LncRNA HAGLROS promotes breast cancer evolution through miR-135b-3p/COL10A1 axis and exosome-mediated macrophage M2 polarization

Abstract

Long non-coding RNAs (lncRNAs) play an important role in breast cancer progression, but the function of lncRNAs in regulating tumor-associated macrophages (TAMs) remains unclear. As carriers of lncRNAs, exosomes play an important role as mediators in the communication between cancer cells and the tumor microenvironment. In this study, we found that lncRNA HAGLROS was highly expressed in breast cancer tissues and plasma exosomes, and its high expression was related to the poor prognosis of breast cancer patients. Functionally, breast cancer cell-derived exosomal lncRNA HAGLROS promotes breast cancer cell proliferation, migration, epithelial-mesenchymal transition (EMT) process and angiogenesis by inducing TAM/M2 polarization. Mechanistically, lncRNA HAGLROS competitively binds to miR-135-3p to prevent the degradation of its target gene COL10A1. Collectively, these results indicated that the lncRNA HAGLROS/miR-135b-3p/COL10A1 axis promoted breast cancer progression, and revealed the interactive communication mechanism between breast cancer cells and TAMs, suggesting that lncRNA HAGLROS may be a potential biomarker and therapeutic target for breast cancer.

Fig. 1. LncRNA HAGLROS upregulation is associated with advanced progression and poor prognosis in breast cancer.

A Heat map showing the top differentially expressing lncRNAs in breast cancer samples compared to normal tissues (log₂ |FC | > 1 and P < 0.05). B Volcano plot showing the expression profiles of lncRNAs. C, D The expression of lncRNA HAGLROS expression in normal and breast cancer tissues was searched by UALCAN and lnCAR databases. E The expression of lncRNA HAGLROS in breast cancer tissues was detected by ISH staining. (a) LncRNA HAGLROS was negative staining in normal breast tissues. (b-d) Weak, moderate, and strong expression of lncRNA HAGLROS in breast cancer tissues (original magnification, a–d: 40 ×; a1–d1: 400 × ). F Representative ISH images of lncRNA HAGLROS expression (brown) in different TNM stages of breast cancer tissues (original magnification, a–b: 40×; a1–b1: 400×). G Representative ISH images of lncRNA HAGLROS expression (brown) in breast cancer tissues with and without LN metastasis (original magnification, a-b: 40×; a1-b1: 400×). H Representative ISH images of lncRNA HAGLROS expression (brown) in breast cancer tissues with and without distant metastasis (original magnification, a-b: 40×; a1-b1: 400×). I Representative ISH images (brown) of lncRNA HAGLROS expression in breast cancer tissues with and without ER expression (original magnification, a-b: 40×; a1-b1: 400×). J Kaplan-Meier plotter survival analysis was performed to assess the impact of lncRNA HAGLROS on overall survival in breast cancer patients. K, M The lnCAR database analyzes the effect of lncRNA HAGLROS on overall survival, Metastasis-free survival and Relapse-free survival in breast cancer patients.

Fig. 2. LncRNA HAGLROS promotes proliferation and metastasis of breast cancer cells in vitro and in vivo.

