Targeted ISCA2 breast cancer inhibition composition and application thereof

Abstract

The invention relates to a composition for inhibiting breast cancer cells by targeting ISCA2 and application thereof, the composition is a composition for inhibiting breast cancer cells by targeting ISCA2, and through at least one of small interference (si-ISCA2) of an ISCA2 gene and overexpression of miRNA-let-7e-3p, the composition targets ISCA2 and inhibits the expression of ISCA2, and further inhibits the expression of AKT and mTOR in an AKT / mTOR signal channel, so that the composition can inhibit breast cancer cells by targeting ISCA2. The target of inhibiting proliferation, migration and invasion ability of breast cancer cells is achieved, and the compound can be applied to preparation of drugs for treating breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition that targets ISCA2 to inhibit breast cancer and its application. Background Technology

[0002] Breast cancer (BC) poses a serious threat to the health of women worldwide due to its high incidence and mortality rates. The combination of traditional surgical treatment with chemotherapy, radiotherapy, targeted therapy, and immunotherapy has made significant contributions to improving the clinical outcomes for BC patients. Despite continuous advancements in BC treatment and significantly enhanced treatment efficacy, some patients still experience adverse outcomes due to recurrence, metastasis, and drug resistance. Therefore, the search for new and more effective treatment approaches is crucial to improving treatment outcomes.

[0003] In recent years, research on molecular mechanisms has received increasing attention, and precise targeted therapy through gene binding has become a new treatment approach. This involves identifying specific targets, such as miRNAs targeting downstream genes, to develop specific targeted drugs. Summary of the Invention

[0004] In view of this, the present invention provides a composition that targets ISCA2 to inhibit breast cancer and its application, and the discovery and confirmation of this new regulatory axis is of great significance for the development of new breast cancer treatment options.

[0005] A first aspect of the invention: a composition that targets ISCA2 to inhibit breast cancer, said composition comprising at least one of the following:

[0006] (i) Small interfering RNAs or miRNAs that target ISCA2 or ISCA2 transcripts and can inhibit the expression of ISCA2 gene expression products or gene transcription;

[0007] (ii) Able to express or form constructs of the small interfering RNA or miRNA described in (i);

[0008] (iii) A construct containing ISCA2 or ISCA2 complementary sequence, which can form an interfering molecule that inhibits the expression of ISCA2 gene expression products or gene transcription after being transformed into the body;

[0009] (iv) Immune-associated cells, their differentiated cells, or constructs after the ISCA2 gene sequence has been suppressed or knocked out.

[0010] Preferably, the miRNA is at least one of miRNA-let-7e-3p or overexpression of miR-let-7e-3p (miR-let-7e-3pmimic), wherein the miR-let-7e-3p binds directly to the 3'-UTR region of ISCA2.

[0011] The miR-let-7e-3p mimicsense sequence of the overexpression of miR-let-7e-3p (miR-let-7e-3p mimic) is shown in SEA ID NO.1, and the miR-let-7e-3p mimic antisense sequence is shown in SEA ID NO.2.

[0012] Preferably, the small interfering RNA is a small interfering RNA (si-ISCA2) of the ISCA2 gene.

[0013] The specific sense and antisense sequences of the si-ISCA2 sequence are any of the following sets:

[0014] Group 1: The si-ISCA2-1 sense sequence is shown in SEA ID NO.3, and the si-ISCA2-1 antisense sequence is shown in SEA ID NO.4.

[0015] The second group: the si-ISCA2-2 sense sequence is shown in SEA ID NO.5, and the si-ISCA2-2 antisense sequence is shown in SEA ID NO.6.

[0016] The third group: the si-ISCA2-3 sense sequence is shown in SEA ID NO.7, and the si-ISCA2-3 antisense sequence is shown in SEA ID NO.8.

[0017] Preferably, the composition targets ISCA2, inhibits ISCA2 expression, and subsequently suppresses the protein expression levels of AKT and mTOR, key proteins in the AKT / mTOR pathway, thereby inhibiting the AKT / mTOR pathway and ultimately suppressing breast cancer cells.

[0018] Preferably, it inhibits the proliferation, migration, and invasion of breast cancer cells.

[0019] The second aspect of the invention: the use of any of the above-described compositions in at least one of (1) to (8),

[0020] (1) Application in the preparation of drugs for the treatment of breast cancer,

[0021] (2) Application in the preparation of compositions for inhibiting the growth and proliferation of breast cancer cells.

[0022] (3) Application in the preparation of compositions that inhibit breast cancer cell migration.

[0023] (4) Application in the preparation of compositions that inhibit breast cancer cell invasion.

[0024] (5) Application in the preparation of compositions that inhibit ISCA2 protein expression.

[0025] (6) Application in the preparation of compositions that inhibit the AKT / mTOR pathway.

[0026] (7) Application in the preparation of compositions that inhibit AKT protein expression.

[0027] (8) Application in the preparation of compositions that inhibit mTOR protein expression.

[0028] The third aspect of the invention: the use of human miRNA, miR-let-7e-3p, in at least one of (1) to (8),

[0029] (1) Application in the preparation of drugs for the treatment of breast cancer,

[0030] (2) Application in the preparation of compositions for inhibiting the growth and proliferation of breast cancer cells.

[0031] (3) Application in the preparation of compositions that inhibit breast cancer cell migration.

[0032] (4) Application in the preparation of compositions that inhibit breast cancer cell invasion.

[0033] (5) Application in the preparation of compositions that inhibit ISCA2 protein expression.

