THRIL mediates endothelial progenitor cells autophagy via AKT pathway and FUS

Abstract

Background: This study focused on the roles of lncRNA THRIL in coronary atherosclerotic heart disease (CAD) through regulating AKT signaling pathway and directly interacting with FUS.

Methods: QRT-PCR was conducted to detect the expression of THRIL in CAD blood samples and endothelial progenitor cells (EPCs). Cell autophagy of EPCs was examined through Cyto-ID Autophagy Detection Kit. CCK-8 assay and flow cytometry were carried out to assess cell viability and apoptosis under various interference conditions. Western blotting was conducted to detect the expression of interest proteins. The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured by qRT-PCR. The direct interactions between HCG18 and FUS was confirmed through RNA electrophoretic mobility shift assay (RNA EMSA) and RNA immunoprecipitation (RIP) assay.

Results: THRIL was upregulated in CAD blood samples and EPCs. Knockdown of THRIL in EPCs promoted cell viability, inhibited cell autophagy and further suppressed the development of CAD. Over-expression of THRIL induced inactivation of AKT pathway, while knockdown of THRIL played reversed effects. THRIL directly interacted with FUS protein and knockdown of FUS reversed the over-expressing effect of THRIL on cell proliferation, autophagy and the status of AKT pathway.

Conclusion: THRIL inhibits the proliferation and mediates autophagy of endothelial progenitor cells via AKT pathway and FUS.

Keywords: AKT; Coronary heart disease; FUS; THRIL.

Fig. 1

LncRNA THRIL induced cell autophagy and promotes CAD progression. (a) THRIL was overexpressed in CAD blood samples and EPCs. *P < 0.05, compared with normal (healthy) group. The number of CAD patients and healthy controls were all twenty. (c) THRIL was depressed in EPCs in transfection with sh-THRIL-1 or sh-THRIL-2 and overexpressed through using pCMV6-THRIL. The RNA expression of THRIL was measured through qRT-PCR. *P < 0.05, compared with NC group. (c) Cell viability was measured through using CCK-8 assays. (d) Flow cytometry results revealed that sh-THRIL reduced apoptotic cell number of EPCs. (f) Autophagy assays results revealed that sh-THRIL reduced EPCs autophagy rate. Normal: EPCs from healthy controls; NC: EPCs isolated from CAD patients; All the transfection experiments were performed in EPCs isolated from CAD patients. *P < 0.05, compared with NC group; #P < 0.01, compared with normal ones. The data shown represent three separate experiments performed in triplicate in each experiment (mean ± the standard deviation [SD])

Fig. 2

The expression of VCAM-1/ICAM-1 and AKT signaling pathway in cells. (a) and (b) QRT-PCR results demonstrated that shTHRIL suppressed the expression of VCAM-1 and ICAM-1 and over-expression of THRIL could promote CAD progress. (c) and (d) AKT signaling pathway expressions in various transfection groups. (d) The expressions levels of ATG1 and LC3-II in various transfection groups. Normal: EPCs from healthy controls; NC: EPCs isolated from CAD patients; All the transfection experiments were performed in EPCs isolated from CAD patients. *P < 0.05, compared with NC group; #P < 0.05, compared with normal ones. The data shown represent three separate experiments performed in triplicate in each experiment (mean ± the standard deviation [SD])

Fig. 3

THRIL inhibits proliferation and promoted apoptosis through binding with FUS (a left) The expression of THRIL in CAD EPCs cell nucleus and cytoplasm was measured through northern blotting. (a right) The expression of FUS in CAD EPCs cell nucleus and cytoplasm was measured through western blotting. (b-d) The direct interactions between THRIL and FUS was confirmed through RNA electrophoretic mobility shift assays (RNA EMSA) and RNA immunoprecipitation (RIP) assays. (e) The effects of THRIL over-expression on cell viability of CAD EPCs was reversed through knockdown of FUS. (f) The effects of over-expression of THRIL on cell apoptosis of CAD EPCs was reversed through knockdown of FUS. NC: EPCs isolated from CAD patients; All the transfection experiments were performed in EPCs isolated from CAD patients. Data are presented as mean ± SD. *P < 0.05. The data shown represent three separate experiments performed in triplicate in each experiment (mean ± the standard deviation [SD])

Fig. 4

The effects of THRIL on AKT pathway and autophagy pathway were reversed through knockdown of FUS. (a) and (b) AKT signaling pathway expressions in various transfection groups. (c) and (d) ATG1 and LC3-II expression levels in various transfection groups. NC: EPCs isolated from CAD patients; All the transfection experiments were performed in EPCs isolated from CAD patients. *P < 0.05, compared with NC group. The data shown represent three separate experiments performed in triplicate in each experiment (mean ± the standard deviation [SD])