Application of circPOK in the treatment of metastatic breast cancer

Abstract

The present invention discloses the application of circPOK in the treatment of metastatic breast cancer. The inventors found that the expression level of circPOK in breast cancer tissue was lower than that in normal tissue, and low expression of circPOK was closely related to the poor prognosis of breast cancer patients, suggesting that circRNAcircPOK may be an important regulatory factor in inhibiting the occurrence and development of breast cancer. The inventors conducted an in vitro cell functional study on circRNAcircPOK and found that high expression of circPOK can inhibit the migration ability of breast cancer cell lines. After overexpression of circPOK, the migration ability of MDA-MB-231 and HCC1806 breast cancer cell lines was weakened, and in vivo experiments confirmed that circPOK can effectively inhibit the formation of lung metastases of breast cancer in nude mice. It can be seen that circPOK plays a role in inhibiting the occurrence and development of tumors in breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of circPOK in treating metastatic breast cancer. Background Art

[0002] The global incidence of breast cancer accounts for about 30% of female malignant tumors. The incidence rate worldwide ranges from 27 / 100,000 (Africa and East Asia) to 97 / 100,000 (North America). The mortality rate to incidence ratio is 15%, which seriously threatens women's health. [1] Tumor metastasis is the main cause of death from breast cancer. The median survival after breast cancer metastasis is less than two years. How to treat metastatic breast cancer is an urgent issue to be solved. [2] .

[0003] There is currently no report of an effective cure for metastatic breast cancer, and most patients with metastatic breast cancer will have to receive treatment for most of the rest of their lives. [3] , and the treatment dose is usually the maximum tolerated dose, which produces serious side effects [4, 5] , which adversely affects the patient's quality of life [6, 7] , leading to delayed treatment [8] , and decreased compliance due to increased toxicity and intolerance [9, 10] , making it impossible to continue treatment.

[0004] Circular RNAs (CircRNAs) discovered in plant viruses for the first time

[11] CircRNAs were subsequently discovered in human cells.

[12] CircRNAs are generated by covalent bonding between the 3' and 5' ends of transcripts.

[13] CircRNAs can regulate many important physiological processes, including aging, insulin secretion, tissue remodeling, and the pathogenesis of some diseases such as malignant tumors.

[14] CircRNAs exert their regulatory effects through their ability to act as molecular sponges for miRNAs, their regulatory roles in transcription or splicing, and their interactions with RNA-binding proteins.

[14] CircRNAs can indirectly affect cancer-related pathways and the malignant behavior of breast cancer cells by regulating miRNAs

[15] In addition, similar to other long noncoding RNAs, CircRNAs may regulate the proliferation and invasion of breast cancer

[16] CircRNAs are mainly found in the cytoplasm, but a small part of them are also found in the nucleus.

[17] About 10% of expressed genes can produce CircRNAs, so CircRNAs are widely present in living organisms.

[18] Although traditionally considered noncoding RNAs, most circRNAs contain exons and are partially associated with ribosomes.

[0005] CircPOK acts independently as a non-coding oncogene RNA in stromal tumors. CircPOK further regulates pro-proliferative and pro-angiogenic factors by co-activating the ILF2 / 3 complex, thereby promoting the occurrence and development of stromal tumors.

[19] However, the role and mechanism of CircPOK in breast cancer need to be further explored.

[0006] References:

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[0008] [2]JIN L, HAN B, SIEGEL E, et al. Breast cancer lung metastasis: Molecular biology and therapeutic implications [J]. Cancer Biol Ther, 2018,19(10): 858-68.

[0009] [3]JONES S E. Metastatic breast cancer: the treatment challenge[J]. Clin Breast Cancer, 2008, 8(3): 224-33.

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[0011] [5]MINASIAN L, ROSEN O, AUCLAIR D, et al. Optimizing dosingofoncology drugs [J]. Clin PharmacolTher, 2014, 96(5): 572-9.

[0012] [6]WOOD R, MITRA D, DE COURCY J, et al. Patient-reported QualityofLife and Treatment Satisfaction in Patients With HR(+) / HER2(-) Advanced / Metastatic Breast Cancer [J]. Clin Ther, 2017, 39(8): 1719-28.

