Application of LINC02568 as target in preparation of ER + breast cancer treatment kit

Abstract

The invention discloses an application of LINC02568 as a target in preparation of an ER < + > breast cancer treatment kit. The reagent targeting LINC02568 can effectively inhibit in-vitro growth of ER < + > breast cancer cells and in-vivo tumor formation, and can be combined with an endocrine therapeutic drug or a small-molecule inhibitor targeting CA12 to achieve a synergistic inhibition effect on growth of cells and tumors.

Description

Technical Field

[0001] The present invention belongs to the field of cancer treatment biotechnology, and specifically relates to the application of LINC02568 as a target in the preparation of an ER+ breast cancer treatment kit. Background Art

[0002] Worldwide, breast cancer ranks first in the incidence and mortality of women, seriously threatening women's lives and health. According to the different receptor expressions in tumor tissues, breast cancer can be divided into four clinically relevant subtypes: luminal A, luminal B, HER2-high expression, and basal-like. Among them, luminal A and luminal B are collectively referred to as ER+ breast cancer due to their high expression of estrogen receptors (ER), accounting for more than 70% of the total number of breast cancer cases. Although a part of breast cancer originates from heritable carcinogenic mutations, a considerable part of breast cancer still originates from epigenetic reprogramming caused by abnormal epigenetic mechanisms. Therefore, finding abnormal epigenetic mechanisms in ER+ breast cancer and finding key regulatory factors are of great significance for understanding the occurrence and development of ER+ breast cancer and developing potential new drug treatment targets.

[0003] The main mechanism of ER+ breast cancer is the abnormal activation of downstream target genes of the ER signaling pathway due to excessive exposure to estrogen (E2). The effect of estrogen in breast cancer is mediated by estrogen receptors ERα and ERβ, of which ERα is considered to be the main driver of ER+ breast cancer. Based on this, endocrine therapy that blocks the function of ERα has become the first choice for ER+ patients. This type of drug blocks the biosynthesis of estrogen or inhibits the function of ERα, thereby inhibiting the abnormal growth of hormone-dependent tumor cells to achieve the purpose of treatment. With the continuous development of endocrine therapy drugs, endocrine therapy has also made great progress. Existing endocrine therapy drugs include selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs), selective estrogen receptor downregulators (SERDs) and ER PROTAC degraders. Although ER+ breast cancer patients have a good 5-year survival rate, about 30% of patients will develop endocrine resistance, recurrence or metastasis after receiving endocrine therapy, which greatly threatens the lives of patients. Studies have shown that ligand-independent ER activation is the main driving factor of endocrine resistance, so inhibiting ER itself or the activation of ER signaling pathways is still an effective means to overcome resistance. Therefore, finding regulatory factors that play a key role in the estrogen signaling pathway and developing new drug targets provide new ideas for the treatment of ER+ breast cancer, especially endocrine-resistant breast cancer.

[0004] The abnormal epigenetic mechanisms of tumor cells are jointly regulated by molecules including nuclear receptors, transcription factors, epigenetic regulators, and non-coding RNA. LncRNA is a class of non-coding RNA molecules with a length of more than 200 nucleotides and functioning in the form of RNA. As a non-coding RNA molecule, the regulatory mechanisms involved in lncRNA are closely related to its intracellular localization. Most lncRNAs localized in the cell nucleus are enriched near the detranscription site and recruit nearby RNA or protein to drive the formation of nuclear compartments, thereby cis-regulating DNA contacts, heterochromatin formation, or gene expression. In addition, some nuclear-localized lncRNAs leave their transcription sites and trans-regulate the transcription of distal genes, or participate in biological processes such as the formation of nuclear structure and alternative splicing. lncRNAs transported to the cytoplasm Long non-coding RNAs found in the cytoplasm often regulate protein modification and intercellular signaling by forming RNP complexes, or directly or indirectly regulate mRNA translation or degradation by interacting with mRNAs or miRNAs. Due to their diverse regulatory functions, lncRNAs have been shown to play an important role in tumor occurrence and development. As a drug target, lncRNA has great potential. The use of small interfering RNA (siRNA) and antisense oligonucleotides (ASO) to recruit RNA-induced silencing complex (RISC) and ribonuclease H (RNaseH) respectively to target and silence specific RNA molecules has brought great hope for RNA therapy. The development of ribonuclease targeting chimera (RIBOTAC) technology has made the use of small molecules to target RNA and recruit ribozymes for degradation another promising targeted intervention strategy. Therefore, the development of drugs targeting lncRNA will provide a new approach for the clinical treatment of ER+ breast cancer.

[0005] Changes in intracellular (pHi) and extracellular (pHe) pH homeostasis are one of the important epigenetic changes in tumor development. Due to increased glucose consumption, metabolic changes and increased glycolysis ratio, tumor cells increase intracellular lactate production and excretion, resulting in decreased cellular pHe and a slight increase in pHi. Abnormal pH gradients in tumor cells provide conditions for tumor growth, drug resistance, metastasis and recurrence. The expression of some key molecules in cells is believed to be related to the unique pH maintenance in tumor cells, including NHE1, V-ATPase and carbonic anhydrase (CAs). Studies have shown that transmembrane CAs can work together with cytoplasmic CAs to create and maintain pH differences in the hypoxic tumor microenvironment, thereby promoting ATP synthesis, cell growth and other malignant phenotypes in the acidic tumor microenvironment. CA12 belongs to membrane-associated CAs, which can reversibly catalyze CO2 to generate HCO3- and a proton in the presence of water. Studies have shown that CA12 plays an important regulatory role in tumor cell signal transduction, survival, migration, invasion and stemness maintenance. In addition, studies have shown that CA12 expression increases on the surface of drug-resistant cells, indicating that it may play a role in drug resistance. However, the potential regulatory mechanisms of differential expression of CA12 in tumor cells remain unclear. Understanding these mechanisms will help people develop therapeutic strategies to inhibit the formation of a unique pH environment in tumors, thereby inhibiting tumor growth, drug resistance, metastasis, and recurrence. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide the use of LINC02568 as a target in the preparation of an ER+ breast cancer treatment kit.

[0007] The technical solution of the present invention is as follows:

[0008] Application of LINC02568 as a target in the preparation of a therapeutic kit for ER+ breast cancer.

[0009] In a preferred embodiment of the present invention, the ER+ breast cancer treatment kit contains an agent that targets and inhibits LINC02568.

[0010] Further preferably, the reagent is an antisense nucleotide.

[0011] More preferably, the ER+ breast cancer treatment kit also contains endocrine therapy drugs and / or small molecule inhibitors targeting CA12.

[0012] Still further preferably, the endocrine therapy drug is selected from tamoxifen and fulvestrant, and the small molecule inhibitor targeting CA12 is U-104.

[0013] An ER+ breast cancer treatment kit comprising an agent for targeted inhibition of LINC02568.

[0014] In a preferred embodiment of the invention, the agent is an antisense nucleotide.

[0015] More preferably, the invention also contains endocrine therapy drugs and / or small molecule inhibitors targeting CA12.

[0016] More preferably, the endocrine therapy drug is selected from tamoxifen and fulvestrant, and the small molecule inhibitor targeting CA12 is U-104.

[0017] The beneficial effects of the present invention are as follows: the reagent targeting LINC02568 in the present invention can effectively inhibit the in vitro growth of ER+ breast cancer cells and the in vivo tumor formation, and can be used in combination with endocrine therapy drugs or small molecule inhibitors targeting CA12 to have a synergistic inhibitory effect on the growth of cells and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The expression result diagram of a large number of mRNAs and adjacent lncRNAs in estrogen-induced ER+ breast cancer cells in Example 1 of the present invention. Among them, (A, B) MCF7 cells were treated with estrogen (E2, 10-7M, 6h), and RNA was extracted for RNA-seq analysis. The Venn diagram shows the number of RefSeq annotated mRNAs (A) and lncRNAs (B) upregulated and downregulated by estrogen at different change multiples (q<0.05). (C) RT-qPCR analysis was performed on the RNA samples in (A) to detect representative lncRNAs (±sem) that were upregulated in RNA-seq. (D, E) UCSC genome visualization analysis of estrogen-induced lncRNAs and adjacent coding genes. (F) RT-qPCR analysis was performed on the RNA samples performed according to the above steps to detect the expression of estrogen-induced lncRNAs and adjacent coding genes (±sem). (G, H) UCSC genome browser visualization analysis shows the density of ERα, H3K4me1, H3K4me2, H3K4me3, H3K27Ac, ​​P300, H3K9me3 and H3K27me3 ChIP-seq tags. Red represents the presence of estrogen, blue represents the absence of estrogen, and the genomic regions of estrogen-induced lncRNAs and adjacent coding genes are marked with dotted boxes. (I, J) MCF7 cells were treated with fulvestrant (1 μM, 6 h) in the presence or absence of estrogen (E2, 10-7 M, 6 h), and then RNA was extracted and RT-qPCR analysis was performed to detect the expression of lncRNAs and adjacent coding genes (± sem, **P < 0.01, ***P < 0.001).

