Lymphometastatic breast cancer treatment target and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a lymphatic metastatic breast cancer treatment target and application thereof. The exosome piR-hsa-28212 disclosed by the invention can synergistically enhance VEGFC / VEGFR3 signal transduction, so that lymphatic vessel generation in breast cancer is promoted, and the potential of the exosome piR-hsa-28212 serving as a treatment target of lymphatic metastatic breast cancer is indicated. Besides, piR-hsa-28212 in the serum exosome of the breast cancer patient with lymphatic metastasis is obviously up-regulated, so that the piR-hsa-28212 can be used as a serum marker for predicting the lymphatic metastasis of the breast cancer, and has the advantages of noninvasive property, in-vitro stability, acquisition of samples and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a lymph node metastatic breast cancer treatment target and application thereof. Background Art

[0002] Breast cancer (BC) is one of the most common cancers worldwide, accounting for 32% of cancers in women. Although increasing evidence suggests that lymph node (LN) metastasis is a poor prognostic factor for breast cancer, the exact mechanisms driving breast cancer LN metastasis remain unclear. Therefore, fully clarifying the molecular mechanisms that trigger breast cancer LN metastasis is of great clinical significance for exploring effective drug targets for therapeutic intervention in breast cancer patients.

[0003] The vascular endothelial growth factor C (VEGFC) / VEGF receptor 3 (VEGFR3) pathway is one of the most recognized lymph node metastasis signals in various tumors. The interaction between VEGFC and VEGFR3 has the potential to activate the formation and expansion of tumor-associated lymphatic vessels, thereby promoting the spread of cancer cells to lymph nodes, promoting tumor progression and facilitating distant metastasis. However, the precise mechanism by which coordinated stimulation of VEGFC / VEGFR3 signaling promotes lymph node metastasis in BC remains to be fully elucidated.

[0004] Exosomes are nanoscale lipid bilayer vesicles with a diameter of 30 to 150 nm that have tissue-specific anchoring proteins that facilitate targeted endocytosis in recipient cells. Tumor-derived exosomes carry bioactive molecules that facilitate communication between tumor cells and the tumor microenvironment (TME). This transport is essential for the establishment of a pre-metastatic niche, a key step in cancer metastasis.

[0005] Exosomes have the ability to penetrate the lymphatic system through the stroma, thus acting as an effective medium for communication between tumor cells and the lymphatic system. PIWI-interacting RNAs (piRNAs) are 24-32 nucleotides in length and have 2'-O-methylated 3' ends, representing the largest class of small ncRNAs. PiRNAs have been shown to play pro- or anti-cancer roles by directly binding to PIWI proteins (highly conserved RNA-binding proteins) to form RNA-induced silencing complexes (RISCs). PiRNAs can be encapsulated into exosomes and released into the circulation, where they are subsequently taken up by recipient cells to exert their biological functions. The widespread distribution of piRNAs through exosomes and their important regulatory functions enhance their potential as key factors in cancer lymphatic metastasis. A large number of previous studies have focused on exosomal miRNAs, lncRNAs, and circRNAs, but the role of exosomal piRNAs in breast cancer lymph node metastasis remains underexplored. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, the present invention provides a lymph node metastatic breast cancer treatment target and application thereof.

[0007] The present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a therapeutic target for lymph node metastatic breast cancer, wherein the target piR-hsa-28212 sequence is shown in SEQ ID NO.1, and piR-hsa-28212 activates the METTL3 / IGF2BP2-m6A signaling pathway, leading to increased VEGFC expression and secretion.

[0008] In a second aspect, the present invention provides the use of the target piR-hsa-28212 in the preparation of a diagnostic product for lymph node metastatic breast cancer.

[0009] Compared with the prior art, the present invention has the following advantages: (1) The exosomal piR-hsa-28212 of the present invention synergistically enhances VEGFC / VEGFR3 signaling, thereby promoting lymphangiogenesis in breast cancer, indicating its potential as a therapeutic target for lymphatic metastatic breast cancer.

[0010] (2) piR-hsa-28212 was significantly upregulated in serum exosomes of breast cancer patients with lymph node metastasis, and can be used as a serum marker for predicting lymph node metastasis of breast cancer. It has the advantages of being non-invasive, stable in vitro, and easy to obtain specimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be further described below in conjunction with the accompanying drawings.