[0013] [7]KUCHUK I, BOUGANIM N, BEUSTERIEN K, et al. Preference weightsforchemotherapy side effects from the perspective of women with breast cancer[J]. Breast Cancer Res Treat, 2013, 142(1): 101-7.

[0014] [8]CARDOSO F, DI L A, LOHRISCH C, et al. Second and subsequentlinesof chemotherapy for metastatic breast cancer: what did we learn in the lasttwo decades [J]. Ann Oncol, 2002, 13(2): 197-207.

[0015] [9]SHAH M, RAHMAN A, THEORET M R, et al. The Drug-Dosing ConundruminOncology - When Less Is More [J]. N Engl J Med, 2021, 385(16): 1445-7.

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[11] SANGER H L, KLOTZ G, RIESNER D,et al. Viroids are single-strandedcovalently closed circular RNA molecules existing as highly base-paired rod-like structures [J]. Proc Natl Acad Sci U S A, 1976, 73(11): 3852-6.

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[12] HSU M T, COCA-PRADOS M. Electronmicroscopic evidence for thecircular form of RNA in the cytoplasm of eukaryotic cells [J]. Nature, 1979,280(5720): 339-40.

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[13] QU S, LIU Z, YANG X, et al. Theemerging functions and roles ofcircular RNAs in cancer [J]. Cancer Lett, 2018,414: 301-9.

[0020]

[14] QU S, ZHONG Y, SHANG R, et al.The emerging landscape of circularRNA in life processes [J]. RNA Biol, 2017,14(8): 992-9.

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[15] ABOGHASEMI M, TEHRANI SS,YOUSEFI T, et al. MicroRNAs in breastcancer: Roles, functions, and mechanism of actions [J]. J Cell Physiol, 2020,235(6): 5008-29.

[0022]

[16] ABOGHASEMI M, TEHRANI SS,YOUSEFI T, et al. Critical roles of long noncoding RNAs in breast cancer [J]. JCell Physiol, 2020, 235(6): 5059-71.

[0023]

[17] MEMCZAK S, JENS M, ELEFSINIOTI A, et al. Circular RNAs are a largeclass of animal RNAs with regulatory potency [J]. Nature, 2013, 495(7441):333-8.

[0024]

[18] SALZMAN J, GAWAD C, WANG PL, etal. Circular RNAs are thepredominant transcript isoform from hundreds of human genes in diverse celltypes [J]. PLoS One, 2012, 7(2): e30733.

[0025]

[19] GUARNERIO J, ZHANG Y, CHELONI G,et al. Intragenic antagonisticroles of protein and circRNA in tumorigenesis [J]. Cell Res, 2019, 29(8):628-40.. Summary of the invention

[0026] The object of the present invention is to overcome at least one deficiency of the prior art and provide a use of circPOK in the treatment of metastatic breast cancer.

[0027] The technical solution adopted by the present invention is:

[0028] The first aspect of the present invention provides:

[0029] Application of reagents for quantifying the expression of circPOK in samples in the preparation of breast cancer prognosis detection reagents.

[0030] In some application examples, the reagent for quantifying the expression level of circPOK in the sample is selected from circRNA tissue chip, immunofluorescence in situ hybridization reagent or PCR reagent.

[0031] In some application examples, the sample is patient serum and / or tissue.

[0032] The second aspect of the present invention provides:

[0033] A breast cancer prognosis analysis system, comprising:

[0034] A circRNA expression detection device, used to detect the expression level of circPOK in a sample;

[0035] The prognostic analysis device determines the prognosis of breast cancer based on the expression level of circPOK;

[0036] The result output device is used to output the result analyzed by the prognosis analysis device.

[0037] In some examples of breast cancer prognosis analysis systems, the circRNA expression detection device is selected from at least one of a circRNA tissue chip, an immunofluorescence in situ hybridization analysis device, and a PCR detection device.

[0038] In some examples of the breast cancer prognosis analysis system, the sample is patient serum and / or tissue.

[0039] In some examples of breast cancer prognostic analysis systems, when the expression level of circPOK increases significantly, it is judged that the risk of breast cancer metastasis is low and the prognosis of breast cancer patients is good.

[0040] The third aspect of the present invention provides:

[0041] Application of a circPOK expression promoter in the preparation of a preparation for the treatment or adjuvant treatment of metastatic breast cancer.