[0019] Figure 2 The results of LINC02568 promoting the malignant phenotype of ER+ breast cancer cells in vivo and in vitro in Example 1 of the present invention are shown in (A) a scatter plot showing 129 lncRNAs induced by estrogen detected in RNA-seq analysis (such as Figure 1(B) (FC≥1.5, n=129). Red dots indicate LINC02568. (B) The TCGA database shows the expression of LINC02568 in breast tumor samples of different subtypes (http: / / tumorsurvival.org / ). (C) Breast tumors and corresponding adjacent and normal tissues from breast cancer patients (ER+, n=39; ER-, n=18) were collected, RNA was extracted and RT-qPCR analysis was performed to detect the expression of LINC02568 (±sd, **P<0.01, ns: not significant). (D) Different breast cancer cell lines were collected, RNA was extracted and RT-qPCR analysis was performed, and the expression of LINC02568 was detected as shown (±sem). (E) MCF7 cells were infected with shCTL or shLINC02568 (shLINC02568(1) and shLINC02568(2)) lentivirus for 48 h, and RNA was extracted and analyzed by RT-qPCR to detect the expression of LINC02568 (±sem, ***P<0.001). (FI) MCF7 cells were infected with shCTL or shLINC02568 (shLINC02568(1) and shLINC02568(2)) lentivirus for 48 h, and estrogen (E2, 10 - 7 M) were treated and subjected to cell proliferation assay (F), clone formation assay (G), wound healing assay (H) and Transwell assay (I) (±sem, ***P<0.001). (J) MCF7 cells were infected with LINC02568 overexpression lentivirus for 48h, RNA was extracted and RT-qPCR analysis was performed to detect LINC02568 expression (±sem, ***P<0.001). (KN) MCF7 cells were infected with LINC02568 overexpression lentivirus for 48h, and estrogen (E2, 10 -7 M) were treated and subjected to cell proliferation assay (K), colony formation assay (L), wound wound assay (M) and Transwell assay (N) (±sem, *P<0.05, **P<0.01, ***P<0.001). (O) MCF7 cells infected with shCTL or shLINC02568 lentivirus were cultured for 48 h and then inoculated subcutaneously into female BALB / C nude mice (n=3). Estrogen (E2, 10 -2M). Tumors were collected and photographed 8 weeks later. (P) shows the weight of the tumor in (O) (±sd, *P<0.05). (Q, R) MCF7-TamR cells were transfected with siCTL or siLINC02568 and then treated with tamoxifen (5 μM), followed by cell proliferation assay (Q) and clone formation assay (R) (±sem, *P<0.05, **P<0.01, ***P<0.001). (S) Quantitative analysis of clones in (R) (±sem, **P<0.001).

[0020] Figure 3 The results of LINC02568 regulating the expression of ESR1 at the transcriptional level and promoting the malignant phenotype of ER+ breast cancer cells in Example 1 of the present invention are shown in Figure 1. In which, (A) MCF7 cells were subjected to nuclear-cytoplasmic separation and RNA was extracted, and the expression of the RNA shown was detected by RT-qPCR analysis. ACTIN and MALAT1 were used as references for the cytoplasm and nucleus, respectively. (B) MCF7 cells were transfected with siCTL or siLINC02568 for 48 hours and then estrogen (E2, 10 -7 M) were treated for 6 h, followed by RNA-seq analysis to count genes positively and negatively regulated by LINC02568 (FC>1.5). (C, D) Heatmap (C) and box plot (D) were used to represent genes regulated by LINC02568 in (B) (FPKM, log2). (E) Hallmark analysis of genes positively regulated by LINC02568 in (B). (F) The expression of mRNAs (n=206) in the ceRNA network in ER+ breast tumors relative to normal tissues and their correlation with LINC02568 were searched in GEPIA2, and a three-dimensional scatter plot was drawn. (G, H) MCF7 cells were transfected with siCTL or siLINC02568 and cultured in phenol red-free medium for 48 h, followed by the addition of estrogen (E2, 10 -7 M, 6 h for RT-qPCR and 24 h for immunoblotting), and RT-qPCR analysis and western blot analysis were performed to detect the expression of ESR1 mRNA (G) and ERα protein (H). ERα-66, ERα-46 and ERα-36 represent three ERα variants (±sem, **P<0.01) (I, J) ERα-inducible MCF7 cells were transfected with siCTL or siLINC02568, and then cultured in phenol red-free medium with Doxycycline (Dox, 1 μg / ml) for 48 h, followed by the addition of estrogen (E2, 10 -7M, 6 h for RT-qPCR and 24 h for immunoblotting) were subjected to RT-qPCR analysis and western blot analysis to detect the expression of ESR1 mRNA (I) and ERα protein (J). (K) MCF7 cells were transfected with siCTL or siLINC02568 and treated with actinomycin D (ActD, 2 μg / ml) for 0, 1, 6 or 12 h, followed by RNA extraction and RT-qPCR analysis to detect the expression of ESR1 (±sem, **P<0.01, ***P<0.001). (L) RNA samples in (G) were subjected to RT-qPCR analysis to detect the expression of ERα target genes (±sem). (M) MCF7 cells were infected with LINC02568 overexpressing lentivirus and cultured in phenol red-free medium for 48 h, followed by the addition of estrogen (E2, 10 -7 M, 6h). Cells were subjected to RNA extraction and RT-qPCR analysis to detect the expression of ERα target genes (±sem). (N) ERα-inducible MCF7 cells (as described in (I)) were subjected to RNA extraction and RT-qPCR analysis to detect the expression of ERα target genes (±sem, *P<0.05, **P<0.01, ***P<0.001). (O, P) MCF7 cells were transfected with siCTL or siLINC02568, infected with CTL or ERα-overexpressing lentivirus, and then estrogen (E2, 10 -7 M) were treated with 4% paraformaldehyde and 1% paraformaldehyde, and then subjected to cell proliferation assay (O) and clone formation assay (P). (Q) Quantitative analysis of clone staining in (P) (±sem, ***P<0.001).

[0021] Figure 4The result diagram of LINC02568 as a sponge of miR-1233-5p in Example 1 of the present invention to regulate ESR1 expression and the malignant phenotype of ER+ breast cancer cells. Among them, (A) After MCF7 cells were transfected with CTL, miR-1233-5p or miR-6778-5p mimics for 48 hours, RNA was extracted and RT-qPCR analysis was performed to detect the expression of LINC02568 and ESR1 (±sem, *P<0.05). (B) Schematic diagram of sequence matching between miR-1233-5p and wild-type LINC02568 (WT) or the corresponding mutant form (MUT). (C) Schematic diagram of sequence matching between miR-1233-5p and wild-type ESR1 3'UTR (WT) or the corresponding mutant form (MUT). Among them, the 3'UTR of ESR1 mRNA is divided into five regions (regions 1 to 5). (D) MCF7 cells were co-transfected with LINC02568 (WT) / (MUT) and miR-1233-5p mimics followed by dual luciferase reporter gene assay (±sem, *P<0.05, ns: no significant difference). (E) MCF7 cells were co-transfected with ESR1 3'UTR (WT) / (MUT) and miR-1233-5p mimics followed by dual luciferase reporter gene assay (±standard error, ***P<0.001, ns: non-significant difference). (FJ) MCF7 cells were co-transfected with siLINC02568 and miR-1233-5p inhibitor for 48 h, and the expression of ESR1 mRNA (F) and ERα protein (G), the expression of ERα target genes (H), cell proliferation (I) and colony formation (J) were detected (±sem, *P<0.05, **P<0.01, ***P<0.001). (K, L) MCF7 cells were transfected with CTL or miR-1233-5p mimics, followed by cell proliferation (K) and clonogenic assays (L) (±sem, **P<0.01).

[0022] Figure 5 The results of LINC02568 in Example 1 of the present invention regulating tumor-specific pH homeostasis by regulating CA12 expression and promoting the malignant phenotype of ER+ breast cancer cells. (AC) MCF7 cells were transfected with siCTL or siLINC02568 and cultured in phenol red-free medium for 48 hours before being treated with estrogen (E2, 10 -7MCF7 cells were treated with siCTL or siLINC02568 for 48 h, and RNA was subsequently extracted and subjected to RT-qPCR analysis to detect the expression of CA12 (A), USP3 (B), and CA12 pre-mRNA (C) (±sem, **P<0.01, ***P<0.001). (D) MCF7 cells were transfected with siCTL or siLINC02568 for 48 h, and RNA polymerase II (Pol II) ChIP-qPCR analysis was performed to detect the binding of Pol II in the CA12 promoter region. Data are expressed as % input (±sem, ***P<0.001). (EL) MCF7 cells were infected with shCTL or sh CA12 (shCA12(1) and shCA12(2)) lentivirus for 48 hours and then subjected to RT-qPCR (E), immunoblot analysis (F), intracellular pH measurement (G), extracellular pH measurement (H), cell proliferation assay (I), colony formation assay (J), scratch assay (K) and Transwell assay (L) (±sem, **P<0.01, ***P<0.001). (M) MCF7-TamR cells were transfected with siCTL or siCA12 and then treated with tamoxifen (5 μM) and subjected to cell proliferation assay (±sem, **P<0.01, ***P<0.001). (N, O) MCF7 cells were infected with shCTL or shLINC02568 lentivirus for 48 hours and then subjected to intracellular (N) or extracellular (O) pH measurement (±sem, **P<0.01, ***P<0.001). (PU) MCF7 cells were transfected with siCTL or siLINC02568 and infected with CA12 overexpression lentivirus, followed by intracellular pH measurement (P), extracellular pH measurement (Q), cell proliferation assay (R), clone formation assay (S), scratch assay (T) and Transwell assay (U) (±sem, *P<0.05, **P<0.01, ***P<0.001). (V) MCF7-TamR cells were transfected with siCTL or siLINC02568 and infected with CA12 overexpression lentivirus, followed by tamoxifen (5 μM) treatment, and cell proliferation assay (±sem, ***P<0.001).