[0012] Figure 1 Figures for the validation experiment of piR-hsa-28212 overexpression; A is a diagram of qRT-PCR analysis confirming the dysregulated expression of piRNA in serum exosomes of 28 LN- and 28 LN+ breast cancer patients; B is a ROC curve diagram of the diagnostic efficacy of piR-hsa-28212 in detecting lymph node metastasis in 56 breast cancer patients; C is a qRT-PCR analysis diagram of piR-hsa-28212 expression in MDA-MB-468 cells and exosomes derived from them (MDA-MB-468-EXO) after inhibiting piR-hsa-28212; D is a qRT-PCR analysis diagram of piR-hsa-28212 expression in MDA-MB-231 cells and exosomes derived from them (MDA-MB-231-EXO) after overexpression of piR-hsa-28212; Figure 2Figure 2 is a graph showing the expression level of piR-hsa-28212 in HLECs incubated under different conditions; A is a graph showing the expression level of piR-hsa-28212 in HLECs incubated with PBS, MDA-MB-231-EXO or MDA-MB-468-EXO; B is a graph showing the qRT-PCR analysis of piR-hsa-28212 expression in HLECs after incubation with MDA-MB-468-EXOsh-28212, MDA-MB-231-EXOmimic-28212 and their respective controls; Figure 3 Quantitative analysis of tube formation and Transwell migration of HLECs; wherein, A is a tube formation analysis diagram of HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212; B is a relative node number diagram of HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212; C is a relative grid number diagram of HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212; D is a quantitative analysis diagram of Transwell migration of HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212; Figure 4 The figures are the quantitative analysis of the popliteal lymph node volume and the percentage of lymph node metastasis in two groups of nude mouse models (n=6); A is the quantitative analysis of the popliteal lymph node volume in two groups of nude mouse models (n=6); B is the quantitative analysis of the percentage of lymph node metastasis in two groups of nude mouse models (n=6); Figure 5 : is an analysis diagram of the effect of piR-hsa-28212 on the expression level of METTL3; wherein, A is a graph showing the protein level of METTL3 in MDA-MB-468 cells with piR-hsa-28212 knockdown and MDA-MB-231 cells with piR-hsa-28212 overexpression; B is a statistical analysis graph showing the protein level of METTL3 in MDA-MB-468 cells with piR-hsa-28212 knockdown and MDA-MB-231 cells with piR-hsa-28212 overexpression; C is a Transwell assay graph showing MDA-MB-468 cells with METTL3 inhibition and MDA-MB-231 cells with METTL3 overexpression; D is a statistical analysis graph showing the Transwell assay of MDA-MB-468 cells with METTL3 inhibition and MDA-MB-231 cells with METTL3 overexpression; Figure 6 The figure is an analysis of the effect of METTL3 on piR-hsa-28212; A is a Transwell experiment analysis diagram showing that METTL3 overexpression can partially eliminate the effect of piR-hsa-28212 knockdown on the migration and invasion of MDA-MB-468 cells; B is a Transwell experiment analysis diagram of METTL3 knockdown; C is a qRT-PCR and protein blot analysis diagram of VEGFC expression in MDA-MB-468 cells with piR-hsa-28212 knockdown; D is a qRT-PCR and protein blot analysis diagram of VEGFC expression in MDA-MB-468 cells with piR-hsa-28212 overexpression; Figure 7 : The expression level analysis graphs of mA or VEGFC in breast cancer cells where piR-hsa-28212 or METTL3 is inhibited or overexpressed; wherein, A is the mA level analysis graph of VEGFC mRNA in breast cancer cells where piR-hsa-28212 is inhibited or overexpressed using MeRIP-qPCR; B is the expression analysis statistical graph of VEGFC after METTL3 knockdown in MDA-MB-468 cells analyzed by qRT-PCR and protein blotting; C is the expression analysis statistical graph of VEGFC after overexpression in MDA-MB-231 cells analyzed by qRT-PCR and protein blotting; D is the expression analysis graph of VEGFC after METTL3 knockdown in MDA-MB-468 cells analyzed by qRT-PCR and protein blotting; E is the expression analysis graph of VEGFC after overexpression in MDA-MB-231 cells analyzed by qRT-PCR and protein blotting; Figure 8 The figure is a study and analysis diagram of the potential binding site between piR-hsa-28212 and TBX1 3'-UTR; wherein, A is a statistical analysis diagram of qRT-PCR and protein blot analysis of VEGFR3 expression in HLECs treated with MDA-MB-468-EXOsh-28212, MDA-MB-231-EXOmimic-28212 and their controls; B is a qRT-PCR and protein blot analysis diagram of VEGFR3 expression in HLECs treated with MDA-MB-468-EXOsh-28212 and their controls; C is a qRT-PCR and protein blot analysis diagram of VEGFR3 expression in HLECs treated with MDA-MB-231-EXOmimic-28212 and their controls; D is a schematic diagram of the potential binding site between piR-hsa-28212 and TBX1 3'-UTR; Fig. 9The figures are dual luciferase reporter gene assay and analysis of TBX1 and VEGFR3 protein expression in HLECs treated with different methods; A is a dual luciferase reporter gene assay; B is a qRT-PCR analysis of TBX1 expression in HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212 and their controls; C is a Western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with MDA-MB-468-EXOsh-28212 and their controls; D is a Western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with MDA-MB-231-EXOmimic-28212 and their controls; Fig.10 The figures are dual luciferase reporter gene assay and qRT-PCR and western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with si-NC and si-TBX1; wherein, A is a dual luciferase reporter gene assay demonstrating luciferase activity in HLECs transfected with VEGFR3 reporter gene containing wild-type TBE or mutant TBE; B is a statistical figure of qRT-PCR and western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with si-NC and si-TBX1; C is a statistical figure of qRT-PCR and western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with si-NC and si-TBX1. DETAILED DESCRIPTION

