Exosomal LINC00460/miR-503-5p/ANLN positive feedback loop aggravates pancreatic cancer progression through regulating T cell-mediated cytotoxicity and PD-1 checkpoint

Abstract

Background: Long non-coding RNA (lncRNA) LINC00460 is an onco-lncRNA in a variety of cancers, including pancreatic cancer (PC). This study is aimed to investigate the regulatory mechanisms of LINC00460 in PC.

Methods: The tumor and adjacent normal tissues were collected from 73 PC patients. The expression of LINC00460, miR-503-5p, and ANLN was detected using qRT-PCR. We then analyzed the proliferation, migration, invasion, and apoptosis/cell cycle of PC cells by performing the MTT/EdU, transwell, and flow cytometry assays, respectively. The xenograft tumor model were utilized to confirm the effect of LINC00460 knockdown on PC through anti-PD-1 therapy in vivo, and the sensitivity of PANC-1 cells to the cytotoxicity of CD8⁺ T cells in vitro. Western blotting was used to determine the protein levels. A co-culture model was utilized to explore the effects of exosomes on macrophages.

Results: LINC00460 was up-regulated in PC tissues and cells. LINC00460 knockdown suppressed cell proliferation, migration, and invasion, facilitated cell apoptosis and G0/G1 phase arrest, and inhibited the tumor growth through anti-PD-1 therapy. Both miR-503-5p down-regulation and ANLN up-regulation reversed the effects of LINC00460 knockdown on inhibiting the proliferation, migration and invasion, and on promoting the apoptosis, G0/G1 phase arrest, and the sensitivity of PC cells to the cytotoxicity of CD8⁺ T cells. Exosomes were uptaken by the ambient PC cells. PANC-1 cells-derived exosomal LINC00460-induced M2 macrophage polarization accelerates the cell migration and invasion.

Conclusions: LINC00460 silencing attenuates the development of PC by regulating the miR-503-5p/ANLN axis and exosomal LINC00460-induced M2 macrophage polarization accelerates the migration and invasion of PANC-1 cells, thus LINC00460 may act as a possible therapeutic target for treating PC.

Keywords: ANLN; Exosomes; LINC00460; M2 polarization; Pancreatic cancer; miR-503-5p.

Fig. 1

LINC00460 is highly expressed in PC tissues and cells. A The expression of LINC00460 in PAAD tissues and normal tissues was analyzed in TCGA. *P < 0.01. B The expression of LINC00460 in PC tissues (n = 73) and adjacent tissues (n = 73) was detected by qRT-PCR. P < 0.01. C The expression of LINC00460 in lymph node metastasis or non-metastasis was detected by qRT-PCR. P < 0.01. D The expression of LINC00460 at different WHO grades was detected by qRT-PCR. P < 0.01. E Correlation between LINC00460 expression and overall survival of PC patients was assessed using Kaplan–Meier analysis. *P < 0.05. F The expression of LINC00460 in H6C7 cells and PC cell lines (ASPC-1, BxPC-3, SW1990, PANC-1 and Mia-PaCa-2) was detected by qRT-PCR. **P < 0.01 vs. the H6C7 cells group

Fig. 2

LINC00460 knockdown inhibits the malignant characteristics of PANC-1 cells in vitro and suppresses the growth of tumor xenograft in vivo. A The expression of LINC00460 in PANC-1 cells was detected by qRT-PCR. B The viability (OD450) of PANC-1 cells was measured by MTT assay. C The proliferation of PANC-1 cells was determined by EdU assay (200 ×). D The apoptosis of PANC-1 cells was analyzed by flow cytometry assay. (E) The migration ability of PANC-1 cells was measured by transwell assay. F The invasion ability of PANC-1 cells was measured by transwell assay. G The protein levels of E-cadherin and N-cadherin were determined by Western blot. H The images of tumor xenograft with LINC00460 knockdown. I The tumor volumes were monitored at different time points. J The tumor weights were measured 4 weeks later. (K) Ki67 staining in PC tissues was measured by IHC assay (400 ×). **P < 0.01 vs. the sh-NC group

Fig. 3

MiR-503-5p is the direct target of LINC00460. A The predicted targets of LINC00460 from databases (StarBase and TCGA) were exhibited by Venn diagram. B Five miRNAs selected from databases were detected by RIP assay in PANC-1 cells. **P < 0.01 vs. the MS2 group. C The expression of miR-503-5p in PAAD tissues and normal tissues was analyzed in TCGA. *P < 0.05. D The predicted binding site between LINC00460 and miR-503-5p. E The luciferase activity in PANC-1 cells transfected with pGL3-LINC00460 WT/pGL3-LINC00460 MUT and miR-503-5p mimics/NC was determined by DLR assay. **P < 0.01 vs. the miR-NC group. F The expression of miR-503-5p after transfection of sh-LINC00460-1/NC into PANC-1 cells was detected by qRT-PCR. **P < 0.01 vs. the sh-NC group. G The expression of miR-503-5p in tumor tissues (n = 73) and adjacent tissues (n = 73) was detected by qRT-PCR. P < 0.01. H The correlation between LINC00460 and miR-503-5p. P < 0.01, r = −0.4115. I The expression of miR-503-5p in H6C7 cells and PC cell lines (ASPC-1, BxPC-3, SW1990, PANC-1 and Mia-PaCa-2) was detected by qRT-PCR. **P < 0.01 vs. the H6C7 cells group

