Use of circular RNA-circuck2 in treatment of hepatocellular carcinoma

Abstract

The present invention provides a use of a circular RNA (circUCk2) in the treatment of hepatocellular carcinoma. Specifically, the content of circUCk2 in cancer cells is increased by means of a circUCk2 overexpression element or by means of direct addition of circUCk2, thereby improving the sensitivity of hepatocellular carcinoma cells to combined treatment with a tyrosine kinase inhibitor and an EGFR inhibitor. Experiments have shown that under the condition of high circUCK2 expression, the combined treatment exhibits a more potent killing effect on liver cancer cells.

Description

Application of circular RNA-circUCK2 in the treatment of hepatocellular carcinoma

[0001] This application claims priority to Chinese application No. 2024117166370, filed on November 27, 2024, the contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, and more specifically, this invention relates to the use of a circular RNA-circUCK2 in the treatment of hepatocellular carcinoma. Background Technology

[0003] Hepatocellular carcinoma (HCC) is a malignant tumor originating from liver parenchymal cells, accounting for more than 85% of primary liver cancer cases. According to the latest epidemiological statistics, the incidence and mortality rates of HCC remain high globally and in China. Because early-stage HCC often presents with no obvious symptoms or only mild symptoms, and because it progresses rapidly, many HCC patients are already at an advanced stage at the time of diagnosis. Furthermore, the inherent insensitivity of HCC to traditional radiotherapy and chemotherapy leads to poor treatment outcomes. Currently, targeted therapy and immunotherapy have become one of the main treatment methods for patients with advanced HCC.

[0004] Lenvatinib is an oral multi-target tyrosine kinase inhibitor primarily used to treat various types of cancer. It exerts its anti-tumor effect by inhibiting multiple tyrosine kinases associated with tumor growth and metastasis, such as vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and other receptors. Currently, lenvatinib has been approved as a first-line targeted therapy for advanced liver cancer. Compared to the traditional first-line targeted therapy sorafenib, lenvatinib has shown non-inferiority in clinical trials (REFLECT trial) and has demonstrated better performance in objective response rate and progression-free survival, making it suitable for patients with high-risk characteristics such as large tumor burden and vascular invasion. However, individual differences exist in lenvatinib treatment (such as the molecular characteristics and gene mutation types of tumors), and with the large-scale use of lenvatinib, clinical resistance has also emerged.

[0005] A high-throughput screening study based on CRISPR-CAS9 gene editing technology found that EGFR overexpression or EGFR pathway activation is one of the main causes of lenvatinib resistance. A clinical trial (NCT04642547) of lenvatinib combined with an EGFR inhibitor showed that this combination therapy demonstrated good efficacy in more than half of patients with advanced liver cancer who had high EGFR expression. However, some patients did not respond to the combination therapy of lenvatinib and an EGFR inhibitor. Therefore, it is urgent to understand the causes of resistance to combination therapy and to solve the problem of drug resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for improving the sensitivity of liver cancer cells to combined therapy with tyrosine kinase inhibitors and EGFR inhibitors.

[0007] In a first aspect of the invention, there is provided the use of an active ingredient for preparing a formulation or pharmaceutical composition for enhancing the sensitivity of liver cancer cells to combined therapy with a tyrosine kinase inhibitor and an epidermal growth factor receptor (EGFR) inhibitor.

[0008] The active ingredients are selected from the following group:

[0009] (a)circUCK2;

[0010] (b) mRNA expressing circUCK2; and / or

[0011] (c) The vector that expresses circUCK2.

[0012] In another preferred embodiment, circUCK2 is a circular RNA transcribed from the second to third exons of the gene UCK2, and its corresponding DNA sequence is shown in SEQ ID NO1.

[0013] In another preferred embodiment, the active ingredient is a circUCK2 overexpression plasmid.

[0014] In another preferred embodiment, the plasmid includes the target gene as shown in SEQ ID NO1.

[0015] In another preferred embodiment, the plasmid comprises primers as shown in SEQ ID NO2 and SEQ ID NO3.

[0016] In another preferred embodiment, the tyrosine kinase inhibitor is selected from the group consisting of: lenvatinib, sorafenib, regorafenib, donafenib, imatinib, sorafenib, sunitinib, lapatinib, and dasatinib.

[0017] In another preferred embodiment, the EGFR inhibitor is selected from the group consisting of: pelitinib, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, and ametinib.

[0018] In another preferred embodiment, the combination therapy of the tyrosine kinase inhibitor and the epidermal growth factor receptor (EGFR) inhibitor is a combination of lenvatinib and peritinib.

[0019] In another preferred embodiment, the liver cancer cells are SNU398 cells.

[0020] In another preferred embodiment, the liver cancer cells are liver cancer cells that express low, normal, or high levels of circUCK2.

[0021] In another preferred embodiment, normal expression means that the expression level of circUCK2 is the average level of circUCK2 expression in all hepatocellular carcinoma patients.

[0022] In another preferred embodiment, circUCK2 binds to miR-149-5p, thereby relieving miR-149-5p's inhibition of CNIH4. The uninhibited CNIH4 enhances TGFa secretion, thereby activating EGFR.