[0013] The present invention will be further described below in conjunction with the accompanying drawings.

[0014] Example 1 To investigate the piRNA expression profile associated with lymph node metastasis in breast cancer, we performed piRNA sequencing of serum exosomes and identified piR-hsa-28212, which was significantly associated with lymph node metastasis, in a cohort of 28 lymph node metastasis-negative (LN-) and 28 lymph node metastasis-positive (LN+) breast cancer patients. The sequence was GGCGGGAGTAACTATGACTCTCTTAAGGTA. The functional role of exosomal piR-hsa-28212 was further investigated using in vitro migration and tube formation assays and an in vivo footpad-popliteal lymph node metastasis model. To elucidate the regulatory mechanism of piR-hsa-28212, we used a series of assays, including RNA pull-down, RNA immunoprecipitation (RIP), luciferase reporter gene, and actinomycin D assay.

[0015] Materials and methods 2.1 Clinical specimens Serum specimens were obtained from patients at the Second Hospital of Shandong University. Breast cancer was confirmed by pathology by two independent professional pathologists. This example was approved by the Research Ethics Committee of the Second Hospital of Shandong University (KYLL-2022-338).

[0016] 2.2 Cell culture Human breast cancer cell lines (MDA-MB-231, MCF-7, BT549, MDA-MB-468) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HLECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Cell lines were maintained using standard media and conditions. MDA-MB-231, MCF-7, and MDA-MB-468 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). BT549 was maintained in Roswell Park Memorial Institute (RPMI, Gibco) 1640 medium supplemented with 10% FBS. HLECs were maintained in endothelial cell medium (ECM, ScienCell, CA, USA) containing 5% FBS and 1% matching growth factors (ScienCell, CA, USA). All cell lines were grown in a 37°C, 5% CO2 cell culture incubator.

[0017] 2.3 RNA sequencing and data analysis Serum samples from breast cancer patients (6 LN+ vs 6 LN-) were used for small RNA sequencing. Total RNA was extracted using miRNeasy MiniKit (Qiagen, Germany) following the manufacturer's procedure. RNA concentration and quality were measured using Qubit 2.0 Fluorometer (LifeTechnologies, USA) and Nanodrop One Spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, USA), and only samples with RNA integrity number (RIN) higher than 7.0 were selected for sequencing. Library preparation and sequencing were performed by Huake Gene Co., Ltd. (Shanghai, China). Sequencing reads for each sample were mapped to piRbank using CLC Genomics Workbench 5.5 to quantify piRNA expression (counts). piRNA levels were expressed as TPM (transcripts per million).

