidated. Thus, first, mimetics or inhibitors of these two miRs were used to overexpress or inhibit the expression of miR-937-5p and miR-937-3p in OVCAR3 and A2780 cells. By CCK8 analysis, we found that overexpression of miR-937-5p significantly increased proliferation rates of both cell lines, whereas miR-937-3p had no effect (FIGS. 3, A and B). Consistently, inhibitors of miR-937-5p, but not miR-937-3p, could inhibit proliferation of OVCAR3 and a2780 cells (fig. 3, c and D). In addition, miR-937-5p can also improve proliferation capacity of TOV21G and SKOV3 cells. Meanwhile, edU determination is also carried out, and the result shows that the overexpression of miR-937-5p can obviously increase the proportion of EdU positive cells. It is highly suggested that miR-937-5p is the major product responsible for MIR937 carcinogenesis.
We then overexpressed miR-937-5p or miR-937-3p in MIR 937-/-cells to further investigate which products could rescue OV cell growth arrest caused by MIR937 deficiency. We found that only transfection of the miR-937-5p mimetic can rescue the growth capacity of MIR 937-/-cells, approaching that of MIR937+/+ cells (FIGS. 3, E and F). Finally, we established subcutaneous xenograft tumors by MIR937+/+ and MIR 937-/-cells of OVCAR3 and A2780, into which were injected five times per three days, each of agoniR-937-5 p and agoniR-937-3 p (FIG. 3G). On day 25, mice were sacrificed and xenograft tumors were measured for size and weight to confirm the effect on both mirs. MIR 937-/-xenograft size increased significantly when agoniR-937-5 p was injected instead of agoniR-937-3 p (FIGS. 3, H and J). In addition, agoniR-937-5 p rescued the proliferation rate and weight of xenograft tumors of MIR937-/-OVCAR3 and A2780 cells (FIG. 3, J to M). Taken together, our data confirm that miR-937-5p is a product of the MIR937 gene, with carcinogenesis.
Tumor suppressor gene of miR-937-5p direct targeting FBXO16
Since miR-937-5p is functional in promoting tumor growth, we hypothesize that miR-937-5p might target some of the Tumor Suppressor Genes (TSGs) in OV. Through literature screening we selected 45 OV TSGs for further study. We also used the TargetScan database to obtain a list of potential miR-937-5p targets. When the genes in the two lists overlap, 11 TSGs were found to be likely targeted by miR-937-5p (FIG. 4A). To verify whether these genes were indeed targeted by miR-937-5p, we transfected miR-937-5p mimics in OVCAR3 cells, quantified the expression of the indicator gene, and found that miR-937-5g only reduced expression of FBXO16 (fig. 4B). In contrast, the lack of MIR937 in OVCAR3 cells greatly increased mRNA levels of FBXO 16. In addition, a binding site was found in the 3' untranslated region (UTR) of FBXO16, and luciferase reporter gene analysis showed that miR-937-5p inhibited luciferase activity of WT 3' UTR, but did not affect mutant 3' UTR (FIGS. 4, C and D). To directly test whether binding of miR-937-5p to FBXO 16' utr inhibited its protein translation, we transfected mimetics and inhibitors and tested the effect of miR-937-5p on FBXO16 protein levels in OVCAR3 and a2780 cells. Studies showed that miR-937-5p mimic was significantly reduced, while the inhibitor enhanced expression of FBXO16 (fig. 4E). Consistently, the loss of MIR937 was combined with an increase in FBXO16 protein. In addition, we have submitted samples of xenograft tumors for western blot analysis and found that agonir-937-5 p can reduce FBXO16 protein levels enhanced by MIR937 deletion. Taken together, these data demonstrate that FBXO16 is a true target for miR-937-5p in OV cancer cells.
Furthermore, the tumor-inhibiting effect of FBXO16 on ovarian cancer has been further demonstrated in our study model. We silenced expression of FBXO16 by three small interfering RNAs (sirnas) and examined the effect of FBXO16 on cell proliferation. By CCK8 analysis, we found that all siRNA targeting FBXO16 could significantly enhance the proliferative activity of OVCAR3 cells. In addition, two short hairpin RNAs (shrnas) targeting FBXO16 were also constructed to generate stable cell lines with FBXO16 silencing. It was also revealed that stable knockdown of FBXO16 can continuously increase proliferation rate of OVCAR3 cells. For in vivo assays, both shRNA can significantly increase tumor size and weight by subcutaneous xenograft models (figures 4,F and G). Taken together, our data indicate that miR-937-5p targets FBXO16, thereby reducing its tumor suppression and exerting carcinogenesis.