[0034] (6) Application in the preparation of compositions that inhibit the AKT / mTOR pathway.

[0035] (7) Application in the preparation of compositions that inhibit AKT protein expression.

[0036] (8) Application in the preparation of compositions that inhibit mTOR protein expression.

[0037] Human miRNA, miR-let-7e-3p, also has at least one of the following applications (1) to (4): (1) Application of preparing miR-let-7e-3p biomarkers in the preparation of kits for breast cancer screening and diagnosis.

[0038] (2) The application of miR-let-7e-3p as a standardized internal control in the preparation of reagent kits for breast cancer screening and diagnosis.

[0039] (3) Application of drugs for restoring drug sensitivity in breast cancer cells.

[0040] (4) Application as a recognition sequence for miR-let-7e-3p in gene therapy vectors.

[0041] A fourth aspect of the present invention: a miR-let-7e-3p overexpression (miR-let-7e-3p mimic), wherein the miR-let-7e-3p overexpression (miR-let-7e-3p mimic) is used in at least one aspect of (1) to (8),

[0042] (1) Application in the preparation of drugs for the treatment of breast cancer,

[0043] (2) Application in the preparation of compositions for inhibiting the growth and proliferation of breast cancer cells.

[0044] (3) Application in the preparation of compositions that inhibit breast cancer cell migration.

[0045] (4) Application in the preparation of compositions that inhibit breast cancer cell invasion.

[0046] (5) Application in the preparation of compositions that inhibit ISCA2 protein expression.

[0047] (6) Application in the preparation of compositions that inhibit the AKT / mTOR pathway.

[0048] (7) Application in the preparation of compositions that inhibit AKT protein expression.

[0049] (8) Application in the preparation of compositions that inhibit mTOR protein expression.

[0050] Specifically, the miR-let-7e-3p mimic sense sequence is shown in SEA ID NO.1, and the miR-let-7e-3p mimic antisense sequence is shown in SEA ID NO.2.

[0051] The fifth aspect of the present invention: a small interfering sequence (si-ISCA2) of the ISCA2 gene, the small interfering sequence (si-ISCA2) of the ISCA2 gene, and its application in at least one aspect of (1) to (8).

[0052] (1) Application in the preparation of drugs for the treatment of breast cancer,

[0053] (2) Application in the preparation of compositions for inhibiting the growth and proliferation of breast cancer cells.

[0054] (3) Application in the preparation of compositions that inhibit breast cancer cell migration.

[0055] (4) Application in the preparation of compositions that inhibit breast cancer cell invasion.

[0056] (5) Application in the preparation of compositions that inhibit ISCA2 protein expression.

[0057] (6) Application in the preparation of compositions that inhibit the AKT / mTOR pathway.

[0058] (7) Application in the preparation of compositions that inhibit AKT protein expression.

[0059] (8) Application in the preparation of compositions that inhibit mTOR protein expression.

[0060] Specifically, the sense sequence and antisense sequence of the si-ISCA2 sequence are any one of the following pairs:

[0061] Group 1: The si-ISCA2-1 sense sequence is shown in SEA ID NO.4, and the si-ISCA2-1 antisense sequence is shown in SEA ID NO.5.

[0062] The second group: the si-ISCA2-2 sense sequence is shown in SEA ID NO.6, and the si-ISCA2-2 antisense sequence is shown in SEA ID NO.7.

[0063] The third group: the si-ISCA2-3 sense sequence is shown in SEA ID NO.8, and the si-ISCA2-3 antisense sequence is shown in SEA ID NO.9.

[0064] The sixth aspect of the present invention: the use of a miR-let-7e-3p inhibitor (miR-let-7e-3p inhibit) in at least one of (1) to (7), (1) in a composition that promotes the proliferation of breast cancer cells, (2) in a composition that promotes the migration of breast cancer cells, (3) in a composition that enhances the invasive ability of breast cancer cells, (4) in a composition that enhances the protein expression of ISCA2, (5) in a composition that enhances the AKT / mTOR pathway, (6) in a composition that enhances the AKT protein expression, and (7) in a composition that enhances the mTOR protein expression. Specifically, the sense sequence of the miR-let-7e-3p inhibitor is as shown in SEA ID NO.9.

[0065] This invention experimentally verified the expression of miR-let-7e-3p in human breast cancer cells and its role and mechanism in cell proliferation, migration, and invasion. Specifically, miR-let-7e-3p was expressed at low levels in the BRCA cell line. Transfection with miR-let-7e-3p mimics inhibited the proliferation, migration, and invasion of MCF7 and MDA-MB-231 cells. Bioinformatics analysis showed that ISCA2 is a potential target gene of miR-let-7e-3p, and ISCA2 is positively correlated with AKT. Dual-luciferase reporter assay confirmed the targeted binding of miR-let-7e-3p to ISCA2. Western blot results showed that transfection with miR-let-7e-3p mimics significantly inhibited the expression of ISCA2, AKT, and mTOR. Knocking down ISCA2 significantly inhibited the expression of AKT and mTOR, as well as the proliferation, migration, and invasion of MCF7 and MDA-MB-231 cells, while reversing the pro-proliferative and metastatic effects of miR-let-7e-3p inhibitors on BRCA cells. The conclusion is that miR-let-7e-3p is expressed at low levels in BRCA cells, and it can inhibit the proliferation, migration, and invasion of BRCA cells by targeting ISCA2 to suppress the AKT / mTOR signaling pathway. Attached Figure Description

[0066] Appendix Figure 1 This study investigated the expression of miR-let-7e-3p in breast cancer cells (BRCA). A: dbDEMC analysis of miR-let-7e-3p expression in BRCA; B: RT-qPCR detection of miR-let-7e-3p expression in BRCA. Compared with the control group, *P < 0.05, **P < 0.01.