[0018] 2.4 Mouse popliteal lymph node metastasis model BALB / c nude mice (4–5 weeks old, 18–20 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (China) and used to construct the popliteal lymph node metastasis model. Then, the footpad tumors and popliteal lymph nodes were excised and embedded in paraffin for further analysis. The experiments were approved by the Research Ethics Committee of the Second Hospital of Shandong University. MDA-MB-468 cells (5 × 10 6 ) was slowly injected into the footpad of mice. Then, the mice were randomly divided into 2 groups (6 mice in each group) and injected with exosomes (20 μg / dose) intratumorally every 3 days. When the tumor reached 200 mm, the footpad tumor and popliteal lymph nodes (lymph node volume = 0.5 × width) were removed. 2 × length). Pathological sections of primary tumors and popliteal lymph nodes were analyzed by in situ hybridization (ISH), HE, and immunofluorescence (IF) staining.

[0019] 2.5 RNA pull-down analysis For RNA pulldown, PierceTM Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, 0164) was used. Biotinylated piR-hsa-28212 and antisense sequences were incubated with cell lysates overnight at 4°C. Then, pre-washed beads were added to capture piR-hsa-28212 binding proteins, which were then further analyzed using Western blotting.

[0020] 2.6 RNA immunoprecipitation (RIP) analysis Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17-700, Millipore, USA) was used to perform RIP assay according to the manufacturer’s instructions. Briefly, 1 × 10 7 MDA-MB-468 cells were incubated with magnetic beads conjugated with 5 μg of anti-MEETL3 antibody (Abcam, ab195352) or anti-IgG (negative control) for 6 h at 4°C. Immunoprecipitated RNA was eluted, purified, and dissolved in RNase-free water and further measured by qRT-PCR assay, in which U6 was used as a nonspecific control.

[0021] 2.7 Tube formation experiment HLECs were treated with exosomes (MDA-MB-468-EXOsh-NC or MDA-MB-468-EXOsh-28212) for 48 h. Pretreated HLECs were then seeded onto 96-well plates coated with reduced growth factor Matrigel (BD Biosciences) and incubated for 6 h at 37°C, 5% CO2. The formed lymphatic vessels were imaged using light microscopy and automatically analyzed using the Angiogenesis Analyzer plugin in ImageJ software.

[0022] 2.8 m6A dot plot analysis The RNA was heated at 95°C for 5 minutes to denature the mRNA and immediately cooled on ice. The samples were then spotted on a nylon membrane, air-dried for 10 minutes, and cross-linked by UV irradiation. After blocking in TBST containing 5% BSA for 1 h, the membrane was incubated with a 1:1000 dilution of anti-m6A antibody and a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. The membrane was then developed by enhanced chemiluminescence. The present invention also applies methylene blue staining to verify whether there are equal amounts of RNA samples on the membrane.

[0023] 2.9 m6A RNA immunoprecipitation and qPCR (MeRIP-qPCR) Intact total RNA was extracted using the Trizol method. RNA fragmentation and m6A RNA immunoprecipitation were performed using the m6A MeRIP kit (5203-2, BersinBio) according to the manufacturer's instructions. IP production RT-qPCR was performed as described above.

[0024] 2.10 Dual luciferase reporter gene assay Dual luciferase reporter gene assay was performed using the Dual Luciferase Reporter Gene Assay Kit (DL 101-01, Vazyme, China) according to the manufacturer's instructions. Briefly, cells with different pretreatments were harvested and washed three times with PBS, and then further lysed in PBL lysis buffer for 20 min. The supernatant was then collected and added to a 96-well plate, 10 μl per well, followed by the addition of 100 μl of luciferase assay reagent II for exactly 2 seconds. Subsequently, the luciferase intensity was recorded by a microplate luminometer (Promego, USA), and the Renilla luciferase intensity was detected as a control.