[0023] Figure 6 The results of the ASO targeting LINC02568 in Example 1 of the present invention can effectively inhibit the growth of ER+ breast tumors. (A) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and cultured in phenol red-free medium for 48 h, followed by the addition of estrogen (E2, 10 -7M, 6h) were treated with ASO-CTL and RNA was extracted and RT-qPCR was performed to detect the expression of target genes of LINC02568, ESR1, CA12 and ERαba (±sem). (BE) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and cultured in phenol red-free medium for 48h, followed by the addition of estrogen (E2, 10 -7 M) were treated with MCF7 cells and subjected to cell proliferation assay (B), colony formation assay (C), scratch assay (D) and Transwell assay (E) (±sem, *P<0.05, **P<0.01, ***P<0.001). (F) MCF7 cells were subcutaneously injected into female BALB / C nude mice, and estrogen (E2, 10 - 2 M) until the tumor size reaches approximately 100 mm 3 . The mice were then randomly divided into two groups (n=4) and injected intratumorally with ASO at a dose of 5 nmol (50 μl, 100 mM dissolved in PBS), once every 2 days, for a total of 8 times. The tumors were removed, photographed and weighed. (G) Weight statistics of the tumors in Figure (F) (±sd, ***P<0.001). (H) RNA extraction and RT-qPCR analysis were performed on the tumors described in (F) to detect the expression of LINC02568, ESR1, CA12 and representative ERα target genes (±sem). (I) RNA extraction and RT-qPCR analysis were performed on the tumors described in (F) to detect the pre-mRNA expression of CA12 (±sd, *P<0.05). (J) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and cultured in phenol red-free medium for 48 h, followed by administration of fulvestrant (ICI, 1 μM, 6 h) and estrogen (E2, 10 -7 M, 6h), RNA was extracted and RT-qPCR analysis was performed to detect the expression of LINC02568, ESR1, CA12 and representative ERα target genes, as shown (±sem). (K, L) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and then treated with fulvestrant (ICI, 2.5μM), followed by cell proliferation assay (K) and clone formation assay (L) (±sem, **P<0.01, ***P<0.001). (M) MCF7 cells were subcutaneously inoculated into female BALB / C nude mice, and estrogen (E2, 10 -2 M) until the tumor size reaches approximately 100 mm 3. The mice were then randomly divided into four groups (n=5) and injected intratumorally with ASO at a dose of 5 nmol (50 μl, 100 mM dissolved in PBS), once every 2 days, for a total of 6 times. ICI was injected subcutaneously once a week at a dose of 250 mg / kg. After the treatment, the tumors were removed, photographed and weighed. (N) Weight statistics of the tumors in Figure (M) (±sd, **P<0.01, ***P<0.001). (O) Weight change curve of the mice described in (M) (±sd). (P) The tumors described in (M) were subjected to RNA extraction and RT-qPCR analysis to detect the expression of LINC02568, ESR1, CA12 and representative ERα target genes (±sem). (Q, R) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and then treated with U-104 (50 μM), followed by cell proliferation assay (Q) and clone formation assay (R) (±sem, **P<0.01, ***P<0.001). (S) MCF7 cells were subcutaneously inoculated into female BALB / C nude mice, and estrogen (E2, 10 -2 M) until the tumor size reaches approximately 100 mm 3 . The mice were then randomly divided into four groups (n=3) and injected intratumorally with ASO at a dose of 5 nmol (50 μl, 100 mM dissolved in PBS), once every 2 days, for a total of 6 times. U-104 was injected intraperitoneally once every two days at a dose of 50 mg / kg. After the treatment, the tumors were removed, photographed and weighed. (T) Statistics of tumor weight in Figure (S) (± sd, *P < 0.05). (U) The weight change curve of mice described in (S) (± sd).

[0024] Figure 7 The figure is a model diagram of LINC02568 in Example 1 of the present invention promoting the development and endocrine resistance of ER+ breast cancer through dual trans and cis mechanisms. Among them, LINC02568 induced by estrogen / ERα acts as a ceRNA, which binds to miR-1233-5p to regulate the expression of ESR1 in the cytoplasm, thereby promoting the activation of ERα target genes. LINC02568 also regulates the transcription of its adjacent coding gene CA12 through cis action, thereby regulating the intracellular and extracellular pH homeostasis. The ERa-target gene activation and intracellular and extracellular pH homeostasis regulated by LINC02568 jointly promote the development and endocrine resistance of ER+ breast cancer.

[0025] Figure 8The results of the expression of a large number of lncRNAs in ER+ breast cancer cells induced by estrogen in Example 1 of the present invention are shown in Figure 1. (A) The correlation of the effect of the entire transcriptome under the action of estrogen (E2) in two biological replicates of RNA sequencing. (B) Based on RNA sequencing analysis of lncRNAs regulated by estrogen in CAMA-1, EFM-19, HCC1500, MDA-MB-134-VI and T-47D (q < 0.05) cells, pie charts are used to represent lncRNAs that are upregulated or downregulated under estrogen stimulation in at least three ER+ breast cancer cell lines. (C) Breast tumors and corresponding normal tissues from ER+ breast cancer patients (n=10) were collected for RNA extraction and RT-qPCR analysis to detect the expression (±sem) of lncRNA / mRNA pairs (DSCAM-AS1 / DSCAM, PVT1 / MYC, IRAIN / IGF1R, LOC100129931 / GRPEL1, KCTD21-AS1 / GAB2, SLC25A25-AS1 / SLC25A25, SH3BP5-AS1 / SH3BP5, C15orf59-AS1 / C15orf59, LOC90784 / POLR1A, and LOC101928837 / TSKU).

[0026] Fig. 9 The result diagram of the estrogen-induced long non-coding RNA, LINC02568, in Example 1 of the present invention, promoting the malignant phenotype of ER+ breast cancer cells. Among them, (A) GEPIA2 database shows the expression of LINC02568 in different cancer types. Among them, LINC02568 is upregulated in BRCA and downregulated in PAAD and SKCM (FC≥2, P≤0.01). (BD) Figure 2 G, 2H, 2I crystal violet dye quantitative analysis (B), scratch healing rate analysis (C), and invasion cell number statistics (D) (±sem, *P < 0.05, **P < 0.01, ***P < 0.001). (EG) Figure 2 Quantitative analysis of crystal violet dye in L, 2M, and 2N (E), analysis of wound healing rate (F), and statistics of the number of invasive cells (G) (±sem, **P<0.01, ***P<0.001). (H) MCF7 cells were transfected with siCTL or siLINC02568 and cultured in phenol red-free medium for 48 h, followed by the addition of estrogen (E2, 10 -7M, 6h) were treated with siCTL or siLINC02568 and then subjected to RNA extraction and RT-qPCR analysis to detect the expression of LINC02568 (±sem, ***P<0.001). (I, J, L, M) MCF7 cells were transfected with siCTL or siLINC02568 and cultured in phenol red-free medium for 48h, followed by the addition of estrogen (E2, 10 -7 M) Treatment and cell proliferation assay (I), clone formation assay (J), scratch assay (L) and Transwell assay (M) (±sem, ***P<0.001). (K, N, O) Figure 2 J, 2L, 2M crystal violet dye quantitative analysis (K), scratch healing rate analysis (N), and invasion cell number statistics (O) (±sem, ***P < 0.001). (P, Q, S, U) T47D cells were infected with shCTL or shLINC02568 lentivirus and cultured in phenol red-free medium for 48 h, followed by the addition of estrogen (E2, 10 -7 M) Treated and subjected to cell proliferation assay (P), clone formation assay (Q), scratch assay (S) and Transwell assay (U) (±sem, *P<0.05, **P<0.01). (R, T, V) Figure 2 Quantitative analysis of crystal violet dye (R), analysis of scratch healing rate (T), and statistics of the number of invasive cells (V) (±sem, ***P<0.001).

[0027] Fig.10 The results of LINC02568 as a miRNA sponge in Example 1 of the present invention promoting ESR1 expression through miR-1233-5p. (A) CeRNA network composed of LINC02568, miRNA and mRNA (genes positively regulated by LINC02568, n=206). The green, yellow and light blue nodes represent LINC02568, miRNA and mRNA, respectively. (B) UCSC genome visualization analysis shows Figure 3 B The expression of ESR1 in the RNA-seq. (C) MCF7 cells were infected with shCTL or shLINC02568 (shLINC02568-1 and shLINC02568-2) lentivirus for 48 h, and then estrogen (E2, 10 -7 M, 6h) and RNA was extracted for RT-qPCR analysis to detect the expression of ERα target genes (±sem). (D) T47D cells were transfected with siCTL or siLINC02568 and cultured in phenol red-free medium for 48h, followed by the addition of estrogen (E2, 10 -7M, 6h). RNA was extracted and RT-qPCR analysis was performed to detect the expression of ERα target genes (±sem). (E) Figure 8 RNA was extracted from breast tumor tissues and corresponding normal tissues in C (n=10) and subjected to RT-qPCR analysis to detect the expression of miR-1233-5p (±sd, ns: no significant difference). (F) Figure 2 RNA was extracted from tumor samples in O and subjected to RT-qPCR analysis to detect the expression of miR-1233-5p (±sd, ns: no significant difference). (G, H) Figure 4 J(G) and Figure 4 Quantification of crystal violet dye in L(H) (±sem, ***P<0.001).