[0067] Appendix Figure 2 Overexpression of miR-let-7e-3p inhibited the proliferation, migration, and invasion of BC cells. A and B: RT-qPCR detection of miR-let-7e-3p overexpression transfection efficiency in different BC cell lines; C: Detection of the effect of miR-let-7e-3p overexpression on BC cell colony formation ability; D and E: Transwell assay detection of the effect of miR-let-7e-3p overexpression on BC cell migration and invasion ability. Compared with the control group, *P < 0.05, **P < 0.01.

[0068] Appendix Figure 3This study used bioinformatics and experimental methods to predict, screen, and validate the downstream target gene ISCA2 of miR-let-7e-3p. A: miRanda, miRDB, and TargetScan websites were used to predict downstream target genes of miR-let-7e-3p, and Venny 2.0 software was used to find the intersection of these downstream target genes. B: RT-qPCR was used to detect the differential expression of ISCA2, AP3D1, and EDEMC3 in BC cells. C: Western blot was used to detect the protein expression of ISCA2 in BC cells. D and E: Dual-luciferase reporter assays were used to detect the targeting relationship between miR-let-7e-3p and ISCA2. F: RT-qPCR was used to detect the differential expression of ISCA2 after miR-let-7e-3p overexpression in different BC cell lines. G and H: Western blot was used to detect the differential expression of ISCA2 after miR-let-7e-3p overexpression in different BC cell lines. Compared with the control group, *P < 0.05, **P < 0.01.

[0069] Appendix Figure 4 Small interfering ISCA2 can inhibit the proliferation, migration, and invasion abilities of BC cells. A and B: qRT-PCR detection of ISCA2 interference efficiency in different BC cell lines; C and D: Western blot detection of ISCA2 interference efficiency in different BC cell lines; E: Detection of the effect of small interfering ISCA2 on BC cell colony formation ability; F and G: CCK-8 assay detection of the effect of small interfering ISCA2 on BC cell viability; H and I: Transwell assay detection of the effect of small interfering ISCA2 on BC cell migration and invasion abilities. Compared with the control group, *P < 0.05, **P < 0.01.

[0070] Appendix Figure 5 The small-interference ISCA2 assay reversed the effects of miR-let-7e-3p on the proliferation, migration, and invasion of BC cells. A and B: qRT-PCR assays of miR-let-7e-3p co-transfection efficiency with ISCA2 in different BC cell lines; C and D: Western blot assays of miR-let-7e-3p co-transfection efficiency with ISCA2 in different BC cell lines; E: assay of the effect of miR-let-7e-3p co-transfection with ISCA2 on colony formation in BC cell lines; F and G: Transwell assays of the effect of miR-let-7e-3p co-transfection with ISCA2 on the migration and invasion of BC cell lines. Compared with the control group, *P < 0.05, **P < 0.01.

[0071] Appendix Figure 6This study investigated the effects of miR-let-7e-3p overexpression and small interfering ISCA2 on the AKT / mTOR signaling pathway in BC cell lines. A and B: Timer prediction of AKT and mTOR expression in breast cancer; C and D: Western blot analysis of the effect of miR-let-7e-3p overexpression on AKT and mTOR protein expression in BC cell lines; E and F: Western blot analysis of the effect of ISCA2 knockdown on AKT and mTOR protein expression in BC cell lines. Compared with the control group, *P < 0.05, **P < 0.01. Detailed Implementation

[0072] Since the molecular pathogenesis of breast cancer (BC) remains largely unclear, there are currently no effective clinical treatments for advanced BC. Therefore, this invention aims to explore new treatment methods and targets for BC by delving into its molecular mechanisms.

[0073] In recent years, many miRNAs have been aberrantly expressed in human cancers and can exert tumor-promoting or tumor-suppressing functions. miR-let-7 was first identified in 2000. miRNA-let-7e-3p, as an important gene in the miRNA-let-7 family, inhibits cell invasion, metastasis, proliferation, and tumorigenesis in pancreatic cancer (PCa) through overexpression. miR-let-7e consists of a main chain miR-let-7e-3p and a guest chain miR-let-7e-5p. The mechanism of action of miR-let-7e-3p in breast cancer remains unclear, particularly regarding its directly regulated downstream target genes. Through bioinformatics analysis, we found significant differences in miR-let-7e-3p expression in BC cells; therefore, we selected miR-let-7e-3p as the target gene for our experimental research.

[0074] This study first analyzed miR-let-7e-3p using bioinformatics, revealing it to be a tumor suppressor gene. Simultaneously, qRT-PCR was used for validation, showing its presence in different subtypes of BC cells, such as HR2. + The expression of tumor suppressor cells differed significantly among MCF-7 cells and triple-negative breast cancer cells (MDA-MB-231, MDA-MB-453, MDA-MB-468, BT-549), suggesting that miR-let-7e-3p may also occur and develop in BC cells, but with varying degrees of influence. Further validation was achieved through cloning experiments, CCK-8 assays, and Transwell migration and invasion phenotypic experiments, with results consistent with the above findings.

