LncRNA-TUSC7/miR-224 affected chemotherapy resistance of esophageal squamous cell carcinoma by competitively regulating DESC1

Abstract

Background: This study aims to clarify the underlying mechanism for the tumor suppressive function of lnc TUSC7 in chemotherapy resistance of esophageal squamous cell carcinoma (ESCC).

Methods: TUSC7, miR-224 and DESC1 expressions in ESCC tissues and cells were detected by qRT-PCR. Protein level of DESC1, EGFR and p-AKT were observed by Western blot. Overall survival was calculated using the Kaplan-Meier method. Dual-luciferase reporter gene assay and RIP assay were used to comfirm TUSC7 binding to miR-224, and miR-224 binding to DESC1. Cell proliferation, apoptosis, and colony formation was detected by MTT, Flow Cytometry and Colony formation assays.

Results: TUSC7 was downregulated in ESCC tissues and cells, and low TUSC7 indicated worse overall survival. The analysis of bioinformatics softwares showed that TUSC7 specifically bound to miR-224, and we proved miR-224 was upregulated in ESCC and negatively correlated with TUSC7 expression. Overexpression of TUSC7/inhibition of miR-224 suppressed cell proliferation, colony formation and chemotherapy resistance of ESCC cells, and promoted cell apoptosis. In addition, we confirmed that miR-224 specifically bound to DESC1, and negatively correlated with DESC1. TUSC7 suppressed the proliferation and chemotherapy resistance of ESCC cells by increasing DESC1 expression via inhibiting miR-224. We also confirmed DESC1 inhibited chemotherapy resistance of ESCC cells via EGFR/AKT. Finally, in vivo experiments demonstrated that overexpression of TUSC7 decreased tumor growth and chemotherapy resistance.

Conclusion: These findings suggested TUSC7 suppressed chemotherapy resistance of ESCC by downregulating miR-224 to modulate DESC1/EGFR/AKT pathway.

Keywords: Chemotherapy resistance; DESC1; Esophageal squamous cell carcinoma; TUSC7; miR-224.

Fig. 1

TUSC7 expression in ESCC tissues and cells. a Sixty-two ESCC tissues and adjacent normal esophageal epithelial tissues were collected. qRT-PCR showed that TUSC7 expression was downregulated in ESCC tissues. b Patients were divided into chemotherapy Response group (n = 22) and Non-response group (n = 40) according to the patient’s response to chemotherapy. qRT-PCR showed that TUSC7 expression was upregulated in chemotherapy Response group. c Kaplan-Meier survival analysis showed that patients with lower TUSC7 levels (n = 31) had poorer overall survival than patients with high TUSC7 levels (n = 31). d qRT-PCR was used to detect TUSC7 expression in ESCC cells (TE-13, KYSE140, EC9706, KYSE30) and human esophageal epithelial cells (Het-1A). Compared with Het-1A cells, TUSC7 expression was downregulated in ESCC cells with lowest expression in KYSE30 cells and highest in TE-13 cells

Fig. 2

TUSC7 regulated the expression of miR-224. a Online bioinformatics software predicted there were two potential binding sites between TUSC7 and miR-224, and we mutated the two sites. b Luciferase reporter gene vector containing TUSC7 WT or TUSC7 mutant 1 or (and) TUSC7 mutant 2, and miR-224 or pre-NC were co-transfected into HEK293T cells. Dual-luciferase reporter gene assay showed that miR-224 mimic decreased the activity of TUSC7 WT, TUSC7 mutant 1 and TUSC7 mutant 2, and there was no significant difference in the activity in TUSC7 mutant 1 + mutant 2, suggesting miR-224 could bind to the two sites of TUSC7. c miR-224 expression was upregulated in ESCC tissue. d-e EC9706 or KYSE30 cells were transfected with si-NC or si-TUSC7-1 or si-TUSC7-2. qRT-PCR showed that si-TUSC7-1 or si-TUSC7-2 downregulated TUSC7 expression (d), and increased miR-224 expression (e). f-g EC9706 or KYSE30 cells were transfected with pcDNA or pcDNA-TUSC7. qRT-PCR showed that pcDNA-TUSC7 upregulated TUSC7 expression (f), and decreased miR-224 expression (g). H. KYSE30 cell lysate was treated with Ago2 antibody for RNA immunoprecipitation (RIP). qRT-PCR showed that TUSC7 was significantly increased in Ago2 than IgG. I. TUSC7 expression and miR-224 expression was negatively correlated in ESCC tissues

Fig. 3

Overexpression of TUSC7 or inhibition of miR-224 inhibited proliferation of ESCC cells and promoted cell apoptosis. a-c EC9706 or KYSE30 cells were transfected with pcDNA or pcDNA-TUSC7. MTT assay showed that overexpression of TUSC7 inhibited proliferation of ESCC cells (a). Colony formation assay showed that overexpression of TUSC7 inhibited colony formation (b). Flow cytometry showed that overexpression of TUSC7 promoted apoptosis of ESCC cells (c). D-E. EC9706 or KYSE30 cells were transfected with NC or miR-224 inhibitor. qRT-PCR showed that miR-224 expression was downregulated (d). MTT assay showed that miR-224 inhibitor inhibited proliferation of ESCC cells (e). Colony formation assay showed that miR-224 inhibitor inhibited colony formation (f). Flow cytometry showed that miR-224 inhibitor promoted apoptosis of ESCC cells (g)