[0023] In another preferred embodiment, circUCK2 is used as a biomarker to predict the sensitivity of liver cancer patients to combination therapy with tyrosine kinase inhibitors and EGFR inhibitors, guiding clinical medication.

[0024] In another preferred embodiment, the expression of circUCK2 is increased in liver cancer cells, which are more sensitive to combined treatment with tyrosine kinase inhibitors and EGFR inhibitors.

[0025] In another preferred embodiment, the expression of circUCK2 is increased and the expression of miR-149-5p is decreased in liver cancer cells, thereby increasing the sensitivity of the liver cancer cells to combined treatment with tyrosine kinase inhibitors and EGFR inhibitors.

[0026] In another preferred embodiment, the expression of circUCK2 is increased, the expression of miR-149-5p is decreased, and the expression of CNIH4 is increased in liver cancer cells, thereby enhancing the sensitivity of the liver cancer cells to combined treatment with tyrosine kinase inhibitors and EGFR inhibitors.

[0027] In another preferred embodiment, the expression of circUCK2 is increased, the expression of miR-149-5p is decreased, the expression of CNIH4 is increased, and the expression of TGFa is increased in liver cancer cells, thereby enhancing the sensitivity of the liver cancer cells to combined treatment with tyrosine kinase inhibitors and EGFR inhibitors.

[0028] In another preferred embodiment, the expression of circUCK2 is increased, the expression of miR-149-5p is decreased, the expression of CNIH4 is increased, the expression of TGFa is increased, and the expression of phosphorylated EGFR is increased in liver cancer cells, thereby enhancing the sensitivity of the liver cancer cells to combined treatment with tyrosine kinase inhibitors and EGFR inhibitors.

[0029] In another preferred embodiment, the increase refers to the circUCK2 content being 1 / 3 higher than the normally expressed circUCK2 content, preferably 1 / 2, and more preferably 2 / 3 or more.

[0030] In another preferred embodiment, the reduction means that the circUCK2 content is 1 / 3 lower than the normally expressed circUCK2 content, preferably 1 / 2, and more preferably 2 / 3 or more.

[0031] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0032] (1) Tyrosine kinase inhibitors;

[0033] (2) Epidermal growth factor receptor (EGFR) inhibitors; and

[0034] (3) circUCK2, mRNA expressing circUCK2, or vector expressing circUCK2.

[0035] In another preferred embodiment, the tyrosine kinase inhibitor is selected from the group consisting of: lenvatinib, sorafenib, regorafenib, donafenib, imatinib, sorafenib, sunitinib, lapatinib, and dasatinib.

[0036] In another preferred embodiment, the EGFR inhibitor is selected from the group consisting of: pelitinib, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, and ametinib.

[0037] In another preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the pharmaceutical composition is used to reverse the drug resistance of liver cancer cells to (A) and / or (B).

[0039] In another preferred embodiment, the pharmaceutical composition is used to enhance the sensitivity of liver cancer cells to combined treatment with tyrosinase inhibitors and EGFR inhibitors.

[0040] In another preferred embodiment, the pharmaceutical composition is used to treat liver cancer.

[0041] In a third aspect of the invention, a method is provided to improve the sensitivity of liver cancer cells to combined therapy with tyrosinase inhibitors and EGFR inhibitors, the method comprising the steps of:

[0042] (Z1) Preparation of circUCK2 overexpression elements; and

[0043] (Z2) The circUCK2 overexpression element was co-cultured with liver cancer cells.

[0044] In another preferred embodiment, the circUCK2 overexpression element is circUCK2, mRNA expressing circUCK2, or a vector expressing circUCK2.

[0045] In another preferred embodiment, the circUCK2 overexpression element is a plasmid that transiently overexpresses circUCK2.

[0046] In another preferred embodiment, the circUCK2 overexpression element is a gene editor that stably overexpresses circUCK2.

[0047] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0048] In another preferred embodiment, the method is also used to reverse the resistance of liver cancer cells to tyrosinase inhibitors and / or EGFR inhibitors.

[0049] In a fourth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0050] (A) A first pharmaceutical composition comprising: a tyrosine kinase inhibitor as an active ingredient, and a pharmaceutically acceptable carrier;

[0051] (B) A second pharmaceutical composition comprising: an EGFR inhibitor as the active ingredient, and a pharmaceutically acceptable carrier; and

[0052] (C) A third pharmaceutical composition comprising: circUCK2, mRNA expressing circUCK2 or a carrier expressing circUCK2 as an active ingredient, and a pharmaceutically acceptable carrier.

[0053] In another preferred embodiment, the kit further includes (D) a detection reagent, the detection reagent comprising:

[0054] (D1) Detection reagent for detecting the content of circUCK2 in liver cancer cells;

[0055] (D2) Detection reagent for detecting the content of hsa-miR-149-5p in liver cancer cells;

[0056] (D3) Detection reagent for detecting the content of CNIH4 in liver cancer cells;

[0057] (D4) Detection reagents for the content of TGFa in liver cancer cells; and / or

[0058] (D5) A reagent for detecting the content of phosphorylated EGFR in liver cancer cells.