[0075] Using bioinformatics analysis and dual-luciferase reporter assays, the downstream target gene ISCA2 (iron-sulfur cluster assembly protein 2) of miR-let-7e-3p was identified. Prior to this study, the role of ISCA2 in the blastocysteine ​​(BC) process was unclear. The iron-sulfur cluster assembly family (ISCA1, ISCA2) are conserved cofactors of iron and sulfur associated with cysteine ​​sulfur in proteins, playing crucial roles in various cell and molecular biological processes, such as electron transport, structural stability, gene regulation, and enzyme catalysis. ISCA2 is involved in DNA replication, RNA transcription, oxidation, and electron transport. Recent findings indicate that, in addition to being an important component of normal mitochondrial function, ISCA2 is essential for erythrocyte differentiation and cell proliferation.

[0076] Literature review revealed that AKT / mTOR is the most common signaling pathway promoting the growth, proliferation, and metastasis of various cancer cells. This study first used the bioinformatics website Timer to predict the correlation between ISCA2 and AKT, initially identifying it as the research target for further experiments. The AKT / mTOR pathway plays a crucial role in tumor metastasis. Besides cell survival, motility, and immune responses, it is frequently involved in physiological and abnormal cell growth and proliferation. AKT has three isotypes: AKT1 (expressed in most tissues), AKT2 (mainly expressed in tissues with high insulin sensitivity: liver, pancreas, and muscle), and AKT3 (expressed in the brain and testes). The AKT / mTOR signaling pathway is also considered a novel target for breast cancer treatment.

[0077] After synthesizing miR-let-7e-3p overexpression and ISCA2 small interference, the expression of AKT and mTOR proteins in the AKT / mTOR signaling pathway was detected by Western blot. The results showed that, compared with the control group, the expression of AKT and mTOR proteins was significantly downregulated after miR-let-7e-3p overexpression and ISCA2 small interference.

[0078] In summary, miR-let-7e-3p inhibits the AKT / mTOR signaling pathway by targeting ISCA2, thereby suppressing the proliferation, migration, and invasion of human breast cancer cells. This provides an experimental basis for miR-let-7e-3p to become a target for the diagnosis and treatment of human breast cancer (BC).

[0079] The specific experiment is as follows:

[0080] The normal human mammary epithelial cells MCF-10A and BC cells MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468, and BT-549 used in the experiment were all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0081] Table 1 shows the details of miR-let-7e-3p mimic (overexpression), miR-let-7e-3p inhibit (inhibitor), ISCA2 siRNA (si-ISCA2), and each negative control sequence.

[0082]

[0083] Experimental methods:

[0084] 1. Cell culture and cell transfection

[0085] HR2 was confirmed by different serum concentrations. + To optimize the survival of MCF-7 cells and triple-negative breast cancer cells (MDA-MB-231, MDA-MB-453, MDA-MB-468, BT-549), high-glucose DMEM medium containing 10% fetal bovine serum was used. MCF-10A mammary epithelial cells were cultured in high-glucose DMEM medium containing 20 ng / mL recombinant epidermal growth factor, 5% horse serum, 0.5 μg / mL hydrocortisone, 10 μg / mL insulin, 1% non-essential amino acids, and 1% penicillin antibiotics. All cells were cultured at 37°C in a 5% CO2 incubator, with medium changes every two days. Cells were passaged and cryopreserved when confluence reached 80%-90%. Cell passages for experiments were limited to no more than 10 generations. Cell transfection: Logarithmically growing cells were seeded into 6-well plates. When the confluence reached 50%–60%, miR-let-7e-3p mimic, inhibitor, si-ISCA2, Wt-ISCA2, Mut-ISCA2, and control cells were transfected into MCF-7 and MDA-MB-231 cells using Lipofectamine 2000 reagent. The final concentration of Lipofectamine 2000 reagent for transfection was 20 nmol / L. Samples were collected 1–2 days after transfection for subsequent experiments.

[0086] 2. Plate cloning experiment

[0087] MCF-7 and MDA-MB-231 cells, transfected for 48 hours, were digested, resuspended, and counted. The cells were seeded at a density of 300 cells / well in 12-well plates and cultured for 8-14 days, with the medium changed every 4 days. Cells were washed 2-3 times with PBS, fixed with 4% paraformaldehyde for 20-30 minutes, and stained with 0.1% crystal violet solution for 4-6 minutes. Cells were washed 2-3 times with PBS, and the number of cell clones was counted and the colony formation rate calculated.

[0088] 3. Transwell experiment

[0089] Collect cells 24-48 h after transfection, digest with trypsin, and resuspend the cells in serum-free medium (5 × 10⁻⁶). 5 Add cells (number of cells / mL) to the upper chamber, maintaining a final volume of 100 μL. Add 600 μL of complete culture medium containing 20% ​​serum to the lower chamber and incubate for 24-48 h. Do not line the chambers for the migration experiment; pre-add 100 μL of diluted matrix gel to each chamber for the invasion experiment. After the experiment, fix cells with 4% paraformaldehyde for 20-30 min, stain with 0.1% crystal violet solution for 4-6 min, rinse cells 2-3 times with PBS, and gently wipe away the upper layer of cells in the chamber with a cotton swab. Take at least 6 different field-of-view photographs for each well under a microscope, and use ImageJ software to count the number of migrating and invading cells. Calculate the migration rate and invasion rate separately: Migration rate = [(number of migrating cells) / (number of seeded cells)] × 100%, Invasion rate = [(number of invading cells) / (number of seeded cells)] × 100%.