[0025] 2.11 Statistical analysis All measurements and data analysis were performed using GraphPad Prism V9.0 (GraphPad prism, Inc., La Jolla, CA, United States), and p < 0.05 was considered statistically significant. The significance of differences between groups was assessed using Student's t-test or chi-square test (χ2 test), as appropriate. The results are expressed as the mean ± standard error of the mean (SEM) of three independent experiments.

[0026] Example 2 like Figure 1 As shown in Figure A, qRT-PCR analysis confirmed the dysregulated expression of piRNAs in serum exosomes from 28 LN- and 28 LN+ breast cancer patients. Figure 1 As shown in Figure B, the ROC curve illustrates the diagnostic efficacy of piR-hsa-28212 in detecting lymph node metastasis in 56 breast cancer patients; Figure 1 As shown in C, after inhibition of piR-hsa-28212, qRT-PCR analysis of piR-hsa-28212 expression in MDA-MB-468 cells and their derived exosomes (MDA-MB-468-EXO) was performed. After inhibition of piR-hsa-28212, the expression of piR-hsa-28212 in MDA-MB-468 cells and their derived exosomes (MDA-MB-468-EXO) was reduced; Figure 1 As shown in D, qRT-PCR analysis of piR-hsa-28212 expression in MDA-MB-231 cells and their derived exosomes (MDA-MB-231-EXO) after piR-hsa-28212 overexpression. The results showed that after piR-hsa-28212 overexpression, the expression of piR-hsa-28212 in MDA-MB-231 cells and their derived exosomes (MDA-MB-231-EXO) was increased.

[0027] Figure 2 Figure A shows the expression level of piR-hsa-28212 in HLECs incubated with PBS, MDA-MB-231-EXO, or MDA-MB-468-EXO. Compared with PBS, the expression level of piR-hsa-28212 in HLECs incubated with MDA-MB-231-EXO or MDA-MB-468-EXO was increased. Figure 2qRT-PCR analysis of piR-hsa-28212 expression in HLECs after incubation with MDA-MB-468-EXOsh-28212, MDA-MB-231-EXOmimic-28212, and their respective controls is shown in B. Compared with the control, piR-hsa-28212 expression in HLECs was decreased after incubation with MDA-MB-468-EXOsh-28212; compared with the control, piR-hsa-28212 expression in HLECs was increased after incubation with MDA-MB-231-EXOmimic-28212.

[0028] Figure 3 AD in the figure indicates the quantitative analysis of tube formation and Transwell migration of HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212. It indicates that the exosomes produced by MDA-MB-468 cells with knockdown of piR-hsa-28212 reduce the tube formation and Transwell migration ability of HLECs; while the exosomes produced by MDA-MB-231 cells with overexpression of piR-hsa-28212 increase the tube formation and Transwell migration ability of HLECs.

[0029] Figure 4 Figures A and B show the quantitative analysis of the popliteal lymph node volume and lymph node metastasis percentage of two groups of nude mouse models (n=6), indicating that the exosomes produced by MDA-MB-468 cells with knockdown of piR-hsa-28212 reduced the popliteal lymph node volume and metastasis percentage of mice.

[0030] Figure 5 Middle A indicates that the protein level of METTL3 in MDA-MB-468 cells with piR-hsa-28212 knockdown and MDA-MB-231 cells with piR-hsa-28212 overexpression was evaluated by Western blot analysis. The protein level of METTL3 in MDA-MB-468 cells with piR-hsa-28212 knockdown decreased; the protein level of METTL3 in MDA-MB-231 cells with piR-hsa-28212 overexpression increased; Figure 5 Zhong BC stated that Transwell assays of MDA-MB-468 cells with METTL3 inhibition and MDA-MB-231 cells with METTL3 overexpression showed that inhibition of METTL3 in MDA-MB-468 cells would lead to decreased migration and invasion abilities of cells; while overexpression of METTL3 in MDA-MB-231 cells would lead to enhanced migration and invasion abilities of cells.

