Long non-coding RNA HOTAIR promotes exosome secretion by regulating RAB35 and SNAP23 in hepatocellular carcinoma

Abstract

Background: Emerging evidence indicates that tumor cells release a large amount of exosomes loaded with cargos during tumorigenesis. Exosome secretion is a multi-step process regulated by certain related molecules. Long non-coding RNAs (lncRNAs) play an important role in hepatocellular carcinoma (HCC) progression. However, the role of lncRNA HOTAIR in regulating exosome secretion in HCC cells remains unclear.

Methods: We analyzed the relationship between HOTAIR expression and exosome secretion-related genes using gene set enrichment analysis (GSEA). Nanoparticle tracking analysis was performed to validate the effect of HOTAIR on exosome secretion. The transport of multivesicular bodies (MVBs) after overexpression of HOTAIR was detected by transmission electron microscopy and confocal microscopy analysis of cluster determinant 63 (CD63) with synaptosome associated protein 23 (SNAP23). The mechanism of HOTAIR's regulation of Ras-related protein Rab-35 (RAB35), vesicle associated membrane protein 3 (VAMP3), and SNAP23 was assessed using confocal co-localization analysis, phosphorylation assays, and rescue experiments.

Results: We found an enrichment of exosome secretion-related genes in the HOTAIR high expression group. HOTAIR promoted the release of exosomes by inducing MVB transport to the plasma membrane. HOTAIR regulated RAB35 expression and localization, which controlled the docking process. Moreover, HOTAIR facilitated the final step of fusion by influencing VAMP3 and SNAP23 colocalization. In addition, we validated that HOTAIR induced the phosphorylation of SNAP23 via mammalian target of rapamycin (mTOR) signaling.

Conclusion: Our study demonstrated a novel function of lncRNA HOTAIR in promoting exosome secretion from HCC cells and provided a new understanding of lncRNAs in tumor cell biology.

Keywords: Exosome; HCC; HOTAIR; RAB35; SNAP23.

Fig. 1

Abnormal expression of HOTAIR is associated with exosome secretion. a Heatmap showing the relative expression values for 31 exosome secretion-related genes in a series of 374 cases of liver cancer generated from TCGA and categorized into subgroups according to their median HOTAIR expression. The list on the right shows 31 genes involved in exosome secretion. b Enrichment plot showing enrichment of exosome secretion-related genes in the HOTAIR high expression group. c-e The mRNA expression of RAB35, SNAP23, and VAMP3 were analyzed in HCC tissues compared with normal tissues. f-h The mRNA expression of RAB35, SNAP23, and VAMP3 were analyzed in HOTAIR relative high group compared with HOTAIR relative low group.t-test *P-value < 0.05

Fig. 2

HOTAIR promotes exosome secretion in HepG2 cells. a–c Isolated exosomes from HepG2 cells were assessed by a transmission electron microscopy, b western blotting and c NTA. d NTA analysis of the effect of HOTAIR on exosome release in HepG2 cells. Data are reported as the mean ± standard error (SD) from three independent experiments, t-test *P-value < 0.05

Fig. 3

HOTAIR enhances the transport of MVBs towards the plasma membrane. a Confocal microscopy analysis of CD63 (red) in HepG2 cells transfected with pcDNA3.1-HOTAIR. Nuclei were stained with DAPI. b Confocal co-localization analysis of CD63 (red) and SNAP23 (green) in HepG2 cells transfected with pcDNA3.1-HOTAIR. The rectangular box indicates the small punctate structures where CD63 and SNAP-23 were co-localized. c Electron microscopy images showing MVBs in HepG2 cells transfected with pcDNA3.1 or pcDNA3.1-HOTAIR. Red arrows indicated MVBs

Fig. 4

HOTAIR regulates the expression and the localization of RAB35. a Real-time PCR analysis of the mRNA expression of RAB5, RAB7, RAB11, RAB27A, RAB27B, and RAB35, which encode GTPases involved in the release of exosomes, in HOTAIR overexpressing HepG2 cells. b Western blotting analysis of RAB35 protein levels from the above cells. c Confocal co-localization analysis of CD63 (red) and RAB35 (green) in HepG2 cells transfected with pcDNA3.1or pcDNA3.1-HOTAIR. The rectangular box indicates the small punctate structures where CD63 and RAB35 were co-localized. d RIP assays were performed using RAB35 antibody or control IgG antibody in HepG2 cells, and then real-time PCR analysis was used to measure the enrichment degrees of HOTAIR coprecipitated RNA. e NTA analysis of exosome secretion in HepG2 cells co-transfected with pcDNA3.1-HOTAIR and si-Rab35. Data are reported as the mean ± standard error (SD) from three independent experiments, t-test *P-value < 0.05

Fig. 5

HOTAIR induces the translocation of VAMP3 and SNAP23. a Confocal microscopy analysis of SNAP23 (green) in HepG2 cells transfected with pcDNA3.1-HOTAIR. Nuclei were stained with DAPI. b Confocal co-localization analysis of VAMP3 (red) and SNAP23 (green) in HepG2 cells transfected with pcDNA3.1-HOTAIR. The rectangular box indicates the small punctate structures where VAMP3 and SNAP23 were co-localized

Fig. 6

HOTAIR induces phosphorylation of SNAP23 via mTOR signaling. The levels of non-phosphorylated SNAP23 in HepG2 cells as assessed using Phos-tag SDS-PAGE analysis. a HepG2 cells were transfected with pcDNA3.1or pcDNA3.1-HOTAIR. b HepG2 cells transfected with pcDNA3.1 or pcDNA3.1-HOTAIR were treated with rapamycin (Rapa, 50 nmol/L) or control vehicle (DMSO), respectively. Data are reported as the mean ± standard error (SD) from three independent experiments, t-test *P-value < 0.05

Fig. 7

Proposed model for the role of HOTAIR in the regulation of exosome secretion. HOTAIR accelerates MVB transport by inducing RAB35 expression and localization to MVBs. In addition, HOTAIR promotes MVB fusion by regulating the location and phosphorylation of SNAP23 to form the SNARE complex