[0090] 4. Dual-luciferase reporter assay

[0091] 293T cells in logarithmic growth phase were taken, digested and resuspended, and then inoculated with 4×10⁻⁶ cells. 5 Cells were seeded at a density of [number] cells / mL in 24-well plates. Once cells reached 60% confluence, miR-let-7e-3p mimic, mimic negative control, Wt-ISCA2, and Mut-ISCA2 were transfected into 293T cells according to the experimental groups. Cell samples were collected after 48 hours. Luciferase activity was measured according to the TransDetect® Dual-Luciferase Reporter Gene Detection Kit instructions.

[0092] 5. qRT-PCR experiment

[0093] Total RNA was extracted from each group using the TRIzol method, and its concentration and purity were determined. Reverse transcription and qPCR detection were performed using the HiScript II Q RTSuperMix and ChamQ SYBR qPCR Master Mix kits on a PCR instrument (Gene Biotechnology International Trading Co., Ltd.). Primer sequences are detailed in Table 2. Reaction conditions: 95°C, 5 min; 95°C, 15 s; 55°C, 15 s; 72°C, 15 s, for 40 cycles. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and U6 (U6 small nuclear RNA) were used as internal controls. -ΔΔCt The relative expression levels of each gene can be calculated.

[0094] 1.7 Western blot experiment

[0095] After culturing cells for 48 hours, total protein was extracted from each group, and its concentration was determined by the BCA method. 30-60 μg of protein was loaded onto the membrane, separated by SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk on a shaker at room temperature for 3 hours, washed three times with TBST (10 min each time), and then the corresponding primary antibody was added (antibody dilution ratios are detailed in Table 3). The membrane was incubated overnight at 4°C, washed three times with TBST, and then incubated with HRP-labeled secondary antibody at room temperature for 1 hour, followed by three more TBST washes. The membrane was developed with ECL chemiluminescence solution, using GAPDH or β-Tubulin as internal controls. Images were taken using an imager (GE-Heolthcare Bio-Sciences AB-Amersham Imager 680), and grayscale values ​​were measured using ImageJ software. Statistical analysis was performed.

[0096] 1.8 Statistical Analysis

[0097] Graphpad Prism 9.5.1 software was used for statistical analysis and graphing of experimental data. Normally distributed continuous data were expressed as mean ± standard deviation (x±s). The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 or p-value < 0.01 was considered statistically significant.

[0098]

[0099] The experimental procedure is as follows:

[0100] I. Differentially expressed miRNAs in breast cancer tissues were screened using the dbDEMC (biosino.org) website, and miR-let-7e-3p was finally identified. Its expression level in different invasive breast cancer cell lines was detected by qRT-PCR.

[0101] Predictive analysis of differentially expressed miRNAs in BC tissues was performed using data from the dbDEMC (biosino.org) website. The results showed that miR-let-7e-3p was expressed at low levels in BC tissues compared to normal tissues. Figure 1 A, P=1.90e-8). The expression of miR-let-7e-3p in normal breast epithelial cells and cancer cells was detected by RT-qPCR. The results showed that compared with breast epithelial cells MCF-10A, the expression of miR-let-7e-3p was significantly reduced in BC cell lines MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-458, and BT-549. Figure 1B, P<0.05). MCF-7 and MDA-MB-231 cells were used to study the function and mechanism of miR-let-7e-3p.

[0102] II. After overexpressing miR-let-7e-3p in breast cancer cells, we verified its regulatory effect on cell phenotype through clonogenic and Transwell (migration and invasion) experiments.

[0103] RT-qPCR was used to detect the expression of miR-let-7e-3p in MCF-7 and MDA-MB-231 cells after transfection with miR-let-7e-3p mimic (overexpression). The results showed that, compared with the control group, the expression of miR-let-7e-3p was significantly increased after transfection with miR-let-7e-3p mimic (overexpression). Figure 2 A, 2B, P<0.05. The plate colony formation assay was used to examine the effect of miR-let-7e-3p overexpression on the proliferation of BC cells. The results showed that, compared with the NC control group, the overexpression group significantly inhibited the colony formation of both cell lines. Figure 2 C, P<0.05. Transwell assay results showed that, compared with the NC control group, transfection with miR-let-7e-3p mimic (overexpression) significantly inhibited the migration and invasion abilities of MCF-7 and MDA-MB-231 cells. Figure 2 DE, P<0.05).

[0104] III. Bioinformatics Prediction: Software such as TargetScan, miRDB, and miRanda were used to predict downstream target genes of miR-let-7e-3p, and to screen and verify the binding sites of miR-let-7e-3p to the 3'-UTR of the ISCA2 gene.

[0105] Downstream target genes of miR-let-7e-3p were predicted using three bioinformatics software programs: Targetscan, miRDB, and miRanda. The Venny2.0 online software, by taking the intersection, identified eight genes as downstream target genes of miR-let-7e-3p: ISCA2, FUBP3, TMEM163, PHOX2B, ZNF503, PLEKHA1, AP3D1, and EDEM3. Figure 3 A). RT-qPCR analysis revealed that ISCA2 expression was upregulated in the BC cell line compared to MCF-10A mammary epithelial cells. Figure 3 B). Western blot analysis further confirmed that ISCA2 expression was upregulated in the BC cell line. Figure 3 C), preliminary screening identified ISCA2 as a downstream target gene of miR-let-7e-3p.

[0106] IV. Dual-luciferase reporter gene experiment: A reporter gene plasmid containing the wild-type ISCA2 gene 3'-UTR and binding site mutation was constructed, and its fluorescence activity was detected after co-transfection with miR-let-7e-3p mimic (overexpression).