[0031] Figure 6Figure A indicates that Transwell experiments showed that METTL3 overexpression could partially eliminate the effects of piR-hsa-28212 knockdown on the migration and invasion of MDA-MB-468 cells; Figure 6 As shown in Figure 2B, Transwell assays demonstrated that knockdown of METTL3 partially alleviated the migration and invasion abilities of MDA-MB-231 cells, an effect that was enhanced by overexpression of piR-hsa-28212. Figure 6 qRT-PCR and Western blot analysis of VEGFC expression in MDA-MB-468 cells with piR-hsa-28212 knockdown and MDA-MB-231 cells with piR-hsa-28212 overexpression as shown in Figures C and D. The mRNA and protein levels of VEGFC in MDA-MB-468 cells with piR-hsa-28212 knockdown were decreased; the mRNA and protein levels of VEGFC in MDA-MB-231 cells with piR-hsa-28212 overexpression were increased.

[0032] Figure 7 A in the middle indicates that MeRIP-qPCR was used to quantify the mA level of VEGFC mRNA in breast cancer cells where piR-hsa-28212 was inhibited or overexpressed. The mA level of VEGFC mRNA in breast cancer cells where piR-hsa-28212 was inhibited decreased; the mA level of VEGFC mRNA in breast cancer cells where piR-hsa-28212 was overexpressed increased; Figure 7 BE in the middle indicates that qRT-PCR and Western blot were used to analyze the expression of VEGFC in breast cancer cells after METTL3 knockdown or overexpression. The expression of VEGFC in breast cancer cells decreased after METTL3 knockdown, and increased after METTL3 overexpression.

[0033] Figure 8 AC represents qRT-PCR and Western blot analysis of VEGFR3 expression in HLECs treated with MDA-MB-468-EXOsh-28212, MDA-MB-231-EXOmimic-28212 and their controls. Compared with the control, VEGFR3 expression in HLECs was decreased after incubation with MDA-MB-468-EXOsh-28212; compared with the control, VEGFR3 expression in HLECs was increased after incubation with MDA-MB-231-EXOmimic-28212; Figure 8 D shows a schematic diagram of the potential binding site between piR-hsa-28212 and TBX1 3'-UTR. There is a potential binding site between piR-hsa-28212 and TBX1 3'-UTR.

[0034] like Fig. 9 As shown in Figure A, dual luciferase reporter gene assay demonstrated the effect of piR-hsa-28212 on the TBX1 reporter gene containing wild-type or mutant sequences. Dual luciferase reporter gene assay demonstrated that piR-hsa-28212 can bind to the 3'-UTR of TBX1; Fig. 9 As shown in B, qRT-PCR analysis of TBX1 expression in HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212 and their controls. Compared with the control MDA-MB-468-EXOsh-NC, the expression of TBX1 in HLECs treated with MDA-MB-468-EXOsh-28212 was decreased; compared with the control MDA-MB-231-EXOmimic-NC, the mRNA expression of TBX1 in HLECs treated with MDA-MB-231-EXOmimic-28212 was increased; Fig. 9 C and D show Western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with MDA-MB-468-EXOsh-28212 and MDA-MB-231-EXOmimic-28212 and their controls. Compared with the control, TBX1 and VEGFR3 protein expression was decreased in HLECs treated with MDA-MB-468-EXOsh-28212 and; compared with the control, TBX1 and VEGFR3 protein expression was increased in HLECs treated with MDA-MB-231-EXOmimic-2821 and.

[0035] like Fig.10 As shown in A, dual-luciferase reporter gene assay demonstrated luciferase activity in HLECs transfected with VEGFR3 reporter gene containing wild-type TBE or mutant TBE. Dual-luciferase reporter gene assay demonstrated that VEGFR3 can bind to the TBE region of TBX1 in HLECs; Fig.10Figures B and C show qRT-PCR and western blot analysis of TBX1 and VEGFR3 expression in HLECs treated with si-NC and si-TBX1. The mRNA and protein levels of TBX1 and VEGFR3 were decreased in HLECs with knockdown of TBX1. Metastasis is the main cause of death in breast cancer patients. The key predictor of distant metastasis and mortality in breast cancer is the extent of axillary lymph node metastasis. Higher lymphatic vessel density around and within tumors is associated with increased lymph node metastasis and patient mortality. As a member of the VEGF family, VEGFC binds to VEGFR3 on the surface of lymphatic endothelial cells (LECs), thereby stimulating lymphangiogenesis, which may promote the entry of tumor cells into the lymphatic drainage system. However, the regulatory mechanism behind the activation of VEGFC / VEGFR3 signaling that triggers breast cancer lymph node metastasis remains largely unknown. The results of the present invention comprehensively elucidate the molecular mechanism of the synergistic activation of VEGFC / VEGFR3 signaling induced by exosomal piR-hsa-28212, thereby enhancing the understanding of the role of VEGFC in tumor lymphatic metastasis in the present invention.

