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

This study was approved by Ethics Committee of Zhengzhou University, and informed consent was obtained from each patient. A total of 62 EC patients with primary ESCC who took Neoadjuvant chemotherapy after esophagectomy in The First Affiliated Hospital of Zhengzhou University were recruited in this study. ESCC tissues and their paired adjacent normal esophageal epithelial tissues were collected and stored at − 80 °C. According to the National Comprehensive Cancer Network esophageal cancer guideline, the normal tissues were at least 5 cm away from the primary lesion.

Combined chemotherapy for the treatment of ESCC was cisplatin, 5-Fu and adriamycin, or cisplatin, 5-Fu and paclitaxel. According to Response Evaluation Criteria in Solid Tumors (RECIST) guideline, patients were divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) after complete chemotherapy. Patients with CR and PR were defned as responders, whereas those with SD and PD were defned as non-responders.

ESCC cell lines (TE-13, KYSE140, EC9706, KYSE30) and human esophageal epithelial cells (Het-1A) were purchased from Cell Bank of TypeCulture Collection of Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI1640 supplemented with 10% fetal bovine serum (Gibco, USA) in 5% CO₂ incubator under 37 °C.

Cells were seeded into a culture plate and grown to 70-80% confluence for cell transfection. si-TUSC7-1, si-TUSC7-2, pcDNA-TUSC7, miR-224 mimic, miR-224 inhibitor, si-DESC1, LV-TUSC7 and their non-specific control were synthesised by Invitrogen (Shanghai, China), and were transfected into cells using Lipofectamine 2000 (Invitrogen, USA).

Total RNA from ESCC tissues, paired adjacent normal esophageal epithelial tissues, ESCC cell lines and Het-1A cells were isolated by TRIzol Reagent (Invitrogen, USA) according to manufacturer’s instructions. The expressions of TUSC7, miR-224 and DESC1 were neasured by PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, USA), and qRT-PCR was performed by QuantStudio® 3 RCR Real-Time PCR systems (Applied Biosystems, USA). The relative TUSC7, miR-224 and DESC1 expressions were determined by comparative method 2^-ΔΔCt.

Luciferase report gene vectors (pRL-TK, Promega) containing TUSC7 Wild Type (WT) or TUSC7 mutant 1 (Mut 1) or TUSC7 mutant 2 (Mut 2) were transfected into HEK293T cells. Luciferase report gene vectors containing DESC1 3’UTR WT or DESC1 3’UTR Mut (400 ng) was transfected into HEK293T cells with 40 ng pRL-TK vectors (Promega, USA). miR-224 mimic or miR-224 inhibitor or NC was co-transfected with reporter plasmids for 48 h. Cells were collected to measure luciferase activity by dual Glo™ Luciferase Assay System (Promega).

Cell proliferation was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. 8 × 10³ EC9706 or KYSE30 cells were seeded in each well of a 96-well plate. After overnight incubation, absorbance value was read at a reference of 450 nm. The inhibition rate (%) = (1-OD value of treated group/OD value of the control group) × 100%.

Colony formation assay was conducted as previously reported [23]. EC9706 or KYSE30 cell suspension (3500 cells) were uniformly dispersed, seeded onto six-well plates and grew over a span of 2 weeks in 5% CO₂ incubator under 37 °C. Then, cells were fixed with 10% formalin (Sigma, USA) and stained with 1 × diluted Giemsa reagent (Sigma, USA).

Apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). EC9706 or KYSE30 cells with different treatments were collected, centrifuged at 1500 rpm for 5 min, and resuspend with PBS. The supernatant was discarded, and 1 × Annexin V binding buffer (195 μL) was used to resuspend the cells. Annexin-V/FITC (5 μL) was added to the cell suspension. Then, PI (10 μL) was added to the cells and incubated at room temperature in the dark for 15 min. FACScalibur cytometer (Becton Dickinson, USA) was used to observe cell apoptosis.

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used for RIP experiments according to the manufacturer’s instructions. Antibody for RIP assays of endogenous polycomb repressive complex2 (EZH2) was purchased from Invitrogen. AGO2 was assessed by IP-western, and TUSC7 level was detected by qRT-PCR.

Cisplatin (DDP) and 5-Fu were purchased from Sigma and dissolved in DMSO. EC9706 or KYSE30 cells transfected with pcDNA or pcDNA-TUSC7 were incubated with cisplatin (0, 1, 2, 4, 8, 16 μM) or 5-Fu (0, 1, 4, 16, 32, 64 μM) for 48 h.