[0107] Dual-luciferase reporter assays were used to verify the targeting relationship between miR-let-7e-3p and ISCA2. Results showed that, compared to the NC group, the luciferase activity was significantly reduced in the group co-transfected with miR-let-7e-3p mimic (overexpression) and ISCA2 3′UTR-Wt (wild-type), while the luciferase activity in the group co-transfected with miR-let-7e-3p mimic (overexpression) and ISCA2 3′UTR-Mut (mutant) showed no significant change. Figure 3 D-3E, P<0.05). Prove that miR--let-7e-3p directly binds to the 3'-UTR region of ISCA2.

[0108] RT-qPCR results showed that, compared with the control group, transfection with miR-let-7e-3p mimic (overexpression) significantly reduced ISCA2 expression in MCF-7 and MDA-MB-231 cells. Figure 3 F, P<0.05. Western blot results showed that, compared with the control group, transfection with miR-let-7e-3p mimic (overexpression) significantly reduced ISCA2 expression in MCF-7 and MDA-MB-231 cells. Figure 3 G-3H, P<0.05).

[0109] V. Interference with ISCA2 can inhibit the proliferation, migration and invasion of BRCA cells.

[0110] A small interfering ISCA2 sequence (si-ISCA2) was designed and synthesized, and transfected into MCF-7 and MDA-MB-231 cells, respectively. RT-qPCR analysis confirmed that the siRNA transfected with ISCA2 significantly reduced the gene expression level of ISCA2. Figure 4 A-4B, P<0.05. Western blot analysis confirmed that transfection with ISCA2 siRNA significantly reduced ISCA2 protein expression levels. Figure 4 The results of the C-4D (P<0.05) plate colony formation assay showed that, compared with the NC control group, siRNA transfected with ISCA2 significantly inhibited the proliferation of BC cells. Figure 4E-4F, P<0.05. Transwell assay results showed that, compared with the control group, siRNA transfected with ISCA2 significantly inhibited the migration and invasion ability of BC cells (E-4F, P<0.05). Figure 4 G-4H, P<0.05).

[0111] VI. Functional Recovery Experiment: Simultaneously with the inhibition of miR-let-7e-3p, the ISCA2 gene was co-transfected. Results showed that interfering with ISCA2 could partially reverse the effects of miR-let-7e-3p on the proliferation, migration, and invasion abilities of BC cells.

[0112] RT-qPCR and Western blot results showed that, compared with the control group, transfection with miR-let-7e-3p inhibitor significantly enhanced the mRNA and protein expression levels of ISCA2 in MCF-7 and MDA-MB-231 cells. Meanwhile, compared with the miR-let-7e-3p inhibitor group, co-transfection with miR-let-7e-3p inhibitor and si-ISCA2 (ISCA2 siRNA) significantly reduced the mRNA and protein expression levels of ISCA2. Figure 5 AD, P<0.05. Plate colony formation assays and Transwell assays further confirmed that, compared with the miR-let-7e-3p inhibitor group, co-transfection with si-ISCA2 significantly reduced the enhanced proliferation, migration, and invasion abilities of BC cells due to miR-let-7e-3p inhibitor transfection. Figure 5 EG, P<0.05).

[0113] VII. The impact of miR-let-7e-3p and ISCA2 on the AKT / mTOR signaling pathway

[0114] Research reports show that the mechanistic targets of the protein kinase B (PKB / AKT) and rapamycin (mTOR) pathways (PAM pathway) are frequently aberrantly activated in human cancers and play important roles in cell survival, proliferation, movement and metabolism.

[0115] Correlation analysis of ISCA2 and AKT / mTOR signaling pathways was performed using the bioinformatics website Timer. Figure 6AB). The results showed that ISCA2 was positively correlated with AKT (including AKT1 and AKT2). Western blot analysis showed that, compared with the control group, transfection with miR-let-7e-3p mimic (overexpression) or ISCA2 siRNA significantly inhibited the protein expression levels of AKT and mTOR, key proteins in the AKT / mTOR pathway, in MCF-7 and MDA-MB-231 cells. Figure 6 CF, P<0.05).

[0116] In summary, this study detected low expression of human miRNA small molecules miR-let-7e-3p in normal human mammary epithelial cells MCF-10A and human BC cell lines using RT-qPCR. Transfection with miR-let-7e-3p mimic (overexpression) inhibited the proliferation, migration, and invasion of MCF7 and MDA-MB-231 cells. Bioinformatics analysis showed that ISCA2 is a potential target gene of miR-let-7e-3p, and ISCA2 is positively correlated with AKT. Dual-luciferase reporter assay confirmed the targeting binding of miR-let-7e-3p to ISCA2. Western blot results showed that transfection with miR-let-7e-3p mimic (overexpression) significantly inhibited the expression of ISCA2, AKT, and mTOR. Knocking down ISCA2 significantly inhibited the expression of AKT and mTOR, as well as the proliferation, migration, and invasion abilities of MCF7 and MDA-MB-231 cells, while reversing the proliferative and metastatic effects of miR-let-7e-3p inhibitor on BC cells.

[0117] The conclusion was that miR-let-7e-3p was expressed at low levels in BC cells. miR-let-7e-3p binds directly to the 3'-UTR region of ISCA2, and it can inhibit the AKT / mTOR signaling pathway by targeting ISCA2, thereby inhibiting the proliferation, migration and invasion of BC cells.