[0028] Fig.11 The results of Example 1 of the present invention show that CA12 is highly expressed in ER+ breast tumor samples and is highly correlated with the expression of LINC02568. Figure 2 RNA was extracted from samples in O and analyzed by RT-qPCR to detect the expression of CA12 (A) and CA12 pre-mRNA (B) (±sd, *P<0.05). (C) The expression of CA12 in different subtypes of breast tumors was analyzed in the TCGA database (http: / / tumorsurvival.org / ). (D) Figure 2 The samples in C were subjected to RNA extraction and RT-qPCR analysis to detect the expression of CA12 in breast tumor tissues, corresponding adjacent tissues and normal tissues (±sem, ***P<0.001, ns: no significant difference). (E) RNA extraction and RT-qPCR analysis were performed on different breast cancer cell lines to detect the expression of CA12 (±sem). (F) The expression correlation of LINC02568 and CA12 in ER+ breast tumor samples was analyzed using the TANRIC database. (G) Figure 2 The expression correlation of LINC02568 and CA12 was detected in ER+ breast tumor tissue samples in C. (H) MCF7 cells were cultured in phenol red-free medium for 48 h and then estrogen (E2, 10 -7 M) were treated for 6 h, followed by RNA extraction and RT-qPCR analysis to detect the expression of CAs (± sem).

[0029] Fig.12 This is a result diagram of LINC02568 in Example 1 of the present invention regulating tumor-specific pH homeostasis by regulating CA12 and promoting the malignant phenotype of ER+ breast cancer cells. Among them, (A, B, C) Figure 5 J. Figure 5 K and Figure 5 Quantitative analysis of crystal violet staining (A), wound healing rate (B), and number of invasive cells (C) in L (±sem, **P<0.01, ***P<0.001). (DG) MCF7 cells were transfected with siCTL or siCA12 for 48 h, and RNA extraction and RT-qPCR analysis (D), immunoblot analysis (E), intracellular pH measurement (F), and extracellular pH measurement (G) were performed (±sem, **P<0.01, ***P<0.001). (H, I, K, M) MCF7 cells were transfected with siCTL or siCA12 and subjected to cell proliferation assay (H), colony formation assay (I), wound healing assay (K), and Transwell assay (M) (±sem, **P<0.01, ***P<0.001). (J, L, N) Quantitative analysis of crystal violet staining (J), wound healing rate (L), and number of invasive cells (N) in Figures (I), (K), and (M) (±sem, **P<0.01, ***P<0.001). (O, P) MCF7 cells were infected with CA12-overexpressing lentivirus for 48 h, followed by immunoblot analysis (O) and extracellular pH measurement (P) (±sem, ***P<0.001). (Q, R, T, V) MCF7 cells were infected with CA12-overexpressing lentivirus, followed by cell proliferation assay (Q), colony formation assay (R), wound healing assay (T), and Transwell assay (V) (±sem, *P<0.05, **P<0.01). (S, U, W) Quantitative analysis of crystal violet staining (S), wound healing rate (U), and number of invasive cells (W) in (R), (T), and (V) (±sem, **P<0.01, ***P<0.001). (X, Y) Intracellular (X) and extracellular (Y) pH were measured in MCF7 cells 48 h after transfection with siCTL and siLINC02568 (±sem, **P<0.01, ***P<0.001). (Z) Extracellular pH was measured in MCF7 cells 48 h after infection with LINC02568 overexpressing lentivirus (±sem, **P<0.01). (X', Y') MCF7 cells were transfected with siCTL or siLINC02568 (X') or infected with lentivirus expressing LINC02568 overexpression (Y') and then re-seeded into Seahorse 96-well assay plates (approximately 10,000 cells per well) 24 hours later. Extracellular acidification rate was measured 24 hours later (±sem). (Z', X", Y") show Figure 5 S. Figure 5 T. Figure 5Quantitative analysis results of crystal violet staining (Z'), scratch healing rate (X") and number of invasive cells (Y") in U (±sem, **P<0.01, ***P<0.001).

[0030] Fig.13 The results of the ASO targeting LINC02568 in Example 1 of the present invention can effectively inhibit the growth of ER+ breast tumors. Among them, (A, B, C) show Figure 6 C. Figure 6 D and Figure 6 Quantitative analysis results of crystal violet staining (A), scratch healing rate (B) and number of invasive cells (C) in E (±sem, ***P<0.001). (D) Figure 6 The tumor samples shown in F were subjected to RNA extraction and RT-qPCR experiments to detect the expression of miR-1233-5p (±sem, ns: no significant difference). (E) MCF7 cells were inoculated subcutaneously into female BALB / C nude mice, and estrogen was applied to the neck every 2 days (E2, 10 -2 M) until the tumor size reaches approximately 100 mm 3 Then the nude mice were randomly divided into two groups (n=3) and injected with 10 nmol ASO (100 μl, 100 mM dissolved in PBS) once a week for a total of 8 times. The tumors were removed, photographed and weighed. (F) (E) Tumor weight statistics (± sem). (G) Figure 6 Quantitative analysis of crystal violet staining in L (±sem, ***P<0.001). (H) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and cultured in phenol red-free medium for 48 h, followed by the addition of tamoxifen (5 μM, 6 h) or estrogen (E2, 10 - 7 M, 6h), followed by RNA extraction and RT-qPCR analysis to detect the expression of LINC02568, CA12 and ERα target genes (±sem). (I, J) MCF7 cells were transfected with ASO-CTL or ASO-LINC02568 and then treated with tamoxifen (5μM, 6h), followed by cell proliferation assay (I) and clone formation assay (J) (±sem, **P<0.01, ***P<0.001). (K) Quantitative analysis results of crystal violet staining in (J) (±sem, ***P<0.001). (L) Figure 6Quantitative analysis results of crystal violet staining in R (±sem, ***P<0.001). (M, N) MCF7 cells were treated with tamoxifen (5μM) or U-104 (50μM) for cell proliferation assay (M) and clone formation assay (N) (±sem, **P<0.01, ***P<0.001). (O) shows the quantitative results of crystal violet dye in (N) (±sem, **P<0.001). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] Clinical specimens and cell lines

[0035] Breast tumor tissue and paired normal tissue samples were obtained from breast cancer patients who underwent surgery at the Second Affiliated Hospital of Shantou University Medical College. Tissue samples were freshly frozen in liquid nitrogen and stored at −80°C before RNA extraction. This study was approved by the Ethics Committee of the Second Affiliated Hospital and was in accordance with government policies and the Declaration of Helsinki. The experiments were conducted with informed and written consent from each subject.

[0036] Human breast cancer cell lines and human embryonic kidney 293T cells were cultured in DMEM or RPMI 1640. All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Tamoxifen-resistant MCF7 (MCF7-TamR) cells were maintained in medium containing tamoxifen (1 μM). When cells were stimulated with estrogen, they were cultured in hormone-deprived phenol red-free medium supplemented with 5% charcoal-stripped FBS.

[0037] RNA extraction and RT-qPCR experiments

[0038] Total RNA was extracted from the samples using Takara's Trizol reagent according to the manufacturer's protocol. The first-strand cDNA was synthesized by All-In-One RT MasterMix (Abm), and then real-time fluorescence quantitative PCR (qPCR) was performed using an AriaMx real-time fluorescence quantitative PCR instrument (Agilent Technologies). The biological experiment was repeated three times, and representative data are shown. The standard error of the mean is also depicted.

[0039] RNA sequencing (RNA-seq) and data computational analysis

[0040] Total RNA was extracted from MCF7 cells using the RNeasy Mini Kit from Qiagen. Ultra TM RNA libraries were prepared using the Directional RNA Library Prep Kit. Paired-end sequencing was performed using the Illumina HiSeq 3000 platform. Sequencing data were aligned to the human reference genome (hg19) using STAR. Expression of RefSeq annotated genes was calculated using Cufflinks, including options -M (masking reads that have been aligned to duplicate regions) and -u (correcting for multiple aligned reads using the 'rescue method'). RNA-seq files have been deposited in the Gene Expression Omnibus database with access number GSE217190. https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE217190(token: kvmfgemclpyxlwr).

[0041] Molecular cloning

[0042] The shRNAs of LINC02568 and CA12 were cloned into the pLKO.1 vector. The target sequences were: shLINC02568-1 (5′-aatatgttctatcctcctagc-3′, SEQ ID NO.01), shLINC02568-2 (5′-gcccagtaataggacaccaag-3′, SEQ ID NO.02), shCA12-1 (5′-gcccagtaataggacaccaag-3′, SEQ ID NO.03), shCA12-2 (5′-gtgataacaagggagtcattt-3′, SEQ ID NO.04).

