Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ABCB1

Abstract Emerging evidence has indicated the important function of long non‐coding RNAs (lncRNAs) in tumour chemotherapy resistance. However, the underlying mechanism is still ambiguous. In this study, we investigate the physiopathologic role of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) on the paclitaxel (PTX) resistance in breast cancer. Results showed that lncRNA FTH1P3 was up‐regulated in paclitaxel‐resistant breast cancer tissue and cells (MCF‐7/PTX and MDA‐MB‐231/PTX cells) compared with paclitaxel‐sensitive tissue and parental cell lines (MCF‐7, MDA‐MB‐231). Gain‐ and loss‐of‐function experiments revealed that FTH1P3 silencing decreased the 50% inhibitory concentration (IC50) value of paclitaxel and induced cell cycle arrest at G2/M phase, while FTH1P3‐enhanced expression exerted the opposite effects. In vivo, xenograft mice assay showed that FTH1P3 silencing suppressed the tumour growth of paclitaxel‐resistant breast cancer cells and ABCB1 protein expression. Bioinformatics tools and luciferase reporter assay validated that FTH1P3 promoted ABCB1 protein expression through targeting miR‐206, acting as a miRNA “sponge.” In summary, our results reveal the potential regulatory mechanism of FTH1P3 on breast cancer paclitaxel resistance through miR‐206/ABCB1, providing a novel insight for the breast cancer chemoresistance.


| INTRODUCTION
Gradually, breast cancer has been one of the most common gynaecological malignant tumours and the leading cause of cancer-related death of females worldwide. 1,2 In spite of the endeavour and progress on clinical therapeutic strategies, the long-term clinical prognosis and mortality rate are still unsatisfactory. 3 Among these influence factors, chemoresistance is a major obstacle and often causes the poor treatment or failure. 4 Increasing evidence has demonstrated that breast cancer cells have developed chemoresistance against multiple first-line chemotherapy drugs, including paclitaxel, cisplatin, docetaxel, gemcitabine and so on. 5 The chemoresistance of multipledrugs has become the major impediment for the clinical effects and application for these drugs.
Paclitaxel is an effective first-line chemotherapy drug in clinical oncology, which is specific microtubule-stabilizing agent. 6 Paclitaxel is isolated from Taxus brevifolia and acts as one of the most effective plant-derived anticancer drugs. 7 The molecular mechanism for paclitaxel chemotherapy is to cause cell cycle arrest at G2/M phase and induce the cell apoptosis. 8 However, the treatment effect of paclitaxel is going worse due to the chemoresistance caused by multifarious factors. Therefore, the most strategy Wang and Zhang contributed equally to the work. of treatment is to clarify the underlying mechanisms of chemoresistance.
Long non-coding RNAs (lncRNAs) are type transcripts with more than 200 nucleotides without non-protein coding capacity. 9 In series of tumour tissue and cells, lncRNAs have been verified to be dysregulated and contribute to the multifaceted molecular regulation. 10,11 Moreover, lncRNAs have been validated to participate in the formation of tumour chemotherapy resistance through regulating critical molecular pathway and protein transcription. 12 For example, in breast cancer cells, lncRNA-ROR down-regulation inhibits the epithelial mesenchymal transition and promotes the sensibility towards tamoxifen by regulating miR-205/ZEB1/ZEB2 expression. 13 LncRNA FTH1P3 is a novel identified ncRNA in cancer. For example, FTH1P3 was up-regulated in uveal melanoma cell lines and tissues, and the elevated expression of FTH1P3 promoted uveal melanoma cell proliferation, cell cycle and migration through suppressing miR-224-5p. 14

| Paclitaxel sensitivity assay
Paclitaxel sensitivity was determined using CCK-8 assay by measuring the IC50 value (paclitaxel concentration causing 50% decrease in absorbance compared with the control). Briefly, cells (MCF-7/PTX, MDA-MB-231/PTX) were seeded in 96-well plates at a density of 1 9 10 4 cells per well for 24-hour incubation after transfection.
After incubation with paclitaxel treatment, the cell viability was measured using cell Counting Kit-8 (Dojindo, Japan) according to the manufacturer's instructions. The relative expression was normalized to the expression of GAPDH using 2 ÀDDCt method. The experiment was performed in triplicate.

| Flow cytometry
Cell cycle analysis was performed using cell cycle analysis kit (Lianke, Shanghai, China). Briefly, cells were digested with trypsin and washed with phosphate-buffered saline (PBS). Cells (2 9 10 5 ) cells were suspended with 50 lL binding buffer and then treated with cell cycle agents for 48 hours. Then, the adherent cells were washed and trypsinized, and centrifuged at 12 000 g for 5 minutes. After centrifugation, DNA staining was performed with 10 mg PI/mL PBS and 2.5 mg Ag DNase-free RNase/mL PBS for 30 minutes. Cell cycle profiles were generated with Modifit software (BD Biosciences).