[0118] In summary, to inhibit the proliferation, migration, and invasion of breast cancer cells, at least one of the following methods can be used: small interfering with the ISCA2 gene (si-ISCA2), overexpression of miRNA-let-7e-3p, targeting ISCA2, inhibiting ISCA2 expression, and subsequently inhibiting the expression of AKT and mTOR in the AKT / mTOR signaling pathway. This aims to suppress the proliferation, migration, and invasion of breast cancer cells. Such methods can be applied in the preparation of drugs for treating breast cancer, as well as in in vivo or in vitro breast cancer-related experiments. Applications include the preparation of compositions to inhibit breast cancer cell growth and proliferation, compositions to inhibit breast cancer cell migration, compositions to inhibit breast cancer cell invasion, compositions to inhibit ISCA2 protein expression, compositions to inhibit the AKT / mTOR pathway, compositions to inhibit AKT protein expression, and compositions to inhibit mTOR protein expression.

[0119] miR-let-7e-3p can serve as a biomarker for predicting treatment efficacy. Specifically, by detecting the level of a specific miR-let-7e-3p in a patient's blood or tissues, it can predict the response and effect to a specific treatment, and thus predict the efficacy of neoadjuvant chemotherapy. miR-let-7e-3p inhibits breast cancer progression by targeting ISCA2 to regulate the AKT / mTOR axis. Its expression level can be used to predict the pathological response of patients to neoadjuvant chemotherapy. miR-let-7e-3p can also help overcome treatment resistance by regulating specific miRNAs or their downstream target genes ISCA2 to inhibit the AKT / mTOR signaling pathway, thereby restoring drug sensitivity in drug-resistant breast cancer cells.

[0120] By regulating the target gene ISCA2 to improve the tumor microenvironment and enhance the efficacy of immunotherapy, it can serve as a biomarker for the diagnosis and prognosis of breast cancer. The expression pattern of the ISCA2 axis of a specific miR-let-7e-3p target gene can serve as a biomarker for the early diagnosis, subtyping, and prognosis of breast cancer, which can assist clinicians in developing individualized treatment plans. Alternatively, by utilizing the specific binding of miR-let-7e-3p to the target gene ISCA2, targeted gene delivery systems can be designed. For example, a recognition sequence of miR-let-7e-3p that is specifically highly expressed in breast cancer can be carried on a vector, allowing the therapeutic gene to be enriched and expressed only in breast cancer cells, reducing toxic side effects on normal cells.

[0121] To summarize the application scenarios of miR-let-7e-3p:

[0122] 1. Develop drugs as novel targets to upregulate the protein expression of miR-let-7e-3p or inhibit its downstream gene ISCA2, thereby inhibiting tumors, especially breast cancer.

[0123] (1) Overexpression of miR-let-7e-3p can directly inhibit the downstream target gene ISCA2, thereby affecting the AKT / mTOR signaling pathway and inhibiting the proliferation, migration and invasion of breast cancer.

[0124] (2) Directly interferes with the ISCA2 gene, thereby inhibiting the AKT / mTOR signaling pathway, and thus inhibiting the proliferation, migration and invasion of breast cancer.

[0125] (3) Overexpression of miR-let-7e-3p and interference with ISCA2 are used in combination to inhibit the AKT / mTOR signaling pathway and thus inhibit breast cancer proliferation, migration and invasion.

[0126] 2. Application of miR-let-7e-3p biomarker in the preparation of kits for breast cancer screening and diagnosis.

[0127] On the one hand, by detecting the level of specific miR-let-7e-3p in a patient's blood or tissues, the response and effect to a specific treatment can be predicted. miR-let-7e-3p inhibits breast cancer progression by targeting ISCA2 to regulate the AKT / mTOR axis, and its expression level can be used to predict the patient's pathological response to neoadjuvant chemotherapy.

[0128] On the other hand, the expression patterns of specific miR-let-7e-3p target genes along the ISCA2 axis can serve as biomarkers for early diagnosis, classification, and prognosis of breast cancer, and can assist clinicians in developing individualized treatment plans.

[0129] 3. miR-let-7e-3p can be used to overcome drug resistance in treatment.

[0130] miR-let-7e-3p can be used to overcome treatment resistance by regulating specific miRNAs or their downstream target genes ISCA2 to inhibit the AKT / mTOR signaling pathway, thereby restoring drug sensitivity to resistant breast cancer cells.

[0131] 4. Application of miR-let-7e-3p as a gene therapy vector

[0132] By utilizing the specific binding of miR-let-7e-3p to the target gene ISCA2, targeted gene delivery systems can be designed. For example, a recognition sequence of miR-let-7e-3p that is specifically highly expressed in breast cancer can be loaded onto a vector, allowing therapeutic genes to be enriched and expressed only in breast cancer cells, reducing toxic side effects on normal cells.

Claims

1. A composition, characterized in that, This is a composition that targets ISCA2 to inhibit breast cancer, the composition comprising at least one of the following: (i) Small interfering RNAs or miRNAs that target ISCA2 or ISCA2 transcripts and can inhibit the expression of ISCA2 gene expression products or gene transcription; (ii) Able to express or form constructs of the small interfering RNA or miRNA described in (i); (iii) A construct containing ISCA2 or ISCA2 complementary sequence, which can form an interfering molecule that inhibits the expression of ISCA2 gene expression products or gene transcription after being transformed into the body; (iv) Immune-related cells, their differentiated cells, or constructs after the ISCA2 gene sequence has been suppressed or knocked out.