The drug-resistant cell lines were established by the increase of the cisplatin concentration. EC9706 or KYSE30 cells at the logarithmic phase were seeded into culture solution containing DDP with the low concentration started from 0.5 μM. 48 h later, the solution was discarded and fresh solution was added. After digestion, 1 μM DDP was added for the treatment for 48 h. With this procedure of changing solution and gradually increasing DDP concentration, cell lines that can resistent to 10 μM DDP were obtained, and named EC9706/DDP or KYSE30/DDP.

Proteins from ESCC tissues, paired adjacent normal esophageal epithelial tissues, ESCC cell lines and Het-1A cells were extracted using RIPA buffer (Thermo Scientific, USA), and protein concentrations were detected by BCA Protein Assay kit (Pierce Biotechnology, USA). Protein samples (50 μg) were isolated in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) and blocked in 5% non-fat dried milk. Then, the membranes were probed with first primary antibody anti-DESC1 (1:1000, Signalway Antibody, USA), anti-EGFR (1:1000, Invitrogen, USA), anti-p-AKT (1:1000, Invitrogen, USA), anti-AKT (1:1000, Cell Signaling, USA) and anti-GAPDH (1:1000, Invitrogen, USA) and the secondary horseradish peroxidase-conjugated antibody (Invitrogen, USA). GAPDH was used as the internal loading control.

This study was approved by the Ethics Committee of Animal Experiments of Zhengzhou University. KYSE30 cells were transfected with LV-NC or LV-TUSC7. 1 × 10⁶ cells in 150 μl of culture medium were inoculated subcutaneously into 24 female nude mice at 5-weeks of age. Seven days later, mice were intraperitoneally injected with 2 mg/kg cisplatin or PBS every 5 days for 20 days. Therefore, the 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. Tumor volumes and weight were recorded. Tumor volume = (length×width²)/2.

LncBase v.2 was used for predicting the potential binding sites between TUSC7 and miR-224. LncBase v.2 provides users with functional information about the microRNAs and their interaction with lncRNAs in many species. MicroRNA.org↗ resource was used for predicting the potential binding sites between miR-224 and 3’UTR of DESC1. MicroRNA.org↗ provides users with functional information about the microRNAs and their interaction with target genes in many species.

All experiments were performed in triplicate. SPSS 18.0 software was used for data analysis, and the result was expressed as mean ± standard deviation. The overall survival was calculated using the Kaplan-Meier method. One-way ANOVA and t test were used for the data analysis, with P < 0.05 considered statistically significant.

To determine whether TUSC7 was abnormally expressed in ESCC tissues and cells, qRT-PCR and Kaplan-Meier survival analysis were used to detect TUSC7 expression and overall survival of ESCC patients. We observed that TUSC7 level was downregulated in ESCC tissues than normal tissues (Fig. 1a), and upregulated in chemotherapy response group than chemotherapy non-response group (Fig. 1b). In addition, patients with lower TUSC7 levels had poorer overall survival (Fig. 1c), and there were significant associations between TUSC7 levels with tumor size and pathological staging (Table 1). 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. 1d).

According to the prediction of online bioinformatics software, there were potential binding sites between TUSC7 and miR-224 (Fig. 2a). Dual-luciferase reporter gene assay showed that miR-224 mimic significantly decreased the activity of TUSC7 WT, TUSC7 mutant 1 and TUSC7 mutant 2, and there was no significant change in the activity in TUSC7 mutant 1 + mutant 2, suggesting miR-224 could bind to the two sites of TUSC7 (Fig. 2b). And we found miR-224 expression was upregulated in ESCC tissue (Fig. 2c). Besides, si-TUSC7-1 or si-TUSC7-2 downregulated TUSC7 expression (Fig. 2d), and increased miR-224 expression (Fig. 2e). pcDNA-TUSC7 upregulated TUSC7 expression (Fig. 2f), and decreased miR-224 expression (Fig. 2g). Moreover, KYSE30 cell lysate was treated with Ago2 antibody for RIP, and TUSC7 was significantly increased in Ago2 than IgG (Fig. 2h). Finally, there was a negative correlation between TUSC7 and miR-224 expressions in ESCC tissues (Fig. 2i). All these findings suggested that TUSC7 negatively regulated the expression of miR-224.

To figure out the effect of TUSC7 and miR-224 on the proliferation and apoptosis of ESCC cells, EC9706 or KYSE30 cells were transfected with pcDNA or pcDNA-TUSC7 or NC or miR-224 inhibitor. We found overexpression of TUSC7 inhibited proliferation of ESCC cells (Fig. 3a), inhibited colony formation (Fig. 3b), and promoted apoptosis of ESCC cells (Fig. 3c). miR-224 expression was downregulated after treated with miR-224 inhibitor (Fig. 3d). In addition, miR-224 inhibitor inhibited proliferation of ESCC cells (Fig. 3e), inhibited colony formation (Fig. 3f), and promoted apoptosis of ESCC cells (Fig. 3g).