[0043] Full-length LINC02568 and CA12 were amplified from MCF7 cells and cloned into the pCDH-CMV-MCS-EF1-puro expression vector using XbaI and NotI restriction enzymes. Full-length ESR1 was amplified from MCF7 cells and cloned into the pBoBi expression vector using BamHI and XhoI restriction enzymes for overexpression.

[0044] LINC02568 (LINC02568(WT)) and its mutant form, LINC02568(MUT) with a mutation in the miR-1233-5p binding site, were cloned into the psiCHECK2 expression vector using NotI and XhoI restriction enzymes.

[0045] The 3′UTR fragment of ESR1, including the binding site of miR-1233-5p (ESR1-3′UTR(WT)) and a mutant form with a mutation in the binding site (ESR1-3′UTR(MUT)), were amplified from MCF7 cells and cloned into the psiCHECK2 expression vector.

[0046] SiRNAs, miRNA inhibitors, miRNA mimics, antisense oligonucleotides (ASOs) and plasmid transfection, lentiviral packaging and infection

[0047] SiRNAs, miRNA mimics, miRNA inhibitors and ASOs were purchased from RiboBio (siCTL (siRNC#1, siN0000001-1-5); ASO-CTL (lncRNA ASO Negative Control#1, lnc6N0000001-1-10)). The targeting sequences of siRNAs and ASOs were: siLINC02568 (5′-ggagatcccaagccatgaa-3′, SEQ ID NO.05), siCA12 (5′-ggagaggaccgctgaatat-3′, SEQ ID NO.06), ASO-L1NC02568 (5′-aagagtataaacagaggcca-3′, SEQ ID NO.07). Transfection of siRNAs, ASOs and miRNAs was performed using Lipofectamine 2000. Plasmid transfection was performed using polyethyleneimine (PEI). For the packaging and infection of lentiviral vectors, PEI was used to transfect HEK293T cells with the packaging vectors pMDL, VSVG and REV at a ratio of 10:5:3:2. After 48 h of culture, the viruses were collected, filtered and added to MCF7 cells containing 10 μg / mL polybrene for infection.

[0048] Immunoblot analysis

[0049] Anti-ERα (F-10) (sc-8002) was purchased from Santa Cruz Biotechnology; anti-Flag (F1804) antibody was purchased from Sigma; anti-CA12 (15180-1-AP) antibody was purchased from Proteintech; and anti-β-actin (66009-1-IG) antibody was purchased from Proteintech.

[0050] Cell proliferation assay, colony formation assay, scratch assay and Transwell assay

[0051] For cell proliferation assays, the CellTiter 96 AQueous one solution cell proliferation kit was used to measure cell proliferation. For cell proliferation assays after siRNA transfection, cells were cultured in phenol red-free medium for two days after transfection and then treated with estrogen (E2, 10 -7 M) and then the cell proliferation assay was performed. For lentiviral infection, cells were infected with the virus in normal growth medium for 24 h, then changed to phenol red-free medium for 48 h, and then treated with estrogen (E2, 10 -7 M) and perform cell proliferation test. When measuring cell proliferation, add 20 μL of CellTiter 96AQueous onesolution reagent to 100 μL of culture medium and add to the cell culture dish. Place the cells in a 37°C incubator for 1-2 hours and measure the absorbance at a wavelength of 490 nm. After 4-5 days of measurement, draw a cell proliferation curve.

[0052] For the colony formation experiment, cells transfected with siRNA or infected with lentivirus were re-plated in 6-well plates at a density of 2,000 cells per well. After 10 days of culture, cells were harvested for detection. Specifically, the clones were first fixed with methanol / acetic acid solution (3:1) for 5 min, followed by staining with 0.1% crystal violet for 15 min. After being thoroughly rinsed with PBS, the clones were photographed. The crystal violet was washed away with 10% acetic acid, and the absorbance was measured at a wavelength of 590 nm.

[0053] For the scratch assay, cells transfected with siRNA or infected with lentivirus were re-seeded in 6-well plates and estrogen (E2, 10 -7 M) treatment. Use a 200μL pipette tip to scratch the cell surface. After rinsing with PBS and removing cell debris, take pictures of the scratches at three different locations. Use image J to measure and record the size of the scratch area and calculate it as A0. Take the image again after 48h, and measure the size of the scratch area as A1. Cell migration is expressed using the healing rate. Healing rate (%) = (healing area (A0-A1) / healing area A0) × 100%.

[0054] For the Transwell experiment, cells transfected with siRNA or infected with lentivirus were resuspended in serum-free medium and inoculated into the upper chamber of the Transwell chamber, and complete medium containing 10% FBS was added to the lower chamber. The chamber was placed in a 37°C cell culture incubator for 36 hours to allow the cells to migrate to the lower chamber. After 36 hours, the chamber was removed and rinsed with PBS, then fixed with 4% paraformaldehyde at 4°C for 15 minutes and then stained with 0.1% crystal violet for 10 minutes. After being thoroughly rinsed with PBS, the cells that did not migrate to the lower chamber were wiped off with a cotton swab, and the number of migrated cells in the field of view was photographed and counted using a microscope.

[0055] Nude mouse xenograft experiments

[0056] BALB / C female nude mice (aged 4–6 weeks) used for xenograft models were housed under pathogen-free conditions at the Xiamen University Animal Center and followed protocols approved by the Xiamen University Animal Ethics Committee. For xenograft experiments, 5 × 10 mice infected with shCTL or shLINC02568 lentivirus were placed in the xenograft chamber. 6 MCF7 cells were suspended in PBS and inoculated subcutaneously into nude mice. To maintain tumor growth, estrogen (10 -2 M). All nude mice were sacrificed after 8 weeks, and the tumors were removed, photographed, and weighed.

[0057] For in vivo ASO treatment, 5 × 10 6 MCF7 cells were inoculated subcutaneously in nude mice, and estrogen (10 -2 M) to stimulate tumor growth until the tumor volume reaches approximately 100 mm 3 Mice were randomly divided into two groups and ASO was injected intratumorally at a dose of 5 nmol (50 μL, 50 mM dissolved in PBS) every 2 days or injected into the tail vein at a dose of 10 nmol (100 μL, 50 mM dissolved in PBS) every 2 days. Tumors were removed after 8 treatments, photographed and weighed.

[0058] For the combination of ASO and ICI or U-104 in vivo, 5×10 6 MCF7 cells were inoculated subcutaneously in nude mice, and estrogen (10 -2 M) to stimulate tumor growth until the tumor volume reaches approximately 100 mm 3Mice were randomly divided into control group, ASO treatment group, ICI / U-104 treatment group, and ASO and ICI or ASO and U-104 treatment group. ASO was injected intratumorally 6 times every 2 days at a dose of 5nmol (50μL, 50mM dissolved in PBS), ICI was injected subcutaneously once a week at a dose of 250mg / kg, and U-104 was injected intraperitoneally once every 2 days at a dose of 50mg / kg. After the treatment, the tumors were removed, photographed, and weighed.

[0059] Isolation of cytoplasmic and nuclear RNA

[0060] The cells were washed twice with pre-cooled PBS, then scraped with a cell scraper and collected in a centrifuge tube. The cell pellet was suspended in 1 mL of pre-cooled lysis buffer (10 mM HEPES (pH 8.0), 1.5 μL M MgCl2, 10 mM KCl, and 1 μL MDTT) and incubated on ice for 15 min to swell the cells. 1% NP-40 was added to the lysate, vortexed for 10 seconds, and then centrifuged at maximum speed for 2-3 min. The supernatant was used as a cytoplasmic isolate and the precipitate was used as a nuclear isolate. Trizol (Takara) was then used to extract the RNA of the cytoplasm and nucleus.

[0061] Competing endogenous RNA (CeRNA) network analysis

[0062] Three different algorithms were used, TarPmiR 60 、miRand 61 and RNAhybrid 62 , predict miRNAs that interact with LINC02568. The mRNAs that these three miRNAs may target were predicted by Targetscan. The mRNA target genes that were positively regulated by LINC02568 were retained and analyzed using Cytoscape 63 Construction of the CeRNA network of LINC02568.

[0063] Luciferase reporter gene assay

[0064] To determine the interaction between miRNA and target genes, the 3'UTR of LINC02568 and ESR1 and the luciferase reporter gene plasmids with mutant forms of the miR-1233-5p binding site were co-transfected into MCF7 cells with miR-1233-5p mimics. Dual luciferase reporter gene assay was performed 48 h later and luciferase activity was determined using Dual Luciferase Assay Kit.

[0065] Chromatin immunoprecipitation (ChIP) assay

[0066] After the cells were treated, they were fixed with 1% formaldehyde for 15 min at room temperature. Fixation was stopped with glycine (0.125 M) and washed twice with PBS. Cells were lysed using lysis buffer (1% SDS, 10 mM EDTA (pH 8.0) and 50 mM Tris-Cl (pH 7.8)), and chromatin DNA was broken to an average size of 300-500 bp using a non-contact sonicator. IgG or anti-RNA polymerase II antibody was then added for immunoprecipitation at 4°C overnight, and then incubated with protein G magnetic beads at 4°C for 4 h. The magnetic beads were washed with TSE1 (0.1% SDS, 1% TritonX-100, 2mM EDTA (pH 8.0), 150mM NaCl and 20mM Tris-Cl (pH 7.8)), TSE2 (0.1% SDS, 1% TritonX-100, 2mM EDTA (pH 8.0), 400mM NaCl and 20mM Tris-Cl (pH 7.8)), and then washed twice with TE buffer (50mM Tris-Cl (pH 8.0) and 10mM EDTA (pH 8.0)). The magnetic beads were resuspended in TE / SDS buffer (TE buffer contains 1% SDS) and cross-linked at 65°C overnight. DNA was extracted using the QIAquick DNA purification kit and qPCR analysis was performed.