Fig. 4

Overexpression of miR-503-5p inhibits the malignant characteristics of PANC-1 cells. A The expression of miR-503-5p after transfection of miR-503-5p mimics/NC into PANC-1 cells was detected by qRT-PCR. B The viability (OD450) of PANC-1 cells was measured by MTT assay. C The proliferation of PANC-1 cells was determined by EdU assay (200 ×). D The apoptosis of PANC-1 cells was analyzed by flow cytometry assay. E The migration ability of PANC-1 cells was measured by transwell assay. F The invasion ability of PANC-1 cells was measured by transwell assay. G The protein levels of E-cadherin and N-cadherin were determined by Western blot. **P < 0.01 vs. the miR-NC group

Fig. 5

Identification of ANLN as the target gene of miR-503-5p. A Three target genes of miR-503-5p from databases (TargetScan, miRDB and TCGA) were exhibited by Venn diagram. B The predicted complementary binding site of miR-503-5p and ANLN. C The luciferase activity in PANC-1 cells transfected with pGL3-ANLN WT/pGL3-ANLN MUT and miR-503-5p mimics/NC was determined by DLR assay. **P < 0.01 vs. the miR-NC group. D The expression of miR-503-5p in tumor tissues (n = 73) and adjacent tissues (n = 73) was detected by qRT-PCR. P < 0.01. E The correlation between miR-503-5p and ANLN. P < 0.01, r = −0.339. F The correlation between LINC00460 and ANLN. P < 0.01, r = −0.3623. G The protein level of ANLN after transfection of miR-503-5p mimics/NC into PANC-1 cells was determined by western blot assay. **P < 0.01 vs. the miR-NC group. H The protein level of ANLN in H6C7 cells and PC cell lines (ASPC-1, BxPC-3, SW1990, PANC-1 and Mia-PaCa-2) were determined by western blot assay. **P < 0.01 vs. the H6C7 cells group

Fig. 6

LINC00460 knockdown inhibits the proliferation, migration and invasion, and promotes the apoptosis and G0/G1 phase arrest of PANC-1 cells by regulating miR-503-5p/ANLN. A The protein level of ANLN after transfection of pcDNA-ANLN/NC into PANC-1 cells was measured by western blot assay. **P < 0.01 vs. the pcDNA-NC group. B The expression of miR-503-5p after transfection of miR-503-5p inhibitor/miR-NC into PANC-1 cells was measured by qRT-PCR. **P < 0.01 vs. the miR-NC group. C The viability (OD450) of PANC-1 cells was measured by MTT assay. D The apoptosis of PANC-1 cells was analyzed by flow cytometry assay. E The cell cycle of PANC-1 cells was analyzed by flow cytometry assay. F The migration ability of PANC-1 cells was measured by transwell assay. G The invasion ability of P PANC-1 cells was measured by transwell assay. H The protein levels of E-cadherin and N-cadherin were determined by Western blot. I The proliferation of PANC-1 cells was determined by EdU assay (200 ×). **P < 0.01 vs. the sh-NC group, ^##P < 0.01 vs. the sh-LINC00460 group

Fig. 7

Silencing of LINC00460 reduces the tumor growth of PC through anti-PD-1 therapy and enhances the sensitivity of PANC-1 cells to cytotoxicity of tumor-reactive T cells via regulating the miR-503-5p/ANLN axis. A The images of tumor xenograft with different treatments. B The tumor volumes were monitored at different time points. C The tumor weight in mice of each group after 2 weeks of injection. **P < 0.01 vs. the IgG + sh-NC group; ^##P < 0.01 vs. the anti-PD-1 + sh-NC group. D Ki67 staining in PC tissues was measured by IHC assay (400 ×). E The sensitivity of PANC-1 cells to the cytotoxicity of tumor-reactive T cells in different treatments. **P < 0.01 vs. the sh-NC group; ^#P < 0.05, ^##P < 0.01 vs. the sh-LINC00460 group

Fig. 8

Exosomal-LINC00460 facilitates M2 macrophage polarization. A The morphological characteristics of exosomes which was round membranous vesicle were observed by a TEM. Scale bar: 200 nm. B The levels of exosomes surface marker (TSG101, CD63 and CD81) were detected by Western blot. C The mRNA level of macrophage marker CD68 was detected by qRT-PCR. **P < 0.01 vs. the THP-1 cells group. D Uptake of exosomes was observed by a confocal microscope (400 ×). E The expression of LINC00460 in exosomes secreted by PANC-1 cells was detected by qRT-PCR. **P < 0.01 vs. the pcDNA-NC group. F The mRNA levels of M2 polarized markers (CD163, CD206, ARG1 and IL-10) were detected by qRT-PCR. *P < 0.01 vs. the control group; ^##P < 0.01 vs. the Exo-pcDNA-NC group. (G) The mRNA levels of M1 polarized markers (iNOS and IL-12) were detected by qRT-PCR. *P < 0.01 vs. the control group; ^##P < 0.01 vs. the Exo-pcDNA-NC group

Fig. 9

M2 macrophage polarization aggravates the migration and invasion of PANC-1 cells. A The migration ability of PANC-1 cells in a co-culture model was measured by transwell assay. B The invasion ability of PC cells in a co-culture model was measured by transwell assay. C The protein levels of E-cadherin and N-cadherin were determined by western blot assay. *P < 0.05, **P < 0.01 vs. the PANC-1 cells group; ^##P < 0.01 vs. the PANC-1 + M + Exo-NC group