[0059] In another preferred embodiment, the medicine box can also be used to determine whether a liver cancer patient is suitable for combination therapy:

[0060] If the levels of circUCK2, hsa-miR-149-5p, CNIH4, TGFa, and / or phosphorylated EGFR in liver cancer patients are compared with the average levels of the corresponding indicators in all liver cancer patients (C01, C02, C03, C04, C05), respectively,

[0061] If the ratios of C1 / C01, C02 / C2, C3 / C03, C4 / C04, and C5 / C05 are >1.5, preferably >2, and even more preferably >3, then it suggests that (A) and (B) in this medicine box can be used directly for combined treatment.

[0062] If the ratios of C1 / C01, C02 / C2, C3 / C03, C4 / C04, and C5 / C05 are ≤2 / 3, preferably ≤1 / 2, and even more preferably ≤1 / 3, it suggests that (C) of this kit should be used first to increase the circUCK2 content in liver cancer patients, followed by combined treatment with (A) and (B) from this kit.

[0063] In another preferred embodiment, the medicine box can also be used to determine the effectiveness of combined treatment:

[0064] If, after treatment with the drug of the present invention, the measured values ​​H1, H2, H3, H4, and H5 of the contents of circUCK2, hsa-miR-149-5p, CNIH4, TGFa, and phosphorylated EGFR in the patient's liver cancer cells are compared with the measured values ​​H01, H02, H03, H04, and H05 before treatment, and the ratios of H1 / H0, H02 / H2, H3 / H03, H4 / H04, and H5 / H05 are ≤2 / 3, preferably ≤1 / 2, and more preferably ≤1 / 3, then it indicates that the drug of the present invention has the therapeutic effect on liver cancer and can be used continuously.

[0065] In another preferred embodiment, the kit further includes an instruction manual for guiding the use of the pharmaceutical composition or test reagent.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 shows the mechanism by which circUCK2 affects the progression of liver cancer and drug treatment.

[0068] Figure 2 shows: (A) a characteristic structural diagram of circUCK2 in a specific embodiment of the present invention, and a first-generation sequencing diagram of the splicing site of circUCK2 amplification products; (B) circUCK2 is highly expressed in hepatocellular carcinoma; (C) the expression level of circUCK2 is related to the age, history of HBV infection, and degree of tumor differentiation of hepatocellular carcinoma patients; (D) the expression level of circUCK2 is negatively correlated with the 5-year disease-free survival rate of hepatocellular carcinoma patients after surgery; (E) the expression level of circUCK2 is negatively correlated with the 1-year disease-free survival rate of hepatocellular carcinoma patients after surgery; (F) univariate and multivariate analyses show that circUCK2 can be used as an independent risk factor for predicting disease-free survival in hepatocellular carcinoma patients after surgery.

[0069] Figure 3 shows that: (AB) knockdown of circUCK2 expression inhibits the growth of hepatocellular carcinoma cells; (C) knockdown of circUCK2 expression inhibits the migration and invasion of hepatocellular carcinoma cells; (DE) knockdown of circUCK2 expression inhibits tumorigenesis of hepatocellular carcinoma cells; and (F) knockdown of circUCK2 expression inhibits lung metastasis of hepatocellular carcinoma cells.

[0070] Figure 4 shows that: (A) Bioinformatics analysis showed that circUCK2 may bind to hsa-miR-149-5p and hsa-miR-580-3p; (B) Circular RNA pull-down assay showed that only hsa-miR-149-5p could bind to circUCK2; (C and D) Luciferase reporter gene assay confirmed that circUCK2 can bind to circUCK2; (EG) Knockdown of circUCK2 reduced the expression of CNIH4 and the secretion of TGFa; (HJ) Overexpression of circUCK2 enhanced the expression of CNIH4 and the secretion of TGFa; (K) Knockdown or overexpression of circUCK2 could reduce or increase the activity of EGFR, respectively.

[0071] Figure 5 shows that: (A) Hepatocellular carcinoma cells overexpressing circUCK2 are sensitive to treatment with pelitinib in combination with lenvatinib; (BE) In four primary hepatocellular carcinoma tumors, PDX#1 and PDX#2 showed high expression of circUCK2, CNIH4, and TGFa and activation of EGFR, while PDX#3 and PDX#4 showed low expression of circUCK2, CNIH4, and TGFa and no activation of EGFR; (F) The PDX model shows that primary hepatocellular carcinoma tissues with high expression of circUCK2 are sensitive to treatment with pelitinib in combination with lenvatinib. Detailed Implementation

[0072] Through extensive and in-depth research, the inventors have developed a use for circular RNA-circUCK2 in the treatment of hepatocellular carcinoma. More specifically, by overexpressing circUCK2 or directly adding circUCK2, the sensitivity of hepatocellular carcinoma cells to combined therapy with tyrosine kinase inhibitors and EGFR inhibitors is improved. Experiments show that, under conditions of high circUCK2 expression, the combined therapy has a stronger killing effect on hepatocellular carcinoma cells. Based on this, the present invention was completed.