[0067] Intracellular and extracellular pH measurements

[0068] For the measurement of intracellular pH, cells transfected with siRNA or infected with lentivirus were re-seeded in 96-well plates. The cells were washed twice with HEPES buffer (150mM NaCl, 5mM KCl, 5mM glucose and 20mM HEPES (pH 7.4)). HEPES buffer containing pH-sensitive dye BCECF-AM (5μM) was added to the culture dish and incubated at 37°C in the dark for 30min. The cells were then washed twice with HEPES buffer, and the fluorescence intensity was measured at an excitation wavelength of 488nm and emission wavelengths of 530nm and 640nm. The ratio of the emission intensity at 530nm to the emission intensity at 640nm was converted to intracellular pH according to the standard curve. For the drawing of the standard curve, Nigericin (10μg / ml) was added to high K+ phosphate buffer at a specific pH (6.6, 7.0, 7.2, 7.4, 7.8 and 8.2) for the determination of the standard.

[0069] For the measurement of extracellular pH, the cell culture medium was collected and measured using a pH meter.

[0070] Extracellular acidification rate (ECAR) measurement

[0071] The extracellular acidification rate (ECAR) of MCF7 cells was measured in the Extracellular Flux AnalyzerXFe96 using the Seahorse XF Cell Glycolysis Stress Test Kit. Specifically, MCF7 cells transfected with siRNA or infected with lentivirus were re-seeded in Seahorse 96-well assay plates with approximately 10,000 cells per well. After cell attachment, the medium was changed to XF DMEM medium without bicarbonate, glucose, pyruvate, and glutamine (pH 7.4) and incubated in an incubator (37°C, without C02) for 1 h. Then, Glucose, oligomycin, and 2-DG (Glucose, 10 mM; oligomycin, 1 μM; 2-DG, 50 mM) were added in sequence. The ECAR was measured three times, each for 6 min, with a measurement time and a mixing time of 3 min between measurements. The data obtained were used to evaluate glycolytic function, including glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification.

[0072] Statistical analysis

[0073] The differences between experimental and control groups were compared using Student's t test, and multiple group comparisons were performed using two-way analysis of variance (ANOVA). The results of xenograft experiments and clinical breast samples were analyzed using GraphPad Prism 7. The results are expressed as the mean ± standard error (sem) of at least three independent experiments. All statistical analyses were performed using two-tailed P values.

[0074] 2. Experimental Results

[0075] Estrogen induces the expression of a large number of mRNAs and adjacent lncRNAs in ER+ breast cancer cells

[0076] In order to find regulatory factors that play a key role in the estrogen / ERα signaling pathway in ER+ breast cancer, this example analyzed the estrogen-regulated transcriptome in MCF7 cells ( Figure 8 A). The results showed that a total of 767 mRNAs and 619 mRNAs were up-regulated or down-regulated under the stimulation of estrogen (q<0.05, FC>1.5) ( Figure 1 A). At the same time, this example found that a large number of lncRNAs are also regulated by estrogen ( Figure 1 B). The effects of estrogen induction on some representative lncRNAs were verified by RT-qPCR analysis ( Figure 1C) In addition, this example also analyzed estrogen-regulated 1ncRNA in other ER+ breast cancer cells, including CAMA-1, EFM-19, HCC1500, MDA-MB-134-VI and T-47D 42 The results showed that 135 and 106 lncRNAs were induced or repressed by estrogen in at least three cell lines, respectively. Figure 8 B).

[0077] It has been reported that lncRNAs are often induced by estrogen together with nearby coding genes. Therefore, this example analyzes the co-expression pattern of estrogen-induced lncRNAs and their nearby coding genes in MCF7 cells. Among all estrogen-induced lncRNAs, 63.27% of lncRNAs were co-induced with their nearby mRNAs. The UCSC genome browser shows a visualization of some representative lncRNA-mRNA pairs, and this co-induction pattern was subsequently further verified by RT-qPCR analysis ( Figure 1 D-1F). In addition, this example also examined the expression of lncRNA-mRNA pairs in normal and ER+ breast tumor tissues (n=10). The results showed that C15orf59-AS1 / C15orf59 were upregulated in all ER+ breast tumors, and the remaining lncRNA / mRNA pairs were upregulated in at least half of the ER+ breast tumors ( Figure 8 C). To test whether these lncRNA-mRNA pairs are directly associated with ERα, this example analyzes the chromatin binding of ERα in the presence or absence of estrogen. The results show that about 73% of lncRNA-mRNA pairs have ERα binding in the vicinity of chromatin. These ERα binding sites are enriched for active enhancer marks, including H3K4me1 / 2, H3K27Ac, ​​p300, and MED1, and lack repressive histone marks such as H3K9me3 and H3K27me3 ( Figure 1 G and 1H). Treatment with the selective estrogen receptor downregulator fulvestrant (ICI) resulted in decreased expression of lncRNAs, such as DSCAM-AS1 and LINC02568, and their mRNA partners, further indicating that estrogen-induced lncRNA-mRNA pairs are regulated by ERα ( Figure 1 I and 1J). These data suggest that a large number of lncRNAs are co-induced with their nearby coding genes and are directly regulated by ERα.

[0078] Estrogen-induced lncRNA-LINC02568 promotes the malignant phenotype of ER+ breast cancer cells in vivo and in vitro

[0079] In order to explore the clinical relevance of estrogen-induced lncRNAs found in MCF7 cells (q < 0.05, FC > 1.5, n = 129), this example used the GEPIA2 database to search for the expression of these lncRNAs in ER+ breast cancer. The results showed that compared with normal breast tissue, LINC02568 was most significantly overexpressed in ER+ breast tumors (P ≤ 0.01) ( Figure 2 A). Further analysis based on the TCGA dataset showed that LINC02568 was highly expressed in ER+ breast tumor tissues (LumA and LumB) compared with other subtypes ( Figure 2 B). Detection in clinical breast cancer tumor samples found that the expression of LINC02568 in ER+ breast tumor tissue was significantly higher than that in normal tissue, adjacent tissue and ER- breast tumor tissue ( Figure 2 C). In addition, LINC02568 was highly expressed in ER+ breast cancer cell lines compared with normal breast epithelial cell lines and other subtypes of breast cancer cell lines such as triple-negative breast cancer ( Figure 2 D). More interestingly, LINC02568 appears to be significantly overexpressed only in breast tumor tissues, but not in other tumor types, compared with corresponding normal tissues ( Fig. 9 A).

[0080] The high expression of LINC02568 in clinical breast specimens prompted this example to explore whether it plays an important role in the growth and tumorigenesis of ER+ breast cancer cells. To this end, two independent shRNAs were designed to interfere with the expression of LINC02568 ( Figure 2 E). Silencing of LINC02568 significantly reduced the proliferation rate of MCF7 cells ( Figure 2 F) and colony forming ability ( Figure 2 G and S2B). In addition, through wound healing and transwell experiments, this example found that LINC02568 deficiency significantly inhibited the migration and invasion of MCF7 cells ( Figure 2 H and 2I, and Fig. 9 C and S2D). In contrast, overexpression of LINC02568 significantly promoted the proliferation, colony formation, migration, and invasion abilities of MCF7 cells, further demonstrating the oncogenic role of LINC02568 ( Figure 2 J-2N and Fig. 9 E-S2G). siRNA targeting LINC02568 also inhibited the proliferation, colony formation, migration and invasion of MCF7 cells ( Fig. 9 H-S2O) and was confirmed in another ER+ breast cancer cell line, T47D ( Fig. 9P-S2V). To explore whether LINC02568 is essential for estrogen-induced tumorigenesis in vivo, in this example, MCF7 cells infected with shCTL or shLINC02568 lentivirus were injected subcutaneously into BALB / C nude mice for nude mouse tumorigenesis experiments. The results showed that silencing of LINC02568 significantly inhibited estrogen-induced tumor growth ( Figure 2 O and 2P). In addition, this example also explores the potential role of LINC02568 in overcoming tamoxifen resistance in tamoxifen-resistant MCF7 cells (MCF7-TamR). Cell proliferation and clone formation results showed that LINC02568 deficiency can restore the sensitivity of MCF7-TamR cells to tamoxifen ( Figure 2 Q-2S). In summary, the data of this example show that LINC02568 promotes malignant expression and resistance to endocrine therapy drugs (such as tamoxifen) in ER+ breast cancer cells.