[0042] In some application examples, the promoter is selected from a circPOK promoter agonist, a plasmid expressing circPOK mRNA, and a vector loaded with circPOK mRNA.

[0043] The beneficial effects of the present invention are:

[0044] The inventors detected the expression of circRNAcircPOK in 15 pairs of breast cancer clinical tissue samples and corresponding normal breast tissues by qRT-PCR technology. The results showed that the expression level of circPOK in breast cancer tissue was lower than that in normal tissue. The expression of circPOK was verified by human breast cancer tissue microarray, which confirmed that circRNAcircPOK was not only lowly expressed in breast cancer tissue, but also that low expression of circPOK was closely related to the poor prognosis of breast cancer patients, suggesting that circRNAcircPOK may be an important regulatory factor in inhibiting the occurrence and development of breast cancer. Then, the inventors conducted an in vitro cell functional study on circRNAcircPOK. Further experimental studies showed that compared with the control group cell line, high expression of circPOK could inhibit the migration ability of breast cancer cell lines. After overexpressing circPOK, the migration ability of MDA-MB-231 and HCC1806 breast cancer cell lines was weakened. In vivo experiments confirmed that circPOK could effectively inhibit the formation of lung metastases of breast cancer in nude mice. It can be seen that circPOK plays a role in inhibiting the occurrence and development of tumors in breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the expression level of circPOK in breast cancer cells and breast cancer tissues and the results related to prognosis.

[0046] Figure 2 This is the result of the effects of circPOK on the invasion and migration of breast cancer cells in vivo and in vitro.

[0047] Figure 3 and Figure 4 These are the experimental results that circPOK exosomes inhibit breast cancer invasion and migration in vivo and in vitro. DETAILED DESCRIPTION

[0048] The first aspect of the present invention provides:

[0049] Application of reagents for quantifying the expression of circPOK in samples in the preparation of breast cancer prognosis detection reagents.

[0050] The reagent for quantifying the expression amount of circPOK in a sample can be various known reagents. In some application examples, the reagent for quantifying the expression amount of circPOK in a sample is selected from circRNA tissue chip, immunofluorescence in situ hybridization reagent or PCR reagent.

[0051] In some application examples, the sample is patient serum and / or tissue.

[0052] The second aspect of the present invention provides:

[0053] A breast cancer prognosis analysis system, comprising:

[0054] A circRNA expression detection device, used to detect the expression level of circPOK in a sample;

[0055] The prognostic analysis device determines the prognosis of breast cancer based on the expression level of circPOK;

[0056] The result output device is used to output the result analyzed by the prognosis analysis device.

[0057] In some examples of breast cancer prognosis analysis systems, the circRNA expression detection device is selected from at least one of a circRNA tissue chip, an immunofluorescence in situ hybridization analysis device, and a PCR detection device.

[0058] In some examples of the breast cancer prognosis analysis system, the sample is patient serum and / or tissue.

[0059] In some examples of breast cancer prognostic analysis systems, when the expression level of circPOK increases significantly, it is judged that the risk of breast cancer metastasis is low and the prognosis of breast cancer patients is good.

[0060] The third aspect of the present invention provides:

[0061] Application of a circPOK expression promoter in the preparation of a preparation for the treatment or adjuvant treatment of metastatic breast cancer.

[0062] In some application examples, the promoter is selected from a circPOK promoter agonist, a plasmid expressing circPOK mRNA, and a vector loaded with circPOK mRNA.

[0063] The technical solution of the present invention is further explained below in conjunction with experimental data.

[0064] Studies have shown that circPOK is lowly expressed in breast cancer tissues and most breast cancer cell lines:

[0065] First, qRT-PCR was used to detect the expression of 2 breast cancer cells and 1 normal breast cell line, and it was found that circPOK was lowly expressed in breast cancer cells ( Figure 1 B). The inventors collected fresh breast cancer samples from the Department of Breast and Thyroid Surgery of the First Affiliated Hospital of University of South China, and detected the expression of circPOK in 15 pairs of breast cancer clinical tissue samples and corresponding normal breast tissues by qRT-PCR. The results showed that the expression level of circPOK in breast cancer tissues was lower than that in normal tissues ( Figure 1 C), the inventors also obtained the same result by qRT-PCR detection of breast cancer samples collected from the Affiliated Cancer Hospital of Sun Yat-sen University ( Figure 1 D).