2. The composition according to claim 1, characterized in that, The miRNA is at least one of miRNA-let-7e-3p or overexpression of miR-let-7e-3p (miR-let-7e-3p mimic), wherein miR-let-7e-3p directly binds to the 3'-UTR region of ISCA2, the miR-let-7e-3p mimic sense sequence of the overexpressed miR-let-7e-3p (miR-let-7e-3p mimic) is shown in SEA ID NO.1, and the miR-let-7e-3p mimic antisense sequence is shown in SEA ID NO.

2.

3. The composition according to claim 1, characterized in that, The small interfering RNA is a small interfering RNA (si-ISCA2) of the ISCA2 gene. The sense and antisense sequences of the si-ISCA2 sequence are any one of the following combinations. Group 1: The si-ISCA2-1 sense sequence is shown in SEA ID NO.3, and the si-ISCA2-1 antisense sequence is shown in SEA ID NO.

4. The second group: the si-ISCA2-2 sense sequence is shown in SEA ID NO.5, and the si-ISCA2-2 antisense sequence is shown in SEA ID NO.

6. The third group: the si-ISCA2-3 sense sequence is shown in SEA ID NO.7, and the si-ISCA2-3 antisense sequence is shown in SEA ID NO.

8.

4. The composition according to claim 1, characterized in that, This composition targets ISCA2, inhibits ISCA2 gene expression, and thereby inhibits the protein expression levels of AKT and mTOR, key proteins in the AKT / mTOR pathway, thus inhibiting the AKT / mTOR pathway and consequently suppressing breast cancer.

5. The composition according to claim 1, characterized in that, It inhibits the proliferation, migration, and invasion of breast cancer cells.

6. The use of the composition according to any one of claims 1 to 5 in at least one aspect of (1) to (8), (1) in the preparation of a medicament for treating breast cancer, (2) in the preparation of a composition for inhibiting the growth and proliferation of breast cancer cells, (3) in the preparation of a composition for inhibiting the migration of breast cancer cells, (4) in the preparation of a composition for inhibiting the invasive ability of breast cancer cells, (5) in the preparation of a composition for inhibiting the expression of ISCA2 protein, (6) in the preparation of a composition for inhibiting the AKT / mTOR pathway, (7) in the preparation of a composition for inhibiting the expression of AKT protein, and (8) in the preparation of a composition for inhibiting the expression of mTOR protein.

7. miR-let-7e-3p is used in at least one of the following (1) to (8): (1) in the preparation of a medicament for treating breast cancer; (2) in the preparation of a composition for inhibiting the growth and proliferation of breast cancer cells; (3) in the preparation of a composition for inhibiting the migration of breast cancer cells; (4) in the preparation of a composition for inhibiting the invasive ability of breast cancer cells; (5) in the preparation of a composition for inhibiting the expression of ISCA2 protein; (6) in the preparation of a composition for inhibiting the AKT / mTOR pathway; (7) in the preparation of a composition for inhibiting the expression of AKT protein; (8) in the preparation of a composition for inhibiting the expression of mTOR protein.

8. The application of miR-let-7e-3p in at least one of the following (1) to (4): (1) the application of preparing miR-let-7e-3p biomarkers in the preparation of kits for breast cancer screening and diagnosis; (2) the application of miR-let-7e-3p as a standardized internal control in the preparation of kits for breast cancer screening and diagnosis; (3) the application of preparing drugs for restoring drug sensitivity in breast cancer cells; and (4) the application as a recognition sequence of miR-let-7e-3p on gene therapy vectors.

9. A miR-let-7e-3p overexpression (miR-let-7e-3p mimic), used in at least one of the following (1) to (8): (1) in the preparation of a medicament for treating breast cancer; (2) in the preparation of a composition for inhibiting the growth and proliferation of breast cancer cells; (3) in the preparation of a composition for inhibiting the migration of breast cancer cells; (4) in the preparation of a composition for inhibiting the invasive ability of breast cancer cells; (5) in the preparation of a composition for inhibiting the expression of ISCA2 protein; (6) in the preparation of a composition for inhibiting the AKT / mTOR pathway; (7) in the preparation of a composition for inhibiting the expression of AKT protein; (8) in the preparation of a composition for inhibiting the expression of mTOR protein, wherein the miR-let-7e-3p mimic sense sequence is shown in SEA ID NO.1 and the miR-let-7e-3p mimic antisense sequence is shown in SEA ID NO.

2.

10. A small interfering sequence (si-ISCA2) of the ISCA2 gene, used in at least one of the following (1) to (8): (1) in the preparation of a medicament for treating breast cancer; (2) in the preparation of a composition for inhibiting the growth and proliferation of breast cancer cells; (3) in the preparation of a composition for inhibiting the migration of breast cancer cells; (4) in the preparation of a composition for inhibiting the invasive ability of breast cancer cells; (5) in the preparation of a composition for inhibiting the protein expression of ISCA2; (6) in the preparation of a composition for inhibiting the AKT / mTOR pathway; (7) in the preparation of a composition for inhibiting the expression of AKT protein; (8) in the preparation of a composition for inhibiting the expression of mTOR protein. The sense and antisense sequences of the si-ISCA2 sequence are any of the following pairs. Group 1: The si-ISCA2-1 sense sequence is shown in SEA ID NO.4, and the si-ISCA2-1 antisense sequence is shown in SEA ID NO.

5. The second group: the si-ISCA2-2 sense sequence is shown in SEA ID NO.6, and the si-ISCA2-2 antisense sequence is shown in SEA ID NO.

7. The third group: the si-ISCA2-3 sense sequence is shown in SEA ID NO.8, and the si-ISCA2-3 antisense sequence is shown in SEA ID NO.9.

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