Linc01014 regulates gefitinib resistance in oesophagus cancer via EGFR‐PI3K‐AKT‐mTOR signalling pathway

Abstract This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway.

LncRNA CCAT2 is associated with poor prognosis of ESCC. 12 High expression of lncRNA TUG1 is identified to be associated with chemotherapy resistance and poor prognosis of patients with ESCC. 13 Kang et al 14 have revealed that lncRNA PART1 acts as a ceRNA to induce gefitinib resistance in ESCC via miR-129/Bcl-2 pathway. Hence, it is significant to reveal the mechanisms of lncRNAs in regulating ESCC gefitinib resistance.
In this study, the role of lncRNA linc01014 involved in gefitinib resistance was explored in ESCC cell lines. According to these investigations, we hope to provide some new insights in solving gefitinib resistance in ESCC and improving the prognosis of patients with ESSC.

| Cell culture
Human ESCC cell lines KYAE-1, TE8, TE5 and FLO-1 were purchased from the Chinese Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in PRIM 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin/streptomycin (Sigma) at 37°C in a humidified incubator with 5% CO 2 in atmosphere. Cells were transfected with linc01014 mimic, si-linc01014 or negative control (NC) using lipofectamine 2000 (Invitrogen) according to manufacturer's protocol.

| Construction of ESCC cell lines with gefitinib resistance
Gefitinib-resistant cell lines of KYAE-1, TE8, TE5 and FLO-1 were constructed via repeatedly subcultured with an increasing concentration of gefitinib (Sigma) starting from 0.1 μM/mL to final concentration of 10.2 μM/mL. Finally, the gefitinib-resistant cell lines were collected and used for the following investigations.

| Morphology study
Morphology of cells after treating with or without gefitinib was

| Quantitative real-time PCR
After treatment, total RNA in cells was isolated using TRIzol reagent (Takara) according to manufacturer's protocol. cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) according to manufacturer's instruction. Then, the expression of linc01014 was determined using SYBR Green Master Mix (Applied Biosystems) with a total of 2 μg cDNA as a template on a BioRad CFX96 Sequence Detection System (Bio-Rad company). Relative expression of linc01014 was analysed using 2 −ΔΔCt method with GAPDH as the internal control. All premier sequences were purchased from RiBio (Guangzhou, China).

| Western blotting
After treatment, cells were lysated with RIPA lysis buffer (Millipore) supplemented with protease inhibitors (Sigma). Protein concentrations were measured using BCA method (Pierce) according to the manufacturer's protocol (Beyotime). Then, proteins were boiled with loading buffer and subjected for 10% SDS electrophoresis followed by transferring to polyvinylidene fluoride membranes (Millipore).
Subsequently, membranes were blocked with 5% milk at room temperature for 1 hour. Following this, membranes were incubated with a 1:1000 solution of primary antibodies (anti-PI3KCA, anti-Bcl-2, anti-Bax and anti-β-actin, Cell Signaling Technology) at 4°C overnight. Then, membranes were incubated with the secondary antibody at room temperature for 1 hour and visualized using ECL method (Pierce).

Highlights
• ESCC gefitinib-resistant cell lines were successfully constructed.
• Overexpression of linc01014 significantly decreased the apoptosis of FLO-1 cells induced by gefitinib.
• Silencing linc01014 obviously promoted the apoptosis of FLO-1 cells induced by gefitinib. SPSS 19.0 (SPSS Incorporation) was used to perform statistical analyses in this study. Data in this study were presented with mean ± standard deviation (SD). Comparisons among groups were analysed using Student's t test or one-way ANOVA analysis. P < .05 was considered statistically significant.