Negative regulation of autophagy of ovarian cancer by FBXO16
To demonstrate the molecular mechanism by which MIR937/FBXO16 regulates ovarian cancer proliferation, we first tested the effect of MIR937 deletion on hnRNPL, the previously reported FBXO16 target. The results show that there is no significant change in hnRNPL in MIR 937-/-compared to MIR937+/+ cells. This suggests that MIR937 may exert its carcinogenic effect through the unique role of FBXO 16. Thus, we sought to reveal a new target for FBXO16 in ovarian cancer. A 4D label-free quantitative proteomic analysis was performed to screen for biological processes that may be affected in OVCAR3 cells stably knocked down with FBXO 16. Gene Ontology (GO) enrichment analysis of the differentially expressed proteins showed that cell cycle and autophagy-related pathways were significantly up-regulated after FBXO16 silencing, whereas apoptosis and amino acid metabolism-related processes were down-regulated (fig. 5A). Since autophagy is an important process for regulating cell proliferation, cell death and cell metabolism, we interrogate whether FBXO16 affects the expression of autophagy-critical regulatory proteins. By western blot analysis, we found that under normal and hypoxic conditions, FBXO16 knockdown increased protein levels of ULK1, but not ATG7, ATG13 or Beclin-1 (fig. 5B). Consistently, stable overexpression of FBXO16 showed a significant decrease in ULK1 protein (fig. 5C). Furthermore, in a2780 and TOV21G cells, the results of FBXO16 targeting ULK1 only were further confirmed by transient knockdown of FBXO16 by three sirnas.
FBXO16 interacts with ULK1 and promotes polyubiquitination of its K48 linkage
FBXO16 always acts as an adaptor, recruiting substrate to SKP1 cullin 1 (CUL 1) -RBX1 complex for its ubiquitination and subsequent degradation (fig. 6A). Therefore, we examined whether FBXO16 can interact with ULK1, potentially affecting its protein stability. By co-immunoprecipitation analysis, we found that the ectopic expressed Flag-tagged FBXO16 interacted strongly with Myc-tagged ULK1 (fig. 6B). Meanwhile, flag-labeled ULK1 may also bind to Myc-labeled FBXO16 (fig. 6C). To determine the binding requirements of the FBXO16-ULK1 complex, we generated three and four deletion constructs of FBXO16 and ULK1, respectively, and performed a co-IP assay. The results indicate that the C-terminal domains (CTDs) of FBXO16 and ULK1 are responsible for their interactions (fig. 6, D and E). We further detected interactions of ULK1 with key components of SCF complex by co-IP analysis and revealed that Flag-labeled ULK1 could bind to Myc-labeled CUL1, SKP1 and RBX 1. This highly suggests that ULK1 is recruited to the SCF complex by FBXO16.
Furthermore, we co-transfected HEK293T cells with Flag-ULK1, myc-FBXO16 and HA-Ubs (WT, K48 and K63). Cell lysates were immunoprecipitated with Flag antibodies and the polyubiquitinated ULK1 was detected by western blotting. The results indicate that FBXO16 can significantly increase K48 of ULK1, rather than K63 linked polyubiquitination (fig. 6F). On the other hand, we transfected Flag-ULK1, myc-FBXO16 and HA-Ubs (WT, K48R) into HEK293T cells and performed co-IP analysis of cell lysates. The results indicate that lysine-48 (K-48) mutation of Ub to arginine (R) blocks FBXO 16-mediated ubiquitination of ULK1, which further verifies that fbxo16 promotes K48-linked polyubiquitination of ULK1 (fig. 6G). To further demonstrate that FBXO16 promotes ULK1 degradation by ubiquitination, we transiently knockdown FBXO16 and treat cells with Cycloheximide (CHX) to examine the stability of ULK1 protein affected by FBXO16. The data show that FBXO16 knockdown increases ULK1 protein levels and slows their degradation. Taken together, our data demonstrate that FBXO16 accelerates degradation of ULK1 by enhancing polyubiquitination of its K48 linkage.
Clinical MIR937 amplification associated with FBXO16 and ULK1 expression
We then investigated whether MIR937 amplification in OV works through the FBXO16/ULK1 axis. First, ULK1 and autophagy levels (expressed by p62 and LC3 II/I) were detected by western blotting in MIR937 deleted OVCAR3 and a2780 cells, which showed that ULK1 and autophagy levels were slightly down-regulated in MIR937 +/-cells and significantly reduced in MIR 937-/-cells compared to MIR937+/+ cells (fig. 7A). To further elucidate the dependence of miR-937-5p in inhibiting autophagy due to miR937 deletion, expression of miR-937-5p was enhanced and inhibited by mimetics and inhibitors, and we consistently found that miR-937-5g overexpression promoted autophagy, while its knockout inhibited autophagy. Furthermore, we examined the expression of ULK1 in xenograft tumors derived from MIR937-/-OVCAR3 and A2780 cells, which were interfered with by agoniR-937-5 p or 3 p. The results show that agoniR-937-5 p restored ULK1 expression by deletion of MIR 937. In addition, we also tested whether the effect of miR-937-5p on ULK1 and autophagy was through its down-regulation of FBXO 16. Thus, OVCAR3 and a2780 cells were first transfected with miR-937-5p inhibitor to transiently increase FBXO16, and then with siRNA targeting FBXO16 to detect whether FBXO16 knockdown could rescue ULK1 and autophagy, which had been inhibited by FBXO16 elevation mediated by miR-937-5p inhibition. The results show that inhibition of ULK1 by miR-937-5p inhibitor and autophagy can be improved by FBXO16 knockdown. Thus, we conclude that MIR937 regulates OV cancer progression through MIR-937-5p/FBXO16/ULK 1.