[0081] LINC02568 promotes ERα target gene activation and the malignant phenotype of ER+ breast cancer by regulating ERα expression

[0082] In order to understand the molecular mechanism by which LINC02568 regulates the malignant phenotype of ER+ breast cancer cells, this example first evaluated the coding potential of LINC02568 by CPAT (http: / / lilab.research.bcm.edu / cpat / ). The results of CPAT analysis showed that LINC02568 had no coding ability (coding probability = 0.0183). Then, this example detected the subcellular localization of LINC02568 by nuclear-cytoplasmic separation and RT-qPCR analysis. The results showed that LINC02568 was localized in both the cytoplasm and the nucleus, but was mainly localized in the cytoplasm ( Figure 3 A). In order to gain a deeper understanding of the molecular mechanism of LINC02568, this example performed RNA-seq analysis on MCF7 cells transfected with siCTL or siLINC02568. The results showed that 1,497 genes were induced by LINC02568 and 2,110 genes were inhibited (q<0.05, FC>1.5) ( Figure 3 B). Heatmap and box plot showing the expression of genes regulated by LINC02568 ( Figure 3 C and 3D). Hallmark analysis results showed that E2F target genes, G2 / M checkpoint and early estrogen response were the most enriched features of the LINC02568 positively regulated gene cluster ( Figure 3E). In particular, this example found that the expression of a large number of estrogen target genes (such as PGR, TFF1, GREB1, NRIP1, SMAD7, SIAH2, CCND1 and BCL2) depends on LINC02568 ( Figure 3 E).

[0083] Next, this example attempts to analyze the molecular mechanism by which LINC02568 regulates the transcriptional activation of these estrogen target genes. Since LINC02568 is mainly localized in the cytoplasm, this example explores whether LINC02568 can regulate target mRNA as a miRNA sponge. To this end, this example constructed a ceRNA network to connect LINC02568 to its positively regulated target genes through miRNA ( Fig.10 A). In the ceRNA network, this example obtained 206 genes positively regulated by LINC02568 and 5 miRNAs, miR-1226-5p, miR-8070, miR-6778-5p, miR-1233-5p and miR-6849-3p ( Fig.10 A). In order to find target genes that are clinically relevant in ER+ breast cancer and highly correlated with LINC02568, this example first searched the expression of these 206 genes in the GEPIA2 database, and obtained 18 genes that were significantly highly expressed in ER+ breast tumor tissues (FC≥2, P≤0.01) ( Figure 3 F). Then, this example searches for the correlation between LINC02568 and the expression of these 18 genes, and finds that the three genes highly correlated with LINC02568 in breast tumor tissues are ESR1, RALGPS2, and GREB1 (R≥0.3) ( Figure 3 F).

[0084] Since the protein product of ESR1, ERα, is the main driver of estrogen target gene transcriptional activation, ER+ breast cancer development, and endocrine drug resistance, this example believes that LINC02568 may play its role in these aspects by regulating the expression of ESR1. This example first confirmed that LINC02568 deficiency can lead to a significant decrease in ERα mRNA and protein levels (ERα-66)( Figure 3 G and 3H). LINC02568 also modulated the protein level of ERα-46, an ERα variant that shares the same 3'UTR region with ERα-66. Another ERα variant, ERα-36, was not affected because it differs from ERα-66 and ERα-46 in the 3'UTR region ( Figure 3 H). The visualization analysis results of RNA-seq also showed that the expression level of ESR1 was downregulated after LINC02568 loss ( Fig.10 B). In addition, this example also tested the effect of LINC02568 on exogenous ERα mRNA and protein levels in the ERα-inducible MCF7 cell line. The results showed that LINC02568 had no significant effect on the expression of exogenous ERα, indicating that LINC02568 does not regulate the expression of ERα at the translation stage ( Figure 3 I and 3J). After LINC02568 deletion, the stability of ESR1 mRNA was also significantly reduced ( Figure 3 K). Consistent with its role in regulating ERα expression, silencing of LINC02568 suppressed transcriptional activation of ERα target genes such as PGR, TFF1, GREB1, NRIP1, SMAD7, and SIAH2 ( Figure 3 L and Fig.10 C). Similar results were obtained with T47D cells ( Fig.10 D). In contrast, overexpression of LINC02568 further promoted the expression of estrogen target genes ( Figure 3 M).

[0085] To further confirm whether LINC02568 exerts its oncogenic effect by regulating ERα, this example complemented ERα in LINC02568-deficient cells. The results showed that overexpressed ERα partially restored the phenomenon of suppressed activation of ERα target genes caused by LINC02568 deficiency ( Figure 3 N). In addition, overexpressed ERα also largely compensated for the effects of LINC02568 knockdown on cell proliferation and clone formation ( Figure 3 O-3Q). In summary, the data of this example show that LINC02568 regulates the activation of ERα target genes and the growth of ER+ breast cancer cells by regulating the expression of ESR1.

[0086] LINC02568 acts as a sponge for miR-1233-5p to promote the expression of ESR1- and ERα-target genes and the malignant phenotype of ER+ breast cancer cells

[0087] Next, this example attempts to investigate how LINC02568 regulates the expression of ESR1. According to the ceRNA network, LINC02568 is predicted to bind to miR-1233-5p and miR-6778-5p, both of which also bind to ESR1 mRNA ( Fig.10A). To test the effects of these two miRNAs on LINC02568 and ESR1 expression, this example used control, miR-1233-5p or miR-6778-5p mimics to transfect MCF7 cells, and then performed RT-qPCR analysis. The results showed that overexpression of miR-1233-5p significantly reduced LINC02568 and ESR1 mRNA levels, while miR-6778-5p had no significant effect ( Figure 4 A). Next, this example detected whether miR-1233-5p could bind to LINC02568 and the 3′UTR of ESR1. This example first predicted the potential binding sites of miR-1233-5p, LINC02568 and ESR1 3′UTR ( Figure 4 B and 4C), and constructed luciferase reporter gene plasmids containing binding sites and binding site mutations, which were then co-transfected into MCF7 cells with control mimic or miR-1233-5p mimic for dual luciferase reporter gene detection. The results showed that miR-1233-5p mimic transfection significantly reduced the fluorescence intensity of LINC02568(WT)-luc, but did not affect the fluorescence intensity of LINC02568(MUT)-luc ( Figure 4 D). Similarly, miR-1233-5p mimic specifically reduced the fluorescence intensity of ESR1 3′UTR1-luc, but did not affect the fluorescence intensity of plasmids that did not contain the binding site (ESR1 3′UTR2-luc, ESR1 3′UTR3-luc, ESR1 3′UTR4-luc, or ESR1 3′UTR5-luc) or plasmids with a binding site mutation (ESR1 3′UTR1(MUT)-luc). Figure 4 E). These data indicate that miR-1233-5p can simultaneously bind to LINC02568 and the 3'UTR region of ESR1 mRNA and regulate their expressions.

[0088] Next, this example tested whether the regulation of ESR1 and ERα target genes by LINC02568 was carried out through miR-1233-5p. In this example, the expression of miR-1233-5p was inhibited in MCF7 cells lacking LINC02568, and then RT-qPCR was performed to detect the expression of ESR1 and ERα target genes. The results showed that the inhibition of miR-1233-5p partially complemented the regulation of ESR1 ( Figure 4 F and 4G) and the expression of ERα target genes ( Figure 4H). To further test whether LINC02568 regulates the malignant phenotype of ER+ breast cancer cells through miR-1233-5p, this example first examined the function of miR-1233-5p itself. Compared with normal tissues, the expression of miR-1233-5p in ER+ breast tumors did not change significantly ( Fig.10 E). Figure 2 In O, there was no significant change in miR-1233-5p in LINC02568 knockout tumors ( Fig.10 F). However, loss of miR-1233-5p led to a significant increase in the proliferation rate and clone number of MCF7 cells, while overexpression of miR-1233-5p showed the opposite effect, indicating the tumor suppressor function of miR-1233-5p ( Figure 4 I-4L and Fig.10 G, S3H). Similarly, the inhibition of cell proliferation and clone formation caused by LINC02568 deficiency was partially restored after miR-1233-5p was inhibited ( Figure 4 I and 4J, and Fig.10 G). In summary, the data of this example show that LINC02568 regulates the expression of ESR1 by exerting a sponge effect on miR-1233-5p, promoting the activation of ERa target genes and the growth of ER+ breast cancer cells.