A The expression of lncRNA HAGLROS in breast epithelial cells and breast cancer cells was detected by qRT-PCR assay. GAPDH was used as an internal control. B The vector and lncRNA HAGLROS overexpression plasmid were transfected in SK-BR3 and MDA-MB-231 cells, and control, sh-lncRNA HAGLROS-1, sh-lncRNA HAGLROS-2, sh-lncRNA HAGLROS-3 silencing plasmids were transfected in Hs578T and MCF-7 cells (lncRNA HAGLROS overexpression group compared with vector, sh-lncRNA HAGLROS group compared with control), and the transfection effect of lncRNA HAGLROS was detected by qRT-PCR assay. GAPDH was used as an internal control. C–E The effect of differentially expressing lncRNA HAGLROS on the proliferation ability of breast cancer cells was detected by MTT, colony formation and EdU assays. F Representative images of xenograft tumors in nude mice with breast cancer cells differentially expressing lncRNA HAGLROS (upper: vector and lncRNA HAGLROS overexpression groups; lower: control and lncRNA HAGLROS silenced group), and the volume and weight of xenograft tumors (n = 5 per group). G The expression of Ki67 in the xenograft tumor tissues was detected by IHC staining. H The expression of lncRNA HAGLROS in the xenograft tumor tissues was detected by qRT-PCR assay. I, J The effect of differentially expressing lncRNA HAGLROS on the migration and invasion ability of breast cancer cells was detected by wound healing and Transwell assays. K Breast cancer cells differentially expressing lncRNA HAGLROS were separately injected via tail vein into nude mice for in vivo metastasis (n = 5 per group). Representative images show the number of metastatic nodules in the lungs (left), and the number of nude mice that developed lung metastases (right).

Fig. 3. LncRNA HAGLROS promotes the EMT process and angiogenesis of breast cancer cells in vitro and in vivo.

A The protein expression levels of EMT-related markers in breast cancer cells differentially expressing lncRNA HAGLROS were detected by western blotting assay. β-actin was used as an internal control. B The fluorescence expression levels of E-Cadherin and Vimentin in breast cancer cells differentially expressing lncRNA HAGLROS were detected by IF staining. C, D The expression of E-Cadherin and Vimentin in the xenograft tumor tissues was detected by IHC staining and western blotting assay. E, F HUVECs or HLECs were cultured with the supernatants of breast cancer cells differentially expressing lncRNA HAGLROS and the microtubule formations were detected by endothelial tube formation assay. G The effect of differentially expressed lncRNA HAGLROS on the angiogenesis ability of breast cancer cells was detected by VM assay. H The protein expression levels of angiogenesis-related markers in breast cancer cells differentially expressing lncRNA HAGLROS were detected by western blotting assay. β-actin was used as an internal control. I The effect of breast cancer cells differentially expressing lncRNA HAGLROS on angiogenesis in vitro was examined by CAM assay.

Fig. 4. LncRNA HAGLROS as a sponge for miR-135b-3p promotes the proliferative ability of breast cancer cells.

A, B The localization of lncRNA HAGLROS in breast cancer cells was determined by the lncATLAS database and FISH assay. C Heat map representation of differentially expressed miRNA after silencing of lncRNA HAGLROS in Hs578T cells (log₂ |FC| > 1 and P < 0.05). D The expression level of miR-135b-3p in breast cancer cells differentially expressing lncRNA HAGLROS was detected by qRT-PCR assay. U6 was used as an internal control. E The expression level of miR-135b in normal and breast cancer tissues was searched by the UALCAN database. F, G The correlation of miR-135b expression levels in breast cancer with lymph node metastasis and TNM stage were searched by the UALCAN database. H Kaplan-Meier plotter survival analysis was performed to assess the impact of miR-135b on overall survival in breast cancer patients. I The transfection effect of breast cancer cells transfected with miR-135b-3p mimics and inhibitors was detected by qRT-PCR assay. U6 was used as an internal control. J Breast cancer cells co-transfected with wild-type or mutant lncRNA HAGLROS and miR-135b-3p or control were detected by dual luciferase reporter assay. K–M The effect of co-transfected lncRNA HAGLROS and miR-135b-3p on the proliferation ability of breast cancer cells was determined by MTT, colony formation and EdU assays.

Fig. 5. LncRNA HAGLROS as a sponge for miR-135b-3p promotes the migration and invasion ability of breast cancer cells.