[0066] Studies have shown that circPOK inhibits breast cancer metastasis:

[0067] In order to explore the clinical significance of circPOK in breast cancer, the inventors collected breast cancer samples from the Department of Pathology of the First Affiliated Hospital of the University of South China and produced human breast cancer tissue chips at Sevier Biotech. Fluorescence in situ hybridization was performed on the human breast cancer tissue chips obtained above, and the results showed that there was a significant difference in the expression of circPOK in breast cancer tissue and adjacent tissue ( Figure 1 E). Next, the inventors analyzed the correlation between circPOK expression level and clinical pathological characteristics of breast cancer patients. The results showed that circPOK expression level was significantly negatively correlated with tumor size and distant metastasis of breast cancer (P < 0.05) (Table 1).

[0068] Table 1

[0069]

[0070] **Significant at the 0.01 level (two-sided test)

[0071] *Significant at the 0.05 level (two-sided test).

[0072] Studies have shown that circPOK inhibits breast cancer cell invasion and migration in breast cancer:

[0073] Given that circPOK is lowly expressed in breast cancer tissues and is closely related to the prognosis of breast cancer patients. To study the role of circPOK in the development of breast cancer, the inventors constructed a circPOK stably overexpressing cell line. CircPOK was stably overexpressed in MCF-7, MDA-MB-231 and HCC1806 breast cancer cell lines, and the effects of overexpression of circPOK on the invasion and migration ability of the two breast cancer cancer cells were detected by scratch and Transwell chamber invasion and migration experiments. It was found that overexpression of circPOK could significantly inhibit the invasion and migration of breast cancer cells ( Figure 2 AB). The above experimental results show that overexpression of circPOK can inhibit the invasion and migration ability of breast cancer cells.

[0074] To further determine whether circPOK also has the effect of inhibiting breast cancer cell invasion and migration in vivo, the inventors constructed a nude mouse metastatic tumor model by injecting circPOK-overexpressing MDA-MB-231 cells and control cells through the tail vein, and set up a control group. Compared with the control group, the circPOK overexpression group can significantly inhibit the formation of metastatic nodules in the lungs of nude mice ( Figure 2CD), we also confirmed that the lung nodules were breast cancer metastases by HE staining of lung nodules. The above experimental results show that circPOK overexpression can effectively inhibit the lung metastasis of breast cancer cells.

[0075] The inventors found that circPOK was significantly upregulated in the conditioned medium of MCF-7, MDA-MB-231 and HCC1806 circPOK stably overexpressing cells ( Figure 3 AB), and the invasion and migration abilities of MDA-MB-231 and HCC1806 cells co-cultured with circPOK overexpression conditioned medium were significantly weakened compared with the control group ( Figure 3 C). The inventors extracted exosomes from the conditioned medium and confirmed the successful extraction of exosomes by electron microscopy and Western Blotting (CD9, TSG101, CD63 are exosome markers) ( Figure 3 DE), the inventors found no significant difference in the exosome uptake rate between circPOK and the control group by immunofluorescence detection (DIO is an exosome marker, and DAPI is a cell nucleus-specific stain) ( Figure 3 F), circPOK overexpression exosomes can significantly inhibit the Transwell chamber invasion and migration ability of MDA-MB-231 cells compared with the control group exosomes ( Figure 4 G). To explore the effect of exosome-loaded circPOK on breast cancer metastasis in vivo, the inventors constructed a nude mouse breast cancer lung metastasis model by using MDA-MB-231 cells labeled with the luciferase gene, and injected exosome-loaded circPOK / control exosomes through the tail vein. In vivo imaging of small animals revealed that exosome-loaded circPOK can significantly inhibit breast cancer lung metastasis ( Figure 4 H).

[0076] In summary, the inventors' research found that circPOK is lowly expressed in breast cancer tissues and breast cancer cell lines, and high expression of circPOK is closely related to a better prognosis of patients with metastatic breast cancer. Functional experiments showed that circPOK can inhibit the invasion and migration of breast cancer cells both in vivo and in vitro. Therefore, circPOK can be used as a new therapeutic target for the treatment of metastatic breast cancer.

[0077] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Use of a circPOK expression promoter in the preparation of a preparation for treating or assisting in treating metastatic breast cancer, wherein the promoter is an expression vector that expresses circPOK.

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