Fig. 4

Overexpression of TUSC7 or inhibition of miR-224 inhibited chemotherapy resistance of ESCC cells. a EC9706 or KYSE30 cells transfected with pcDNA or pcDNA-TUSC7 were treated with cisplatin (0, 1, 2, 4, 8, 16 μM) for 48 h. Inhibition rate increased with the increase of concentration of cisplatin, and overexpression of TUSC7 increased the inhibition effect of cisplatin on ESCC cells, indicating overexpression of TUSC7 inhibited chemotherapy resistance of ESCC cells. b EC9706 or KYSE30 cells transfected with pcDNA or pcDNA-TUSC7 were treated with 5-Fu (0, 1, 4, 16, 32, 64 μM) for 48 h. Inhibition rate increased with the increase of concentration of 5-Fu, and overexpression of TUSC7 increased the inhibition effect of 5-Fu on ESCC cells. c TUSC7 level was detected in EC9706 and drug-resistant EC9706/DDP cells or KYSE30 and drug-resistant KYSE30/DDP cells. TUSC7 level was downregulated in drug-resistant ESCC cells. d EC9706 or KYSE30 cells transfected with pcDNA or pcDNA-TUSC7 were treated with 2 μM cisplatin for 48 h. Overexpression of TUSC7 promoted cisplatin induced apoptosis of ESCC cells. e EC9706 or KYSE30 cells transfected with NC or miR-224 inhibitor were treated with cisplatin (0, 1, 2, 4, 8, 16 μM) for 48 h. Inhibition rate increased with the increase of concentration of cisplatin, and miR-224 inhibitor increased the inhibition effect of cisplatin on ESCC cells, suggesting miR-224 inhibitor inhibited chemotherapy resistance of ESCC cells. f EC9706 or KYSE30 cells transfected with NC or miR-224 inhibitor were treated with 5-Fu (0, 1, 4, 16, 32, 64 μM) for 48 h. Inhibition rate increased with the increase of concentration of 5-Fu, and miR-224 inhibitor increased the inhibition effect of 5-Fu on ESCC cells. g miR-224 level was detected in EC9706 and drug-resistant EC9706/DDP cells or KYSE30 and drug-resistant KYSE30/DDP cells. miR-224 level was upregulated in drug-resistant ESCC cells. h EC9706 or KYSE30 cells transfected with NC or miR-224 inhibitor were treated with 2 μM cisplatin for 48 h. miR-224 inhibitor promoted cisplatin induced apoptosis of ESCC cells

Fig. 5

miR-224 targetedly regulated DESC1 expression. a Online bioinformatics software microrna.org predicted there was potential binding site between miR-224 and 3’UTR of DESC1. b Luciferase reporter gene vector containing DESC1 3’UTR WT or DESC1 3’UTR MUT, miR-224 mimic or pre-NC or miR-224 inhibitor or NC were co-transfected into HEK293T cells. Dual-luciferase reporter gene assay showed that miR-224 mimic or miR-224 inhibitor decreased or increased the activity of DESC1 WT, and did not significantly change the activity of DESC1 MUT. c-e EC9706 or KYSE30 cells were transfected with miR-224 mimic or miR-224 inhibitor or controls. qRT-PCR and Western blot showed that miR-224 mimic or miR-224 inhibitor significantly decreased or increased mRNA level (c) and protein level (d) of DESC1, and increased or decreased protein level of EGFR and p-AKT (e). f mRNA level of DESC1 was downregulated in ESCC tissues

Fig. 6

TUSC7 inhibited cell proliferation and chemotherapy resistance via miR-224/DESC1. EC9706 or KYSE30 cells were divided into four groups: pcDNA, pcDNA-TUSC7, pcDNA-TUSC7 + NC, and pcDNA-TUSC7 + miR-224 mimic groups. a MTT assay showed that miR-224 mimic reversed the inhibition effect of pcDNA-TUSC7 on cell proliferation. b Colony formation assay showed that miR-224 mimic reversed the inhibition effect of pcDNA-TUSC7 on colony formation. c Flow cytometry showed that miR-224 mimic reversed the promotion effect of pcDNA-TUSC7 on cell apoptosis. d After the treatment of cisplatin (0, 1, 2, 4, 8, 16 μM) for 48 h, miR-224 mimic reversed the inhibition effect of pcDNA-TUSC7 on chemotherapy resistance. e After the treatment of 5-Fu (0, 1, 4, 16, 32, 64 μM) for 48 h, miR-224 mimic reversed the inhibition effect of pcDNA-TUSC7 on chemotherapy resistance. f pcDNA-TUSC7 increased protein level of DESC1 and decreased the expression of EGFR and p-AKT, while miR-224 mimic reversed these effects

Fig. 7

DESC1 inhibited chemotherapy resistance of ESCC cells via EGFR/AKT. EC9706 or KYSE30 cells were divided into four groups: si-NC, si-DESC1, si- DESC1 + DMSO, and si-DESC1 + AST1306 (EGFR inhibitor, 1 μM,24 h) groups. a After the treatment of cisplatin (0, 1, 2, 4, 8, 16 μM) for 48 h, si-DESC1 promoted chemotherapy resistance of ESCC cells, while AST1306 reversed this effect. b After the treatment of 5-Fu (0, 1, 4, 16, 32, 64 μM) for 48 h, si-DESC1 promoted chemotherapy resistance of ESCC cells, while AST1306 reversed this effect. c si-DESC1 upregulated the expressions of EGFR and p-AKT, while AST1306 reversed this effect

Fig. 8

Observation of subcutaneous transplanted tumor of ESCC cells in nude mice. Five-week female BALB/c nude mice were divided into four groups with six mice in each group: LV-NC + PBS, LV-TUSC7 + PBS, LV-NC + cisplatin, and LV-TUSC7 + cisplatin groups. a-c Tumor volume, size and weight were measured. Overexpression of TUSC7 inhibited tumor growth and chemotherapy resistance. d Cascade diagram of signaling pathways