MNX1-AS1 suppresses chemosensitivity by activating the PI3K/AKT pathway in breast cancer

Abstract

Long noncoding RNAs (lncRNAs) critically regulate tumorigenesis and chemosensitivity. Despite the pivotal role of lncRNAs in breast cancer (BC), their specific functions and underlying mechanism, particularly in the context of drug resistance, remain largely unexplored. We discovered that MNX1-AS1 is significantly elevated in BC and contributes to paclitaxel resistance through the PI3K/AKT pathway. Moreover, elevated MNX1-AS1 expression exhibits close association with unfavourable prognosis in BC. Mechanistically, MNX1-AS1 interacts with YBX1, preventing its SMURF2-mediated ubiquitination and subsequent degradation, thereby increasing YBX1 protein levels. Upregulated YBX1 transcriptionally activates the expression of ITGA6 by binding to its promoter in the nucleus. Furthermore, MNX1-AS1 binds to IGF2BP2, promoting the stability of ITGA6 mRNA in an m6A-dependent manner within the cytoplasm. MNX1-AS1 increases ITGA6 expression at transcriptional and post-transcriptional levels, thereby activating the PI3K/AKT pathway. Notably, lipid nanoparticles were implicated to effectively deliver MNX1-AS1 siRNA to tumor-bearing mice, resulting in significant antitumor effects. These findings underscore the role of MNX1-AS1 in activating the ITGA6/PI3K/AKT pathway, which facilitates tumor progression and induces chemoresistance in BC. Targeting MNX1-AS1 may represent a promosing therapeutic strategy to enhance chemotherapy efficacy in BC patients.

Keywords: ITGA6/PI3K/AKT pathway; LncRNA; breast cancer; chemosensitivity; lipid nanoparticles.

Figure 1

Levels of MNX1-AS1 is significantly increased in BC tissues and associated with poor prognosis. (A) MNX1-AS1 expression in BC tissues (n =113) and paired normal tissues (n = 113) in the TCGA dataset. (B) Representative ISH results of MNX1-AS1 expression from the BC tissue microarray. (C) Statistical analysis of ISH expression in BC tissues (n=90) and paired normal tissues (n=90). (D-E) The expression of MNX1-AS1 exhibited obvious upregulation in BC patients with a higher pathological stage according to TCGA data. (F) Kaplan-Meier analysis revealed the overall survival in BC patients based on the relative MNX1-AS1 expression. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 2

MX1-AS1 promotes BC cell proliferation. (A) MNX1-AS1 knockdown efficiency was analyzed by qRT-PCR assays in BC cells. (B) Cell viability examinations of BC cells with MNX1-AS1 knockdown. (C) Colony-forming assays were conducted to determine the proliferation of BC cells with MNX1-AS1 knockdown. (D) EdU staining assays were used to determine the proliferation of BC cells with MNX1-AS1 knockdown. (E) MNX1-AS1 overexpression efficiency was analyzed by qRT-PCR assays in BC cells. (F) Cell viability examinations of BC cells with MNX1-AS1 overexpression. (G) Colony-forming assays were conducted to determine the proliferation of BC cells with MNX1-AS1 overexpression. (H) EdU staining assays were used to determine the proliferation of BC cells with MNX1-AS1 overexpression. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 3

ITGA6/PI3K/AKT is a key downstream pathway of MNX1-AS1 in BC. (A) The heatmap of RNA transcription sequencing of the control group and the sh-MNX1-AS1 group. (B) KEGG analysis for all altered genes after knockdown of MNX1-AS1. (C) The scatter plot of RNA transcription sequencing with the genes related to PI3K/AKT pathway were labeled. (D) The correlation between ITGA6 mRNA (detected by ISH) and MNX1-AS1 mRNA expression levels (detected by ISH) in BC tissue samples. (E) The correlation between ITGA6 protein expression (detected by IHC) and MNX1-AS1 mRNA expression levels (detected by ISH) in BC tissue samples. (F) ITGA6 showed obvious decrease in BC cells with MNX1-AS1 knockdown at mRNA level. (G-H) Western blot assays confirmed that MNX1-AS1 knockdown inhibited the ITGA6/PI3K/AKT pathway, while its overexpression activated it. (I) The inhibition PI3K/AKT pathway was partially reversed by ITGA6 overexpression. (J) The inhibition of BC proliferation was partially reversed by ITGA6 overexpression. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 4