A, B The effect of co-transfected lncRNA HAGLROS and miR-135b-3p on the migration and invasion ability of breast cancer cells was determined by wound healing and Transwell assays. C, D HUVECs or HLECs were cultured with the supernatants of breast cancer cells co-transfected with lncRNA HAGLROS and miR-135b-3p, and the microtubule formations were detected by endothelial tube formation assay. E The effect of co-transfected lncRNA HAGLROS and miR-135b-3p on the angiogenesis ability of breast cancer cells was detected by VM assay. F The protein expression levels of EMT and angiogenesis-related markers in breast cancer cells co-transfected with lncRNA HAGLROS and miR-135b-3p were detected by western blotting assay. β-actin was used as an internal control.

Fig. 6. LncRNA HAGLROS acts as a sponge for miR-135b-3p to regulate COL10A1 expression and promotes the proliferative ability of breast cancer cells.

A The potential target genes of miR-135b-3p were predicted by RNA22, miRWalk, mirDIP, miRDB, and TargetScan databases. B Volcano plot showing the expression profile of differential genes in breast cancer (log₂ |FC| > 1 and P < 0.05). C The expression level of ULBP1, SIX4, SHISA9, CKAP2L, COL10A1 and DYNAP in normal and breast cancer tissues were searched by the GEPIA database. D The mRNA expression level of ULBP1, SIX4, SHISA9, CKAP2L, COL10A1 and DYNAP in breast cancer cells differentially expressing miR-135b-3p was detected by qRT-PCR assay. GAPDH was used as an internal control. E Breast cancer cells co-transfected with wild-type or mutant COL10A1 and miR-135b-3p or control were detected by dual luciferase reporter assay. F The interaction between lncRNA HAGLROS and miR-135b-3p and COL10A1 was analyzed by RIP assay. G, H The control and COL10A1 overexpression plasmid were transfected in SK-BR3 and MDA-MB-231 cells, and control, si-COL10A1-1, si-COL10A1-2, si-COL10A1-3 were transfected in Hs578T and MCF-7 cells (COL10A1 overexpression group compared with control, si-COL10A1 group compared with control), and the transfection effect of COL10A1 was detected by western blotting and qRT-PCR assays. I–K The effect of co-transfected miR-135b-3p and COL10A1 on the proliferation ability of breast cancer cells was determined by MTT, colony formation and EdU assays.

Fig. 7. LncRNA HAGLROS acts as a sponge for miR-135b-3p to regulate COL10A1 expression and promotes the migration and invasion ability of breast cancer cells.

A, B The effect of co-transfected miR-135b-3p and COL10A1 on the migration and invasion ability of breast cancer cells was determined by wound healing and Transwell assays. C, D HUVECs or HLECs were cultured with the supernatants of breast cancer cells co-transfected with miR-135b-3p and COL10A1, and the microtubule formations were detected by endothelial tube formation assay. E The effect of co-transfected miR-135b-3p and COL10A1 on the angiogenesis ability of breast cancer cells was detected by VM assay. F The protein expression levels of EMT and angiogenesis-related markers in breast cancer cells co-transfected with miR-135b-3p and COL10A1 were detected by western blotting assay. β-actin was used as an internal control. G, H The expression of COL10A1 in the xenograft tumor tissues was detected by IHC staining and western blotting assays.

Fig. 8. Exosomal lncRNA HAGLROS promotes TAM/M2 polarization.

A, B Exosomes isolated from supernatants of breast cancer cells were detected by TEM and NTA. C The expression of exosome markers TSG101 and CD9 was detected by western blotting assay. D The expression of lncRNA HAGLROS in exosomes derived from breast cancer cells differentially expressing lncRNA HAGLROS was detected by qRT-PCR assay. E Representative fluorescence microscopy showing the uptake of PKH26-labeled exosomes (red fluorescent dye) derived from MDA-MB-231 cells by recipient macrophages. F–H Macrophages were cultured with exosomes derived from breast cancer cells differentially expressing lncRNA HAGLROS and the expression of TAM/M1, TAM/M2-related markers and lncRNA HAGLROS in macrophages was detected by qRT-PCR, western blotting and flow cytometry assays.