[0073] the term

[0074] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0075] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0076] As used in this invention, “circUCK2” and “circUCK2(2,3)” can be used interchangeably to represent the RNA circularized in the second to third exons of the gene UCK2.

[0077] As used in this invention, "hepatocellular carcinoma", "HCC" and "liver cancer" can be used interchangeably.

[0078] circUCK2

[0079] circUCK2 is a circular RNA containing the second and third exons of the gene UCK2, and its circularized nucleotide sequence has 257 bases.

[0080] Circular RNAs (circRNAs) and their linear host mRNAs are generated from the same precursor mRNA via backsplicing and classical splicing, respectively. This suggests that their expression may exhibit a competitive splicing pattern, and theoretically, their expression levels should be negatively correlated. This hypothesis assumes that circRNA generation is determined solely by backsplicing efficiency. However, in reality, multiple factors can influence circRNA expression, including backsplicing efficiency, the transcriptional elongation rate (TER) of RNA polymerase II (Pol II), the presence of intron backsplicing signals (ICSs), spliceosomes, RNA-binding proteins, and epigenetic modifications. Under the combined influence of all these specific regulatory factors, the correlation between circRNA and its host gene expression is generally weak. This invention found a weak correlation between circUCK2(2,3) and its host gene UCK2.

[0081] miR-149-5p

[0082] The function of miR-149-5p has been studied in various diseases, including cancer. In hepatocellular carcinoma (HCC), miR-149-5p has been reported to inhibit tumor progression. This invention validates the direct binding between circUCK2(2,3) and miR-149-5p through bioinformatics analysis, ChIRP assays, and dual-luciferase reporter assays.

[0083] Furthermore, miR-149-5p mimics or inhibitors reversed the promoting or inhibiting effects of circUCK2(2,3) overexpression or knockdown on HCC cells, respectively. In addition, circUCK2(2,3) mutants with a mutation at the miR-149-5p binding site lost their ability to promote cell proliferation, migration, and invasion in vitro, as well as tumor growth and metastasis in vivo. Therefore, these results strongly suggest that circUCK2(2,3) exerts its pro-cancer role in HCC by acting as a "sponge" for miR-149-5p.

[0084] CNIH4

[0085] CNIH4, a member of the CORNICHON family, plays an evolutionarily conserved role in the transport and secretion of TGFα and the intracellular transport of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) to the plasma membrane. Although high expression of CNIH4 has been observed in mouse germ cells, gene knockout of CNIH4 does not affect germination or fertility in mice. The role of CNIH4 in hepatocellular carcinoma (HCC) and its underlying mechanisms remain to be explored.

[0086] This invention demonstrates that CNIH4 expression is upregulated in HCC tissues and is positively correlated with circUCK2(2,3) expression. In HCC patients, high CNIH4 expression is associated with lower overall survival and disease-free survival. Furthermore, downregulation of CNIH4 inhibits HCC cell proliferation and migration, suggesting that CNIH4 may play an important role in HCC progression and could serve as a potential therapeutic target.

[0087] TGFα

[0088] TGFα is a membrane-bound or soluble ligand of EGFR. Overexpression of TGFα or activation of its signaling pathway is closely associated with the initiation and progression of hepatocellular carcinoma (HCC). For example, spontaneous HCC has been observed in transgenic mice expressing human TGFα; co-expression of TGFα significantly accelerated HCC formation in c-Myc transgenic mice. Although both TGFα and EGF are high-affinity ligands of EGFR, increased TGFα secretion was observed in 65% of HCC patients, while increased EGF secretion was observed in only 16% of HCC patients. This suggests that TGFα may play a major role in EGFR signaling activation in HCC.

[0089] This invention demonstrates that circUCK2(2,3) releases CNIH4 by adsorbing miR-149-5p, thereby protecting it from the inhibition of miR-149-5p, which in turn increases the secretion of TGFα and activates EGFR signaling.

[0090] circUCK2-hsa-miR-149-5p-CNIH4-TGFa-EGFR pathway

[0091] This invention demonstrates a protein pathway associated with liver cancer: circUCK2 is highly expressed in liver cancer and, by binding to miR-149-5p, relieves miR-149-5p's inhibition of CNIH4. The uninhibited CNIH4 then enhances TGFa secretion, thereby activating EGFR. This can promote liver cancer proliferation, migration, and invasion, and also make liver cancer more sensitive to treatment with EGFR inhibitors combined with lenvatinib.

[0092] Tyrosine kinase inhibitors

[0093] The tyrosine kinase inhibitors described in this invention refer to multi-target tyrosine kinase inhibitors, primarily used to treat various types of cancer. By inhibiting multiple receptor tyrosine kinases associated with tumor growth and metastasis, such as vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), and other receptors, they inhibit cell signal transduction, thereby suppressing tumor cell growth and proliferation and promoting apoptosis.

[0094] The lenvatinib used in the embodiments of the present invention is one of the multi-target tyrosine kinase inhibitors and has been approved as a first-line targeted therapy for advanced liver cancer.