[0036] The results of the present study showed that silencing of exosomal piR-hsa-28212 significantly inhibited VEGFC / VEGFR3 signaling-induced breast cancer lymphangiogenesis and lymph node metastasis in vitro, and promoted lymphangiogenesis and lymph node metastasis of mouse footpad tumors in vivo. This suggests that targeting exosomal piR-hsa-28212 with a specially designed safe oligonucleotide (miR2210914093907 (RiboBio, China)) may represent a promising therapeutic strategy to alleviate pathological overactivation of VEGFC / VEGFR3 signaling. Therefore, this approach may provide a new treatment and intervention measure for patients with lymphatic metastatic breast cancer.

[0037] Early detection and dynamic assessment of the metastatic status of breast cancer patients are crucial for effective treatment. Currently, ultrasonography and mammography combined with histopathology are considered the gold standard for breast cancer diagnosis and metastatic status assessment. However, these methods have several limitations, emphasizing the need for continued research and development of alternative diagnostic methods for the assessment of breast cancer and its metastasis.

[0038] Compared to traditional tumor tissue biopsies, liquid biopsies using molecular classifiers identified in patient blood samples offer a minimally invasive alternative with reduced complications and enhanced longitudinal monitoring capabilities. The billions of exosomes circulating in the blood may constitute a key element of liquid biopsies. Notably, the membrane encapsulation of exosomes preserves their structural integrity, and the exosomal contents exhibit greater stability than other serological components due to protection from degradation by circulating proteases and other enzymes. Given the ease of detection and ubiquitous presence of exosomes in serum, they provide a wealth of material for downstream analysis for cancer detection, prognosis, and treatment monitoring, making them a promising non-invasive liquid biopsy approach. In the field of diagnostics, many studies have proposed the application of exosomes as diagnostic tools because they contain disease-specific biomarkers.

[0039] Due to the accessibility and key functions of exosomal noncoding RNAs, they have great potential in BC diagnosis as a supplement and auxiliary modality to the current clinical gold standard. PiRNA sequencing showed that piR-hsa-28212 was overexpressed in serum exosomes from breast cancer patients with lymph node metastasis. This finding was confirmed in serum exosomal piRNAs from 28 paired breast cancer patients (28 LN+ and 28 LN-).

[0040] PiR-hsa-28212 was significantly upregulated in serum exosomes from breast cancer patients with lymphatic metastasis. In vitro, exosomal piR-hsa-28212 promoted migration and tube formation of human lymphatic endothelial cells (HLECs), while in vivo, it enhanced lymphangiogenesis and lymph node metastasis. Mechanistically, piR-hsa-28212 in breast cancer cells activated the METTL3 / IGF2BP2-m6A signaling pathway, leading to increased VEGFC expression and secretion. In addition, piR-hsa-28212 was transferred into HLECs by exosomes, stabilizing TBX1 mRNA, leading to elevated VEGFR3 expression and promoting lymph node metastasis.

[0041] Conclusion: The results of this study reveal a new mechanism by which exosomal piR-hsa-28212 synergistically enhances VEGFC / VEGFR3 signaling-induced breast cancer lymph node metastasis. This piRNA may become a promising therapeutic target for breast cancer patients.

Claims

1. A therapeutic target for lymph node metastatic breast cancer, characterized in that: The target piR-hsa-28212 sequence is shown in SEQID NO.

1. piR-hsa-28212 activates the METTL3 / IGF2BP2-m6A signaling pathway, leading to increased VEGFC expression and secretion.

2. Use of the target piR-hsa-28212 as described in claim 1 in the preparation of a diagnostic product for lymph node metastatic breast cancer.

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