| Evaluation for gefitinib-resistant cell lines
The sensitivity of ESCC and ESCC gefitinibR cells to gefitinib was determined by MTS viability assay and cell morphology. The MTS assay showed that FLO-1-gefitinibR cell lines showed remarkably higher relative MTS activity than FLO-1 cells at concentration of 0.10-10.0 μmol/L, indicating that FLO-1-gefitinibR cell lines presented a higher gefitinib resistance that FLO-1 cells ( Figure 1A). According to these, the KYAE-1-gefitinibR, TE8-gefitinibR and TE5-gefitinibR cell lines were successfully constructed ( Figure 1A). When FLO-1 and FLO-1-gefitinibR cells were cultured in the medium with 10 μmol/L gefitinib, FLO-1 cells presented a shrank and rounded-up morphology and increased detachment after 48-hours culturing, but FLO-1-gefitinibR cells showed increase in confluence from 0 to 48 hours and a slight detachment at 72 hours, indicating that FLO-1-gefitinibR cells presented a higher gefitinib resistance that FLO-1 cells ( Figure 1B).

| ESCC gefitinibR cell lines presented higher proliferation than ESCC
To further confirm the property of gefitinibR cells, the proliferation of ESCC and ESCC gefitinibR cells were determined after treated with 10μM of gefitinib. The results showed that the proliferation of ESCC cell lines and ESCC gefitinib cell lines were significantly inhibited after treating with gefitinib, while the inhibitive extent in FLO-1, KYAE-1, TE8 and TE5 cells were significantly higher than that in FLO-1-gefitinibR, KYAE-1-gefitinibR, TE8-gefitinibR and TE5-gefitinibR cells, indicating that ESCC gefitinibR cells showed more gefitinib resistance than their parental ESSC cells (Figure 2).

| Confirmation the expression of linc01014 in FLO-1 cells
To further reveal the mechanism of gefitinib resistance, the expression of linc01014 was altered in FLO-1 cells and assessed using F I G U R E 1 MTS50 and morphology of ESCC gefitinib-resistant cell lines RT-qPCR. The result showed that linc01014 was significantly decreased/increased in FLO-1 cells (Figure 3), indicating that these cell lines could be used for the following investigations.

| D ISCUSS I ON
In recent decades, several chemotherapy drugs have been explored and applied for cancer therapy, but drug resistance is becoming a new challenge for clinical therapy, which is one of the major cause for failure treatment in clinic. 15 Although many efforts have been made, the mechanism of drug resistance remains unclear and need to be further explored. Along with the research of lncRNA, the regulatory mechanism of lncRNA in cancer provides us a new insight to understand the mechanism of drug resistance. 16 Gefitinib is a common chemotherapy choice for patients with ESSC. 17 The previous study has revealed that gefitinib could sensitize mutations in ESCC to chemotherapy treatment. 18 Mechanism research has identified that gefitinib could sensitize mutation in ESCC cell line KYSE450 via EGFR. 19 However, a phase II study about gefitinib in treating ESCC showed that although the overall response rate could reach 90%, the complete response was only 40% and median progression-free survival was 7.0 months, 7 suggesting there might be a large room to improve this treatment.
The previous studies revealed that PI3K/AKT/mTOR signalling pathway play crucial role in the pathogenesis of ESCC. 20,21 PIK3CA is a subunit of PI3K and often responsible for the activity of PI3K/ Akt/mTOR signalling pathway. 22 Mutation of PI3KCA could cause neoplastic transformation and accelerate cancer progression. 23,24 Shigaki et al 25 27 In addition, EGFR mutant actives PI3K/AKT signalling pathway via ERBB3 and inhibiting the ERBB3/PI3K/AKT signalling pathway is essential for gefitinib anti-cancer effect. 28 These findings

ACK N OWLED G EM ENTS
This work is support by the First Affiliated Hospital of Anhui Medical University.

CO N FLI C T O F I NTE R E S T
All of the authors have no conflict of interest in this research.

AUTH O R CO NTR I B UTI O N S
Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.

CO N S E NT TO PU B LI S H
All of the authors have consented to publish this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included in the article.