[0095] EGFR inhibitors

[0096] EGFR is a member of the transmembrane receptor tyrosine kinase family, composed of three distinct domains: an extracellular domain, a transmembrane domain, and an intracellular domain. EGFR readily binds to its ligands, typically leading to receptor dimerization, activating the activity of intracellular tyrosine protein kinases, phosphorylating C-terminal tyrosine residues, and triggering downstream enzymes such as Ras, Raf, and PI3K, thereby initiating a complex and interconnected series of signal transduction pathways downstream. EGFR regulates cell growth, differentiation, and apoptosis by mediating these signaling pathways.

[0097] The peritinib used in this invention is one type of EGFR inhibitor. Because lenvatinib has developed resistance, and studies have found that high EGFR expression or EGFR pathway activation is one of the main causes of lenvatinib resistance, combining an EGFR inhibitor with lenvatinib enhances the cancer cell killing ability.

[0098] Combination therapy

[0099] The combination therapy of this invention refers to the combined treatment of hepatocellular carcinoma (HCC) patients with tyrosine kinase inhibitors and EGFR inhibitors. In a specific embodiment of this invention, the combination therapy uses the tyrosine kinase inhibitor lenvatinib and the EGFR inhibitor peritoneinib. Lenvatinib is effective as a first-line treatment for unresectable HCC, but it suffers from problems such as drug resistance, significant side effects, and limited efficacy in certain patient subgroups. Studies have shown that combining lenvatinib with an EGFR inhibitor may enhance the treatment effect of HCC.

[0100] In this invention, overexpression of circUCK2(2,3) can further enhance the synergistic killing effect of lenvatinib when used in combination with multiple EGFR inhibitors. This effect has been verified in HCC cells and PDX clinical models.

[0101] Compared with the prior art, the advantages of the present invention are as follows:

[0102] 1. This invention discovers a novel molecular mechanism regulating liver cancer progression: circUCK2 is highly expressed in liver cancer. By binding to miR-149-5p, it relieves the inhibition of CNIH4 by miR-149-5p. The uninhibited CNIH4 enhances TGFa secretion, thereby activating EGFR and promoting the proliferation, migration and invasion of liver cancer.

[0103] 2. This invention also demonstrates that although high expression of circUCK2 is associated with promoting liver cancer proliferation, its high expression also increases the sensitivity of liver cancer cells to combined treatment with EGFR inhibitors and lenvatinib, providing a new approach for liver cancer treatment.

[0104] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0105] Experimental methods

[0106] 1. Construction of circUCK2 overexpression plasmid

[0107] circUCK2 contains the second and third exons of the gene UCK2, and its circulated nucleotide sequence has 257 bases.

[0108] The DNA sequence corresponding to circUCK2 is:

[0109] The reagents used for the quantitative detection of circular RNA include primers for amplifying the circular RNA, the primers comprising:

[0110] F: 5'-TTGAATTCCAGGATAGCTTCTACCGTGTCC-3'

[0111] R: 5'-TTGGATCCGATAGTCCACCTCATTCTGCCC-3'.

[0112] Plasmid construction: The circUCK2 DNA fragment was amplified by PCR using Phanta Max Super-Fidelity DNA Polymerase. The specific PCR reaction system is as follows:

[0113] Water: 18ul, 2×Phanta Max Buffer: 25ul, dNTP Mix (10mM each): 1ul, upstream primer (F, 10μM): 2ul, downstream primer (R, 10μM): 2ul, Phanta Max Super-Fidelity DNA Polymerase: 1ul, cDNA template: 1ul.

[0114] The specific PCR amplification procedure is as follows:

[0115] ① 95℃ for 3 min, ② 95℃ for 15 s, ③ 60℃ for 15 s, ④ 72℃ for 15 s, repeat steps ② to ④ 35 times. ⑤ 72℃ for 5 min, ⑥ cool to room temperature.

[0116] After obtaining the PCR amplification product, the PCR product and the circRNA overexpression plasmid pLC5-ciR were digested with EcoR1 and BamH1, respectively, and the circUCK2 fragment was ligated into pLC5-ciR using T4 ligase.

[0117] The siRNA sequence used to downregulate circUCK2:

[0118] siNC sequence: UUCUCCGAACGUGUCACGUTT,

[0119] si-1 sequence: CCAUUCCCGUCUUCCGUGUTT

[0120] si-2 sequence: UCCGUCUUCCGUGUGUGCTT.

[0121] 2. circUCK2 pull-down experiment

[0122] CircUCK2(2,3) pull-down assays were performed using the ChIRP method. HCC cells were first cross-linked with 4% glutaraldehyde for 10 minutes, followed by a 5-minute cross-linking reaction with 1 / 10 volume of 1.25M glycine at room temperature. 1 ml of cell lysis buffer (50 mM Tris-Cl pH 7.0, 10 mM EDTA, 1% SDS, fresh protease inhibitor, PMSF, and RNase inhibitor) was added per 100 mg of cell particles. After cell lysis and sonication, 1% of the cell lysis buffer (10 μL) was removed.