[0089] LINC02568 cis-regulates CA12 expression, modulates tumor-specific pH homeostasis, and promotes the malignant phenotype of ER+ breast cancer cells

[0090] Based on the above results, LINC02568 is distributed in both the cytoplasm and the nucleus ( Figure 3 A), while under estrogen stimulation, LINC02568 and its neighboring gene CA12 are induced to express together ( Figure 1 H). Therefore, this example speculates that LINC02568 may function in the nucleus through cis regulation in addition to exerting its carcinogenic effect in the cytoplasm through trans action. To verify this hypothesis, this example detected whether the two genes adjacent to LINC02568 in the upstream and downstream of the genome were regulated by it. The results showed that knockdown of LINC02568 weakened the expression of CA12, while another neighboring gene USP3 was not affected ( Figure 5 A and 5B). It has been well documented that lncRNAs are often located in the same “topologically associated domains” (TADs) as nearby genes to regulate chromatin accessibility and thus transcriptional activation of nearby gene promoters. 43-45When LINC02568 was knocked down, the pre-mRNA level of CA12 was reduced, indicating that the regulatory effect of LINC02568 on CA12 occurred at the transcriptional stage ( Figure 5 C) Figure 2 In LINC02568 knockdown tumors shown in O, CA12 mRNA and pre-mRNA levels were also decreased ( Fig.11 A and S4B). To further demonstrate the transcriptional regulation of CA12 by LINC02568, this example used ChIP-qPCR to detect that after LINC02568 was knocked down, the binding of RNA polymerase II in the CA12 promoter region was significantly reduced ( Figure 5 D). Similar to LINC02568, CA12 is highly expressed in ER+ breast tumors (LumA and LumB) ( Fig.11 C). The detection of clinical samples showed that the expression of CA12 in ER+ tumor tissues was significantly higher than that in adjacent tissues and normal tissues, as well as in ER- breast cancer tissues ( Fig.11 D). In addition, LINC02568 was highly expressed in ER+ breast cancer cell lines compared with normal breast epithelial cells and other subtypes of breast cancer cells (e.g., triple-negative breast cancer). Fig.11 E). In addition, analysis based on the TANRIC database (https: / / www.tanric.org) showed that in ER+ breast tumor tissues, CA12 expression was most correlated with LINC02568 expression ( Fig.11 F); this correlation was also confirmed in ER+ tumor tissue samples ( Fig.11 G). CA12 is the most abundant member of the CA family and is only induced by estrogen, which enhances its remarkable effect ( Fig.11 H). In summary, the data of this example revealed that LINC02568 cis-regulates the transcriptional activity of the adjacent gene CA12, and the expression of the two genes is highly correlated in ER+ breast cancer.

[0091] Next, this example explores whether LINC02568's regulation of CA12 is involved in its carcinogenic effect on ER+ breast cancer. CA12 is a member of the membrane-associated carbonic anhydrase protein family, which plays an important role in generating and maintaining pH differences in the hypoxic tumor microenvironment and can promote the growth and metastasis of tumor cells. This example first detected whether CA12 has an effect on the pH homeostasis of ER+ breast cancer cells. This example designed two shRNAs specifically targeting CA12 to knock down CA12 in MCF7 cells ( Figure 5 E and 5F). The results showed that after knocking down CA12, the intracellular pH of MCF7 cells was significantly decreased ( Figure 5 G), while the extracellular pH increases ( Figure 5 H). Next, this example detected whether CA12 had an effect on the malignant phenotype of MCF7 cells. The results showed that CA12 deficiency significantly inhibited the cell proliferation, colony formation, migration and invasion ability of MCF7 cells ( Figure 5 I-5L and Fig.12 A-S5C). When CA12 was knocked down using siRNA, similar effects were found on intracellular and extracellular pH, cell proliferation, migration, and invasion ( Fig.12 D-S5N). In contrast, overexpression of CA12 showed the opposite effect, further reinforcing the importance of CA12 in regulating tumor-specific pH homeostasis and promoting the malignant phenotype of MCF7 cells ( Fig.12 O-S5W). In addition, knockdown of CA12 also inhibited cell proliferation of MCF7-TamR cells ( Figure 5 M).

[0092] After demonstrating that LINC02568 plays an important role in controlling tumor-specific pH homeostasis and promoting the malignant manifestation of ER+ breast cancer cells, this example next examined whether CA12, as a downstream target of LINC02568, is involved in the carcinogenic effect of LINC02568. First, this example found that knocking down LINC02568 would lead to a decrease in the intracellular pH of MCF7 cells and an increase in the extracellular pH ( Figure 5 N and 5O, Fig.12 X and S5Y). In contrast, overexpression of LINC02568 resulted in a decrease in extracellular pH ( Fig.12 Z). Meanwhile, knockdown of LINC02568 reduced the extracellular acidification rate (ECAR) and non-glycolytic acidification in MCF7 cells, while overexpression of LINC02568 had the opposite effect, indicating that LINC02568 is not only involved in regulating extracellular acidification under basal conditions, but also in regulating downstream cellular metabolic activities, such as glycolysis ( Fig.12 X′-S5Y′). Next, in order to test whether CA12 is involved in the regulation of LINC02568 on cell pH and the effect on the malignant phenotype of ER+ breast cancer, this example replenished CA12 in LINC02568 knockdown cells. The results showed that overexpressed CA12 significantly compensated for the adverse effects of LINC02568 deficiency on intracellular and extracellular pH, cell proliferation, clone formation, migration and invasion ( Figure 5 P-5U, Fig.12 Z′-S5Y″). In addition, overexpression of CA12 in MCF7-TamR cells also partially reversed the effect of LINC02568 knockdown on tamoxifen sensitivity ( Figure 5V). In summary, the data of this example revealed that LINC02568 controls tumor-specific pH homeostasis and promotes the malignant phenotype of ER+ breast cancer cells by regulating the expression of CA12.

[0093] LINC02568 as a potential therapeutic target for ER+ breast cancer

[0094] The important regulatory functions of LINC02568 on ERa target gene activation and pH homeostasis, as well as the regulation of tumor growth and drug resistance in ER+ breast cancer cells, prompted this example to explore the potential of LINC02568 as a therapeutic target. In this example, antisense oligonucleotides (ASOs) specifically targeting LINC02568 were designed and synthesized to interfere with the expression of LINC02568 ( Figure 6 A). ASO targeting LINC02568 significantly inhibited the expression of downstream target genes ( Figure 6 A). In addition, ASO also significantly inhibited the proliferation, colony formation, migration and invasion of MCF7 cells ( Figure 6 B-6E and Figures S6A-S6C). To further explore the effect of ASO in animals, in this example, MCF7 cells were subcutaneously inoculated in nude mice and the mice were treated with cholesterol-modified ASO. The results showed that intratumoral injection of ASO targeting LINC02568 significantly inhibited estrogen-induced tumor growth ( Figure 6 F and 6G). The expression of LINC02568-related target genes was also significantly reduced in tumor tissues after ASO treatment, while the expression of miR-1233-5p did not change significantly ( Figure 6 H and 6I, and Fig.13 D). Although not as significant as intratumoral injection, intravenous injection of ASO targeting LINC02568 also inhibited tumor growth in mice ( Fig.13 E and S6F).

[0095] The positive feedback regulation of LINC02568 on ERα suggests that the combination of ASO targeting LINC02568 and endocrine therapy drugs (such as tamoxifen and fulvestrant) may produce a synergistic effect on tumor suppression. To verify this hypothesis, this example used ASO in combination with endocrine therapy drugs ICI and tamoxifen in MCF7 cells. Compared with the use of ASO or ICI alone, the combined treatment of ASO and ICI produced a better inhibitory effect on target gene expression ( Figure 6 J). The combination of ASO and ICI also produced a synergistic inhibitory effect on cell proliferation and clone formation ( Figure 6 K and 6L, and Fig.13 G). Similar effects were observed when ASO targeting LINC02568 was combined with tamoxifen ( Fig.13H-S6K). This example further examined the effect of combined ASO and ICI therapy on tumor growth in a mouse xenograft model. Consistent with the results observed in cultured cells, combined therapy also had a synergistic inhibitory effect on tumor growth of MCF7 cells in mice ( Figure 6 M and 6N), while the body weight of mice was not significantly affected ( Figure 6 O). In the tumors of mice treated with the combined therapy, the expression of LINC02568-related target genes also showed a synergistic decrease ( Figure 6 P). In addition, this example also tested the effect of ASO targeting LINC02568 in combination with U-104, a small molecule inhibitor targeting CA12. The results showed that the combination of ASO and U-104 also produced a synergistic effect on the proliferation and clone formation of MCF7 cells ( Figure 6 Q and 6R, and Fig.13 L), and similarly, it also had a synergistic inhibitory effect on the growth of MCF7 cells in mice ( Figure 6 Similar to the ASO targeting LINC02568, treatment with U-104 also made MCF7 cells more sensitive to tamoxifen ( Fig.13 In summary, the results of this example indicate that LINC02568 can be used as a potential therapeutic target for ER+ breast cancer, and ASO targeting LINC02568 alone or in combination with endocrine therapy drugs or CA12 inhibitors has a potential synergistic effect in inhibiting the growth of ER+ breast cancer.

[0096] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Application of LINC02568 as a target in the preparation of a therapeutic kit for ER+ breast cancer.

2. The use according to claim 1, characterized in that: The ER+ breast cancer treatment kit contains reagents for targeted inhibition of LINC02568.

3. The use according to claim 2, characterized in that: The reagent is an antisense nucleotide.

4. The use according to claim 3, characterized in that: The ER+ breast cancer treatment kit also contains endocrine therapy drugs and / or small molecule inhibitors targeting CA12.

5. The use according to claim 4, characterized in that: The endocrine therapy drug is selected from tamoxifen and fulvestrant, and the small molecule inhibitor targeting CA12 is U-104.

6. A kit for treating ER+ breast cancer, characterized in that: Agents with targeted inhibition of LINC02568.

7. A kit for treating ER+ breast cancer as claimed in claim 6, characterized in that: The reagent is an antisense nucleotide.

8. A kit for treating ER+ breast cancer according to claim 7, characterized in that: There are also endocrine therapy drugs and / or small molecule inhibitors targeting CA12.

9. A kit for treating ER+ breast cancer according to claim 8, characterized in that: The endocrine therapy drug is selected from tamoxifen and fulvestrant, and the small molecule inhibitor targeting CA12 is U-104.

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