MNX1-AS1 accelerates BC proliferation and enhances the resistance of BC cells to paclitaxel. (A) Representative FISH results of MNX1-AS1 expression from the BC tissue with or without recurrence. (B) Changes in IC50 values of the paclitaxel in BC cells after MNX1-AS1 knockdown. (C-E) MDA-MB-231 cells stably expressing sh-CTRL and sh-MNX1-AS1 were inoculated into BALB/c female nude mice. After 6 days of injection, DMSO or paclitaxel (5 mg/kg, dissolved in DMSO) was administered by intraperitoneal injection every 2 days. Representative tumor images (C), tumor weight (D), and tumor volume (E) were shown. (F) Tumor tissue samples were immunostained for H&E, Ki-67, ITGA6 and p-PI3K. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 5

MNX1-AS1 binds with YBX1 and decreases the ubiquitination of YBX1. (A) FISH assays were used to determine the distribution of MNX1-AS1 in MDA-MB-231 and MCF-7 cells. (B) The RIP assays revealed the enrichment of MNX1-AS1 in YBX1 RIP, as compared with its matched IgG group. (C) Western blot analysis of the expression of YBX1 in BC cells with MNX1-AS1 knockdown and overexpression. (D) Relative YBX1 protein levels in MNX1-AS1-silenced cells following CHX treatment. (E) Relative YBX1 protein levels in MNX1-AS1-silenced cells with or without MG132 treatment. (F) Immunoblotting for ubiquitin following immunoprecipitation with YBX1 antibody in MNX1-AS1 knockdown or control cells treated with MG132. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 6

MNX1-AS1 disrupts the binding of SMURF2 with YBX1. (A) Immunoblotting for YBX1 and SMURF2 following immunoprecipitation with SMURF2 and YBX1 antibody. (B) Immunoblotting for ubiquitin following immunoprecipitation with YBX1 antibody in cells stably overexpressing SMURF2 and controls, treated with MG132. (C) Relative levels of YBX1 protein in SMURF2 overexpressing cells or control cells. (D) Western blot analysis of SMURF2 and YBX1 in Co-IP assays performed with control and MNX1-AS1-silenced BC cells. (E) The qRT-PCR analysis of ITGA6 expression in BC cells with YBX1 knockdown. (F-G) ChIP assays demonstrated that knockdown of MNX1-AS1 reduces YBX1 enrichment in the promoter region of ITGA6. (H) Dual-luciferase reporter assays revealed that YBX1 binds to the E1, E2 and E4 promoter region of ITGA6. Error bars show the SD from three independent experiments. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 7

MNX1-AS1 cooperates with IGF2BP2 to regulate ITGA6 expression in an m6A-dependent manner. (A) The RIP assays revealed the enrichment of MNX1-AS1 in IGF2BP2 RIP, as compared with its matched IgG group. (B-C) Relative IGF2BP2 and ITGA6 mRNA levels in cells with IGF2BP2 knockdown. (D) The correlation between IGF2BP2 and ITGA6 was assessed using TCGA data analysis. (E) RNA stability assays were performed using Actinomycin D (2μg/ml) to disrupt RNA synthesis in BC cells, and the degradation rates of the ITGA6 mRNAs were measured every 3 h by qRT-PCR. (F) MeRIP-qPCR assays were performed to quantify the relative m6A modification level of ITGA6 upon MNX1-AS1 knockdown in BC cells. (G) The RIP assays was performed in MNX1-AS1-silenced and control BC cells. The coprecipitated RNA was analyzed by qRT-PCR for ITGA6. The fold enrichment of ITGA6 in IGF2BP2 RIP was compared to the IgG group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 8

Therapeutic efficacy and toxicity evaluation of systemic injection of LNP (si-MNX1-AS1). (A) The size distribution profile of LNPs (si-MNX1-AS1). (B) The qRT-qPCR assays were performed to evaluate the expression level of MNX1-AS1 between LNP-si-MNX1-AS1 and LNP-si-CTRL. (C-E) MDA-MB-231 cells were inoculated into BALB/c female nude mice. Three days after injection, LNP-si-CTRL or LNC-si-MNX1-AS1 was injected peritumorally every three days. Representative tumor images (C), tumor volume (D), and tumor weight (E) are shown. (F) Tumor tissue samples were immunostained with H&E and Ki-67. (G) The schematic model of the contributor and regulatory mechanism of MNX1-AS1 in the occurrence and chemotherapy resistance of BC. (Figure was created with BioRender.com). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01.