[0123] Biotin-labeled DNA probes targeting circUCK2 (circ probe) or linear UCK2 (lin probe) (Ribobio Co., Shanghai, China) were added and incubated with cell lysis buffer at 37°C for 4 h. Then, streptavidin-conjugated M-280 immunomagnetic beads (Thermo Scientific, M-280) were added to the mixture of cell lysis buffer and probes and incubated at 37°C for 30 min. After incubation, half of the immunomagnetic beads were collected, washed and digested with proteinase K to remove proteins, and used for subsequent qRT-PCR to detect circUCK2 enrichment or TaqMan miRNA detection of circUCK2-binding miRNA. The other half of the immunomagnetic beads were boiled in loading buffer at 95–100°C for 5 min, followed by Western blotting to detect the binding of circUCK2(2,3) to AGO2.

[0124] 3. Establishment of patient-derived xenotransplantation (PDX) models

[0125] Hepatocellular carcinoma (HCC) samples were removed and cut into 3mm pieces. 3 Small pieces were then implanted under anesthesia into the anterior subcutaneous tissue of 6- to 8-week-old female BALB / c nude mice. The entire procedure was to be completed within 2 hours. When the tumor volume reached approximately 200 mm², [the procedure was initiated]. 3 Mice were randomly assigned to receive treatment every five days, with treatment regimens including: a control group, lenvatinib (orally administered by gavage at a dose of 4 mg / kg), pelitinib (orally administered by gavage at a dose of 10 mg / kg), or a combination of both. Each single drug in the combination therapy was administered at the same dose and regimen as when used alone. Tumor volume was measured with calipers and calculated using the modified ellipsoid formula: tumor volume = 1 / 2 × length × (width)². In survival curve analysis, treatment continued until the tumor volume reached 2000 mm². 3 .

[0126] 4. Clinical HCC tissue samples

[0127] The research on human tissues used in this invention was approved by the institutional ethics review committees of Jinling Hospital of Nanjing University School of Medicine (DZQH-KYLL-24-01) and Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (XHEC-D-2023-109), and was conducted in accordance with the principles of the Declaration of Helsinki.

[0128] Forty paired hepatocellular carcinoma (HCC) tissues and surrounding tissues were collected from the Department of Transplant Surgery at Xinhua Hospital, Shanghai Jiao Tong University, termed the Xinhua Cohort. Seventy paired HCC tissues and surrounding tissues were collected from the Department of Surgical Oncology at Jinling Hospital, Nanjing University School of Medicine, termed the Jinling Cohort. None of the patients had received prior anticancer treatment. Written informed consent was obtained from all patients for the use of clinical information. All fresh tissues were rapidly frozen in liquid nitrogen and then stored at -80°C for further research.

[0129] 5. circRNA sequencing

[0130] Five paired HCC tissues and peritumoral tissues were selected for circRNA sequencing. Total RNA was isolated using TRIzol reagent. Ribosomal RNA was removed from the samples using the Ribo Zero Magnetic kit. Non-circular RNA was removed by RNase R treatment, and circular RNA was broken up by magnesium RNA cleavage. Subsequently, RNA-seq libraries were prepared using the truseqtmstrand Total RNA Library Prep Kit and then sent to Majorbio for deep sequencing on an Illumina HiSeq4000 system.

[0131] FASTQ reads were aligned with the human reference genome (hg38 / GRCh38). CIRI2 was used to identify circRNAs. In the first scan of the SAM alignment, CIRI2 detected join reads with PCC signals, which reflected a circRNA candidate. Initial filtering employed end-to-end mapping (PEM) and GT-AG splicing signals. After clustering the reads and recording each circRNA candidate sequence, CIRI scanned the SAM alignment again to detect additional join reads, while further filtering eliminated false positive candidates due to misplaced reads from homologous genes or repetitive sequences.

[0132] To identify differentially expressed circRNAs (DEcircRNAs) between HCC tissues and surrounding tumor tissues, the expression level of each circRNA was calculated using the reads per million mapped reads (RPM) method. circRNAs that were detectable in all five HCC tissue types were selected, and differential expression analysis was performed using DEGseq. circRNAs with |log2(fold change)| ≥ 1 and p-adjust ≤ 0.05 were considered to have significantly different expression levels.

[0133] 6. RNA Immunoprecipitation (RIP)

[0134] AGO2RIP assays were performed using the Magana RIP RNA-binding protein immunoprecipitation kit (Millipore, USA), following the manufacturer's protocol. 2x10 7 Cells were lysed in 200 μL of lysis buffer containing protease and RNase inhibitors. 0.5% cell lysis buffer (1 ml) was transferred to a clean tube as input. Magnetic beads were pre-incubated with anti-ago2 antibody (CST#2897) or isotype control (CST#3900) at room temperature for 1 hour, followed by incubation with cell lysis buffer overnight at 4°C. RNA precipitate was purified using the miRNeasy Mini Kit and analyzed by qRT-PCR.