To investigate the effect of TUSC7 and miR-224 on chemotherapy resistance of ESCC cells, EC9706 or KYSE30 cells transfected with pcDNA or pcDNA-TUSC7 were treated with cisplatin or 5-Fu. As shown in Fig. 4a and b, inhibition rate increased with the increase of concentration of cisplatin or 5-Fu, and overexpression of TUSC7 increased the inhibition effect of cisplatin or 5-Fu on ESCC cells, indicating overexpression of TUSC7 inhibited chemotherapy resistance of ESCC cells. We also detected TUSC7 level in EC9706 and drug-resistant EC9706/DDP cells or KYSE30 and drug-resistant KYSE30/DDP cells, and found TUSC7 level was downregulated in drug-resistant ESCC cells (Fig. 4c). And overexpression of TUSC7 promoted cisplatin-induced apoptosis of ESCC cells (Fig. 4d). According to Fig. 4e and f, inhibition rate increased with the increase of concentration of cisplatin or 5-Fu, and miR-224 inhibitor increased the inhibition effect of cisplatin or 5-Fu on ESCC cells, suggesting miR-224 inhibitor inhibited chemotherapy resistance of ESCC cells. We further detected miR-224 level in EC9706 and drug-resistant EC9706/DDP cells or KYSE30 and drug-resistant KYSE30/DDP cells, and observed miR-224 level was upregulated in drug-resistant ESCC cells (Fig. 4g). Also, miR-224 inhibitor promoted cisplatin induced apoptosis of ESCC cells (Fig. 4h).

Regulation of miR-224 on DESC1 was confirmed by luciferase reporter gene in HEK293T cells. Online bioinformatics software microrna.org↗ predicted there was potential binding site between miR-224 and 3’UTR of DESC1 (Fig. 5a). Dual-luciferase reporter gene assay showed that miR-224 mimic or miR-224 inhibitor significantly decreased or increased the activity of DESC1 WT, and there was no significant difference in the activity of DESC1 MUT (Fig. 5b). Moreover, miR-224 mimic or miR-224 inhibitor significantly decreased or increased mRNA level (Fig. 5c) and protein level (Fig. 5d) of DESC1, and increased or decreased protein levels of EGFR and p-AKT (Fig. 5e). We also found mRNA level of DESC1 was downregulated in ESCC tissues (Fig. 5f). These findings proved that DESC1 was a direct target of miR-224.

To figure out whether TUSC7 inhibited cell proliferation and chemotherapy resistance via miR-224/DESC1, EC9706 or KYSE30 cells were transfected with pcDNA, pcDNA-TUSC7, pcDNA-TUSC7 + NC, and pcDNA-TUSC7 + miR-224 mimic. We observed that miR-224 mimic reversed the inhibition effect of pcDNA-TUSC7 on cell proliferation (Fig. 6a), the inhibition effect of pcDNA-TUSC7 on colony formation (Fig. 6b), the promotion effect of pcDNA-TUSC7 on cell apoptosis (Fig. 6c), and the inhibition effect of pcDNA-TUSC7 on chemotherapy resistance (Fig. 6d and e). In addition, pcDNA-TUSC7 increased protein level of DESC1 and decreased the expression of EGFR and p-AKT, while miR-224 mimic reversed these effects (Fig. 6f).

To figure out whether DESC1 inhibited chemotherapy resistance of ESCC cells via EGFR/AKT, EC9706 or KYSE30 cells were transfected with si-NC, si-DESC1, si- DESC1 + DMSO, and si-DESC1 + AST1306 (EGFR inhibitor) [24]. As shown in Fig. 7a and b, si-DESC1 promoted chemotherapy resistance of ESCC cells, while AST1306 reversed this effect. Also, si-DESC1 upregulated expressions of EGFR and p-AKT, while AST1306 reversed this effect (Fig. 7c). These results indicated that DESC1 inhibited chemotherapy resistance of ESCC cells via EGFR/AKT.

In order to confirm the regulatory role of TUSC7 in chemotherapy resistance in vivo, KYSE30 cells were transfected with LV-NC or LV-TUSC7 and subcutaneously injected into female nude mice. We observed that overexpression of TUSC7 inhibited tumor volume, size and weight (Fig. 8a–c). And overexpression of TUSC7 further decreased tumor volume and weight in cisplatin-treated mice (Fig. 8a and b). These results suggested that overexpression of TUSC7 inhibited tumor growth and chemotherapy resistance.

This project was supported by the National Natural Science Foundation of China (No. 81472605).

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This study was approved by Ethics Committee of Zhengzhou University, and informed consent was obtained from each patient. Animal studies were approved by the Ethics Committee of Animal Experiments of Zhengzhou University.

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The authors declare that they have no competing interests.

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