Next, we clinically examined whether MIR937 amplification was associated with FBXO16 and ULK1 in OV patients. In our study, 7 of all enrolled HGSOC patients (n=45) received verification of MIR937 amplification. And comparing representative data of FBXO16 and ULK1 protein expression in MIR937 amplified patients to normal or MIR937 diploid patients. The results showed that FBXO16 expression was relatively high in normal ovarian tissue, slightly reduced in MIR937 diploid OV samples, and significantly reduced in MIR927 amplified patients (fig. 7B). In contrast, ULK1 expression was slightly increased in MIR937 diploid OV samples, but was further increased in MIR937 amplified patients (fig. 7B). Immunohistochemical (IHC) assays also confirmed the results, i.e., MIR937 amplification enhanced ULK1 abundance more than MIR937 diploids in HGSOC patients (fig. 7C). Finally, we review the clinical significance of FBXO16 down-regulation or ULK1 up-regulation in HGSOC patients. We used the Kaplan plotter database to study the correlation of FBXO16 and ULK1 with HGSOC patient prognosis. Separately, ULK1 high expression predicts poor survival in GSE3149 dataset, whereas FBXO16 high expression favors survival in GSE63885 dataset in HGSOC patients. Furthermore, survival analysis of GSE26193 dataset showed the same results (fig. 7,D and E). When patients of GSE26193 dataset were grouped according to FBXO16 and ULK1 expression, we can see that survival for ULK1 low expressing/FBXO 16 high expressing patients was greatly improved, while survival for ULK1 high expressing/FBXO 16 low expressing patients was the worst (fig. 7F). Taken together, our study showed that MIR937 amplification-induced high ULK1 expression and low FBXO16 expression simultaneously resulted in malignant progression in HGSOC patients.
The invention is not a matter of the known technology.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (10)
1. Use of a reagent for detecting MIR 937-related biological element expression in the preparation of a product for screening, (co) diagnosing, detecting, monitoring or predicting the progression of ovarian cancer;
wherein the MIR937 related biological element expression conditions include, but are not limited to, changes in MIR937 gene copy number, changes in miR-937-5p expression levels, changes in FBXO16 expression levels, and changes in ULK1 expression levels.
2. The use of claim 1, wherein the ovarian cancer is high grade serous ovarian cancer; the product is a kit, a detection device or detection equipment.
3. A system for screening, (aiding) diagnosis, detection, monitoring or prediction of the progression of ovarian cancer, the system comprising:
an acquisition unit configured to: acquiring the expression condition of MIR937 related biological elements of a sample to be tested of a subject;
An evaluation unit configured to: judging the disease condition of the subject according to the expression condition of the biological element related to the MIR937 of the subject obtained by the obtaining unit;
wherein the MIR937 related biological element expression conditions include, but are not limited to, changes in MIR937 gene copy number, changes in miR-937-5p expression levels, changes in FBXO16 expression levels, and changes in ULK1 expression levels.
4. The system of claim 3, wherein the ovarian cancer is high grade serous ovarian cancer;
the sample to be tested is an ovarian (cancerous) tissue or cell.
Application of MIR937 related biological element as target in preparation and/or screening of ovarian cancer drugs; wherein the MIR937 related biological element comprises at least one of MIR937, miR-937-3p, FBXO16 and ULK 1.
6. Use of a substance that inhibits MIR937 expression or decreases its activity in any one or more of:
a) Inhibiting miR-937-5p expression or preparing a product for inhibiting miR-937-5p expression;
b) Inhibiting ULK1 expression or preparing a product that inhibits ULK1 expression;
c) Promoting FBXO16 expression or preparing a product that promotes FBXO16 expression;
d) Inhibiting autophagy of ovarian cancer cells or preparing a product for inhibiting autophagy of ovarian cancer cells;
e) Inhibiting the proliferation of ovarian cancer cells or preparing a product for inhibiting the proliferation of ovarian cancer cells;
f) Inhibiting ovarian cancer growth or preparing a product for inhibiting ovarian cancer growth;
g) Products for treating ovarian cancer.
7. The use of claim 6, wherein the agent that inhibits or reduces MIR937 expression comprises an RNA interfering molecule or antisense oligonucleotide, a small molecule inhibitor, shRNA, siRNA directed against MIR937, an agent that performs a lentiviral infection or gene knockout (including agents required for knocking out MIR937 based on CRISPR-Cas9 technology, further including sgRNA).
8. The use of claim 6, wherein the ovarian cancer is high grade serous ovarian cancer.
9. The use according to claim 6, wherein the product is a pharmaceutical or experimental agent for use in basic research.
10. The use of claim 6, wherein when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
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