[0135] Example 1. The influence of circUCK2 on liver cancer progression and drug treatment

[0136] Figure 1 illustrates a novel molecular mechanism regulating liver cancer progression: circUCK2 is highly expressed in liver cancer. By binding to miR-149-5p, it relieves the inhibition of CNIH4 by miR-149-5p. The uninhibited CNIH4 enhances TGFa secretion, thereby activating EGFR. On the one hand, this can promote the proliferation, migration and invasion of liver cancer, and on the other hand, make liver cancer more sensitive to the treatment of EGFR inhibitors combined with lenvatinib.

[0137] Example 2. High expression of circUCK2 is associated with liver cancer.

[0138] Analysis of the relationship between circUCK2 expression and clinicopathological features of hepatocellular carcinoma (HCC) patients revealed that circUCK2 is highly expressed in HCC, and that high circUCK2 expression is correlated with age, history of HBV infection, and tumor differentiation degree in HCC patients. (Figure 2A-C)

[0139] Survival analysis showed that the expression level of circUCK2 in hepatocellular carcinoma (HCC) patients was significantly negatively correlated with recurrence-free survival. Furthermore, univariate and multivariate analyses revealed that elevated circUCK2 expression was considered an independent risk factor for recurrence-free survival, indicating that high circUCK2 expression levels can serve as an important prognostic indicator for HCC patients. (Figures 2D-F)

[0140] Example 3. circUCK2 promotes the proliferation and metastasis of liver cancer cells.

[0141] Functional studies have shown that circUCK2 promotes the proliferation and metastasis of liver cancer cells. In vitro experiments showed that knocking down circUCK2 significantly inhibited the growth, migration, and invasion of liver cancer cells (Figures 3A-C), while in vivo experiments showed that knocking down circUCK2 inhibited tumorigenesis and lung metastasis of liver cancer cells (Figures 3D-F).

[0142] The above results indicate that circUCK2 can serve as a therapeutic target for liver cancer.

[0143] Example 4. CircUCK2-HSA-MiR-149-5p-CNIH4-TGFa-EGFR pathway analysis

[0144] Using public databases: ENCORI, circBank, and CircInteractome, we analyzed the miRNAs that circUCK2 may bind to. By taking the intersection of the predictions from the three databases, we found that circUCK2 may bind to hsa-miR-149-5p and hsa-miR-580-3p (Figure 4A).

[0145] circUCK2 was enriched using an RNA pull-down assay targeting circUCK2. Using the TaqMan miRNA assay kit (Thermo Fisher, USA), it was found that hsa-miR-149-5p could be enriched in the pull-down product of circUCK2, while hsa-miR-580-3p could not, indicating that hsa-miR-149-5p can bind to circUCK2 (Figure 4B).

[0146] To further demonstrate that hsa-miR-149-5p binds to circUCK2, luciferase reporter genes were constructed with either the normal circUCK2 sequence (WT) or the circUCK2 sequence (MUT149) with a mutation at the hsa-miR-149-5p binding site (Figure 4C).

[0147] Luciferase reporter gene assays showed that hsa-miR-149-5p inhibited luciferase activity with the normal circUCK2 sequence (WT), but did not affect luciferase activity with the circUCK2 sequence (MUT149) containing a mutation at the hsa-miR-149-5p binding site (Figure 4D).

[0148] Since CNIH4 is a target of hsa-miR-149-5p, knockdown of circUCK2 at both the cellular and tumor levels revealed that knockdown of circUCK2 reduced the expression of CNIH4 (Figures 4E and 4F).

[0149] Overexpression of circUCK2 at both the cellular and tumor levels revealed that overexpression of the normal circUCK2 sequence (WT) upregulated CNIH4 levels, but overexpression of the circUCK2 sequence with a mutation at the hsa-miR-149-5p binding site (149-mut) had no effect on CNIH4 levels (Figures 4H and 4I).

[0150] Since CNIH4 can help with TGFa secretion, an ELISA kit (Neobioscience, EHC125a.96, China) was used to detect whether circUCK2 could affect TGFa secretion. The results showed that knocking down circUCK2 significantly reduced TGFa secretion (Figure 4G), while overexpression of circUCK2, rather than overexpression of circUCK2 with the hsa-miR-149-5p binding site mutation, significantly enhanced TGFa secretion (Figure 4J).

[0151] Since TGFa is a high-affinity ligand for EGFR, we began by investigating whether circUCK2 could affect EGFR activation. The results showed that knockdown or overexpression of circUCK2 inhibited or activated EGFR, respectively (Figure 4K).

[0152] The above experimental results show that high expression of circUCK2 can relieve the inhibitory effect of hsa-miR-149-5p on CNIH4, thereby enhancing the secretion of TGFa by liver cancer cells (detected by ELISA), and TGFa can activate EGFR.

[0153] Example 5. High expression of circUCK2 enhances the sensitivity of liver cancer cells to combined treatment with the EGFR inhibitors pelitinib and lenvatinib.

[0154] High expression of circUCK2 makes liver cancer cells more sensitive to treatment with the EGFR inhibitor pelitinib in combination with lenvatinib.

[0155] Using a clonogenic assay, we investigated whether circUCK2 could affect the sensitivity of hepatocellular carcinoma cells to the EGFR inhibitor pelitinib combined with lenvatinib. The results showed that without circUCK2 expression, the hepatocellular carcinoma cell line SNU398 was insensitive to pelitinib combined with lenvatinib, but overexpression of circUCK2 significantly enhanced the sensitivity of SNU398 to this treatment (Figure 5A).