| Western blot
The total protein was lysed by RIPA buffer (Sigma-Aldrich) added with protease inhibitors cocktail (Roche). After that, the isolated protein was transferred to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then to PVDF membrane (Millipore, Billerica, MA, USA). The membranes were washed with TBST and blocked with 5% non-fat milk powder and incubated with primary antibody, anti-ABCB1 (1:1000 dilution, Abcam), at room temperature for 2 hour and overnight at 4°C. The membrane was incubated with second antibody (horseradish peroxidase-conjugated goat anti-rabbit) at room temperature for 2 hours. Finally, these blots were detected using an EZ-ECL chemiluminescence detection kit for HRP (Biological Industries, Beit-Haemek, Israel). Tumour volume was measured using caliper following the formula length 9 width 2 /2. The tumour weight was weighed after the mice were killed.

| Statistical analysis
All data were generated from tripartite independent experiments and presented as the mean AE SD. Statistical analyses were performed using SPSS 18.0 and GraphPad software. Differences between groups were analysed using Student's t test or chi-square test analysis. Statistical significance was set as P < .05.

| LncRNA FTH1P3 silencing suppressed the tumour growth of paclitaxel-resistant breast cancer cells and ABCB1 protein in vivo
It had been verified that lncRNA FTH1P3 was up-regulated in paclitaxel-resistant breast cancer cells and FTH1P3 regulated the paclitaxel sensitivity of breast cancer cells in vitro. Then, xenograft mice assay in vivo was performed using MCF-7/PTX cells to test the role of FTH1P3 on tumour growth ( Figure 3A). Results showed that FTH1P3 knockdown significantly decreased the tumour volume compared to empty vector group ( Figure 3B). Besides, FTH1P3 knockdown significantly down-regulated tumour weight compared to empty vector group ( Figure 3C). In the tumour tissue sample, Western blot showed that ABCB1 protein was decreased in FTH1P3 knockdown group compared to empty vector group ( Figure 3D and E). RT-PCR showed that miR-206 expression was increased in FTH1P3 knockdown group compared to empty vector group ( Figure 3F). In summary, results indicated that FTH1P3 silencing suppressed the tumour growth of paclitaxel-resistant breast cancer cells and ABCB1 protein in vivo.

| Bioinformatics tools revealed that miR-206 targeted 3 0 -UTR of FTH1P3
Previous experiments revealed that lncRNA FTH1P3 and ABCB1 protein were closely correlated. To explore the potential relationship within

| DISCUSSION
Breast cancer is one of the most common gynaecological malignancies for women and causes significant morbidity and mortality worldwide. 2,15,16 Clinically, chemotherapy is the most effective assistant  17 However, the occurrence of chemoresistance causes the poor prognosis, high recurrence rate and low 5-year survival rate. 18 In the present study, we investigate the role of lncRNA FTH1P3 in breast cancer paclitaxel resistance and explore the underlying mechanism for the drug resistance generation.   25 For instance, lncRNA XIST is overexpressed in human lung adenocarcinoma A549 cells and increases the chemosensitivity to cisplatin, proposing the function through the let-7i/BAG-1 axis for the responsible for cisplatin resistance. 26 With the rapid development of bioinformatics tools and highthroughput sequencing, more and more potential lncRNAs have been identified and the detailed mechanisms are disclosing. The most canonical theory for lncRNAs is the miRNAs "sponge," performing as miRNAs absorbers to specifically attenuate the miRNAs abundance.
Our team performed bioinformatics predictive tools to investigate the potential regulatory pathway of FTH1P3 in the paclitaxel-resistant breast cancer cells. Results indicate that miR-206 binds with the 3 0 -UTR of FTH1P3, which is confirmed using luciferase reporter assay. Moreover, miR-206 also targets with 3 0 -UTR of ABCB1 mRNA. Because FTH1P3 and ABCB1 are both up-regulated in paclitaxel-resistant breast cancer cells, we could conclude that miR-206 targets simultaneously with FTH1P3 and ABCB1. ABCB1 (P-glycoprotein) is one of most canonical chemoresistance protein that involving multiple drug resistance in cancer. 27,28 Our results reveal that FTH1P3 might promote ABCB1 protein expression through sponging mmiR-206 and subsequently activates the paclitaxel resistance in breast cancer.
In conclusion, above evidence reveals the vital role of lncRNA FTH1P3 in the paclitaxel resistance of breast cancer. FTH1P3 promotes the paclitaxel resistance in human breast cancer tumorigenesis by targeting miR-206/ABCB1 axis, suggesting the novel molecular mechanism of breast cancer chemotherapy resistance.

ACKNOWLEDG EMENT
This work was supported by The First Affiliated Hospital of Zhengzhou University.

CONFLI CTS OF INTEREST
All the authors declare that they have no competing interests.