MiR-590-3p suppresses epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma by inhibiting SIP1 expression

Abstract

The functional roles and clinical significances of miR-590-3p in ICC remain unclear. In the current study, we investigated the expression of miR-590-3p in tissues and sera of ICC by real-time quantitative polymerase chain reaction. We found miR-590-3p was significantly down-regulated in the sera and tissues of ICC patients, especially in those patients with lymph node metastasis or distant metastasis. AUC curves and Cox proportional hazards mode revealed serum miR-590-3p could be novel diagnostic and prognostic biomarker for ICC patients. MiR-590-3p dramatically suppressed epithelial-mesenchymal transition, cell migration, and invasion of ICC cells. SIP1 was identified as direct and functional target of miR-590-3p in ICC cells by luciferase assays. Finally, we found SIP1 expression was inversely correlated with miR-590-3p and closely related to diminished survival in ICC patients. These findings reveal functional and mechanistic roles of miR-590-3p and EMT activator SIP1 in the pathogenesis of ICC.

Keywords: EMT; SIP1; intrahepatic cholangiocarcinoma; metastasis; miR-590-3p.

Figure 1. Expression of miR-590-3p in tissues, sera and cell lines of ICC

(A) Relative expression of miR-590-3p in the tissues of Healthy control, ICC paitents, and ICC patients with lymph node metastasis or distant metastasis; (B) Serum levels of miR-590-3p in healthy controls and ICC patients. Boxes represent interquartile range, and the horizontal line across each box indicates median value; (C) Spearman's correlation analyses show a significantly inverse correlation between miR-590-3p expression level in tissues and sera of ICC patients; (D) Relative expression of miR-590-3p in ICC cell lines; The expression of miR-590-3p was quantified by qRT-PCR and normalized to RNU6B. *P<0.05, **P<0.01,***P<0.001. Serum miR-21 yielded an area under the curve (AUC) value of 0.9081 in distinguishing ICC patients from normal control subjects.

Figure 2. Diagnostic and prognostic role of serum miR-590-3p in ICC patients

(A) Serum miR-590-3p yielded an area under the curve (AUC) value of 0.879 in distinguishing ICC patients from healthy control subjects; (B) and (C) Kaplan-Meier plots representing probabilities of progression-free and overall survival in ICC patients according to expression level of serum miR-590-3p.

Figure 3. MiR-590-3p inhibits the migration and invasion of ICC cells

(A) Expression of miR-590-3p in HUCCT1/RBE transfected with miR-590-3p inhibitors/mimics was validated by RT-qPCR. RNU6B was used as an endogenous control; (B) Effects of miR-590-3p on cell migration of RBE. (C) Effects of miR-590-3p on cell migration of HUCCT1 cells; (D) Effects of miR-590-3p on cell invasion of RBE cells. (E) Effects of miR-590-3p on cell invasion of HUCCT1 cells.**P<0.01.

Figure 4. MiR-590-3p suppresses EMT of ICC cells

(A) Morphological changes of HUCCT1 cells infected with miR-590-3p inhibitors; (B) Western blotting analyses of EMT markers in HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p NC; (C) RT-qPCR analyses of ZEB1, ETS1, SNAIL1, TWIST1, and FN in HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p NC.

Figure 5. SIP1 are direct target of miR-590-3p

(A) MiR-590-3p and its putative binding sequence in the 3’-UTR of SIP1; diagrammatic representation of the luciferase reporter plasmids with WT and MT SIP1 3’-UTR. (B) Relative luciferase activity in 293T cells after transfection with WT or MT SIP13’-UTR plasmids co-transfected with miR-21 inhibitors. (C) and (D) MiR-21 inhibitors/mimics promoted/inhibited the expression level of SIP1 at the mRNA level and protein level in HUCCT1 and RBE cells. Three independent experiments were performed in duplicate. Data are presented as mean ± SD. Two-tailed Student's t test was used. * P< 0.05.

Figure 6. Loss-of-function studies showed that SIP1 siRNA abrogate the of miR-21 inhibitors-induced EMT, cell migration and invasion in ICC in vitro

(A) Morphology assays of HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p inhibitors+SIP1 siRNAs; (B) Migration assays of HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p inhibitors+SIP1 siRNAs; (C) Invasion assays of HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p inhibitors+SIP1 siRNAs; (D) Western blotting analyses of E-cadherin, N-cadhenrin, and Vimentin in HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p inhibitors+SIP1 siRNAs; (E) RT-qPCR analyses of ZEB1, ETS1, SNAIL1, TWIST1, and FN in HUCCT1 cells infected with miR-590-3p inhibitors or miR-590-3p inhibitors+SIP1 siRNAs.

Figure 7. Clinical significance of SIP1 in ICC

(A) Relative mRNA expression of SIP1 in the tissues of Healthy control, ICC paitents, and ICC patients with lymph node metastasis or distant metastasis. (B) Spearman's correlation analyses show a significantly inverse correlation between miR-590-3p expression level and SIP1 mRNA level in ICC tissues; (C) and (D) Kaplan-Meier plots representing probabilities of progression-free and overall survival in ICC patients according to expression level of SIP1. ***P<0.001.