[0156] Furthermore, this phenotype was confirmed in patient-derived tumor xenograft models (PDX). Four human liver cancer xenograft models were constructed, and qPCR results showed that circUCK2 expression levels were high in PDX#1 and PDX#2, while they were low in PDX#3 and PDX#4 (Figure 5B).

[0157] Correspondingly, qPCR results showed that the CNIH4 expression level was high in PDX#3 and PDX#4, while the CNIH4 expression level was low in PDX#3 and PDX#4 (Figure 5C).

[0158] The ELISA kit further demonstrated that TGFa was highly expressed in PDX#1 and PDX#2, but lowly expressed in PDX#3 and PDX#4 (Figure 5D).

[0159] Immunoblot assays demonstrated that EGFR was activated in PDX#1 and PDX#2, but less activated in PDX#3 and PDX#4 (Figure 5E).

[0160] Finally, these four PDX cases were subcutaneously inoculated into nude mice, and the tumors were treated with lenvatinib, peritinib, and lenvatinib combined with peritinib, respectively. The results showed that, compared with lenvatinib alone, PDX#1 and PDX#2, which showed high expression of circUCK2, CNIH4, and TGFa and high EGFR activation, were more sensitive to lenvatinib combined with peritinib, while PDX#3 and PDX#4, which showed low expression of circUCK2, CNIH4, and TGFa and weak EGFR activation, were not very sensitive to lenvatinib combined with peritinib (Figure 5F).

[0161] The above results indicate that the expression levels of circUCK2, CNIH4, and TGFa, as well as the activation level of EGFR in primary hepatocellular carcinoma, can help clinicians develop personalized treatment strategies (whether to use lenvatinib monotherapy or EGFR inhibitors in combination with lenvatinib).

[0162] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an active ingredient, characterized in that, This is used to prepare formulations or pharmaceutical compositions for improving the sensitivity of liver cancer cells to combined therapy with tyrosine kinase inhibitors and epidermal growth factor receptor (EGFR) inhibitors. The active ingredients are selected from the following group: (a)circUCK2; (b) mRNA expressing circUCK2; and / or (c) The vector that expresses circUCK2.

2. The use as described in claim 1, characterized in that, The tyrosine kinase inhibitors are selected from the following group: lenvatinib, sorafenib, regorafenib, donafenib, imatinib, sorafenib, sunitinib, lapatinib, and dasatinib.

3. The use as described in claim 1, characterized in that, The EGFR inhibitors are selected from the following group: pelitinib, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, and ametinib.

4. The use as described in claim 1, characterized in that, The combination therapy of the tyrosine kinase inhibitor and EGFR inhibitor is the combined use of lenvatinib and peritrinib.

5. The use as described in claim 1, characterized in that, The circUCK2 binds to miR-149-5p, thereby relieving miR-149-5p's inhibition of CNIH4. The uninhibited CNIH4 enhances TGFa secretion, thus activating EGFR.

6. The use as described in claim 1, characterized in that, circUCK2 is used as a biomarker to predict the sensitivity of liver cancer patients to combination therapy with tyrosine kinase inhibitors and EGFR inhibitors.

7. The use as described in claim 1, characterized in that, The circUCK2 is a circular RNA transcribed from the second to third exons of the UCK2 gene, and its corresponding DNA sequence is shown in SEQ ID NO:

1.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (A) Tyrosine kinase inhibitors; (B) Epidermal growth factor receptor (EGFR) inhibitors; and (C)circUCK2, mRNA expressing circUCK2, or vector expressing circUCK2.

9. A method for improving the sensitivity of liver cancer cells to combined therapy with tyrosinase inhibitors and EGFR inhibitors, characterized in that, The method includes the following steps: (Z1) Preparation of circUCK2 overexpression elements; and (Z2) The circUCK2 overexpression element was co-cultured with liver cancer cells.

10. A medicine box, characterized in that, The medicine box includes: (A) A first pharmaceutical composition comprising: a tyrosine kinase inhibitor as an active ingredient, and a pharmaceutically acceptable carrier; (B) A second pharmaceutical composition comprising: an EGFR inhibitor as the active ingredient, and a pharmaceutically acceptable carrier; and (C) A third pharmaceutical composition comprising: circUCK2, mRNA expressing circUCK2 or a carrier expressing circUCK2 as an active ingredient, and a pharmaceutically acceptable carrier.

11. The medicine box as described in claim 10, characterized in that, The kit also includes (D) a test reagent, the test reagent comprising: (D1) Detection reagent for detecting the content of circUCK2 in liver cancer cells; (D2) Detection reagent for detecting the content of hsa-miR-149-5p in liver cancer cells; (D3) Detection reagent for detecting the content of CNIH4 in liver cancer cells; (D4) Detection reagents for the content of TGFa in liver cancer cells; and / or (D5) A reagent for detecting the content of phosphorylated EGFR in liver cancer cells.

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