LINC00160 mediated paclitaxel‐And doxorubicin‐resistance in breast cancer cells by regulating TFF3 via transcription factor C/EBPβ

Abstract Chemoresistance represents a major challenge in breast cancer (BC) treatment. This study aimed to probe the roles of LINC00160 in paclitaxel‐ and doxorubicin‐resistant BC cells. Three pairs of BC and adjacent normal tissue were used for lncRNA microarray analysis. Paclitaxel‐resistant MCF‐7 (MCF‐7/Tax) and doxorubicin‐resistant BT474 (BT474/Dox) cells were generated by exposure of parental drug‐sensitive MCF‐7 or BT474 cells to gradient concentrations of drugs. Correlation between LINC00160 expression and clinical response to paclitaxel in BC patients was examined. Short interfering RNAs specifically targeting LINC00160 or TFF3 were designed to construct LINC00160‐ and TFF3‐depleted BC cells to discuss their effects on biological episodes of MCF‐7/Tax and BT474/Dox cells. Interactions among LINC00160, transcription factor C/EBPβ and TFF3 were identified. MCF‐7/Tax and BT474/Dox cells stable silencing of LINC00160 were transplanted into nude mice. Consequently, up‐regulated LINC00160 led to poor clinical response to paclitaxel in BC patients. LINC00160 knockdown reduced drug resistance in MCF‐7/Tax and BT474/Dox cells and reduced cell migration and invasion. LINC00160 recruited C/EBPβ into the promoter region of TFF3 and increased TFF3 expression. LINC00160‐depleted MCF‐7/Tax and BT474/Dox cells showed decreased tumour growth rates in nude mice. Overall, we identified a novel mechanism of LINC00160‐mediated chemoresistance via the C/EBPβ/TFF3 axis, highlighting the potential of LINC00160 for treating BC with chemoresistance.


| INTRODUC TI ON
Breast cancer (BC) represents the most frequently occurring carcinoma among females globally. 1 The treatment regimens include surgical resection, chemotherapy, radiation therapy and integrative therapies. 2,3 Resistance to chemotherapy continues to hinder the treatment of patients suffering from BC. 4 Paclitaxel and doxorubicin are widely used first-line chemotherapeutic agents, which substantially reduce the risk of recurrence and mortality in BC. 5,6 Unfortunately, the chemoresistance to anticancer drugs has been widely observed in BC management. When the cells are exposed to accumulating doses of cytotoxic doxorubicin, the multidrug-resistant phenotype of cells will develop, and the doxorubicin-resistant cells commonly manifest notable cross-resistance to other chemotherapeutic drugs. 7 The resistance to the chemotherapeutic agent paclitaxel also contributes to unfavourable treatment outcomes. 8 Recently, long non-coding RNAs (lncRNAs) have emerged as gene expression regulators of phenotypes of cancer cells and represent promising therapeutic targets in the treatment of chemoresistance in BC. 9 LncRNAs are abnormally expressed in various human diseases, playing critical roles in promoting pathogenesis or maintaining progression. 10 Recent evidence has revealed that lncRNAs have the potential to serve as promising biomarkers for the resistance of cancer cells to chemotherapeutic agents, including BC cells. 11,12 LINC00160 has been shown as a prognostic factor linking to poor BC survival. 13 LncRNAs-guided recruitment of transcription factors (TFs) has been recently studied in the chemotherapeutic drug resistance of BC cells. In the LncMAP database (http://bio-bigda ta.hrbmu.edu.cn/ LncMA P/), CCAAT-enhancer binding protein β (C/EBPβ) is a putative TF regulated by LINC00160, and trefoil factor 3 (TFF3) is the downstream target gene of C/EBPβ. C/EBPβ, mostly observed in oestrogen receptor-negative and highly proliferative and metastatic mammary tumours, appears to be a contributor to tumorigenesis in breast and is usually associated with a poor prognosis. 14 Additionally, the mRNA expression of TFF3 has been observed to be increased in the blood from metastatic BC patients as compared to that from non-metastatic patients. 15 Abnormally expressed TFF3 is involved in the progression of cancers, which accelerates the oncogenic characteristics of prostate cancer cells and diminishes sensitivity to radiation. 16 Therefore, we hypothesized that the LINC00160/C/EBPβ/ TFF3 axis has a regulatory role in the chemoresistance of BC cells.

| Tissue specimens
BC biopsy specimens and adjacent normal tissue were obtained from 47 female patients (aged 45.60 ± 3.52) (See Table S1 for other basic clinical characteristics) admitted into the Anhui No.2 Provincial People's Hospital between January 2012 and January 2013, and the end time of follow-up was from January 2017 to January 2018. Inclusion criteria: (1) no radiotherapy or chemotherapy before admission; (2) appropriate for surgical treatment; and (3) no systemic inflammatory response.
Exclusion criteria: (1) patients with recurrent BC; (2) with other tumours; and (3) received anti-inflammatory treatment and anti-infection treatment 5 months before admission. Each patient was given 175 mg/ m 2 paclitaxel once every three weeks. The follow-up lasted for a period of 60 months. The overall survival (OS) of patients was defined as the time from the initial date of treatment to date of death. Three pairs of BC and adjacent normal tissue were used for lncRNA microarray analysis. Total RNA from each sample was quantified using the NanoDrop ND-1000 (Thermo Scientific, Wilmington, USA).

| mRNA and lncRNA real-time qPCR (RT-qPCR)
Total RNA from tissue and cells was extracted using the method

| Western blot analysis
Western blot analysis was performed as early reported 18

| Subcellular localization
Fluorescence in situ hybridization (FISH) was performed to further examine the subcellular localization of LINC00160. 19

| Fractionation of nuclear/cytoplasmic RNA
The nuclear and cytoplasmic RNA fractions were isolated using a PARIS ™ kit (Life Technologies, Inc, Gaithersburg, MD, USA) according to the manufacturer's instructions. LINC00160 expression was determined by RT-qPCR, with U6 as the internal control for nuclear RNA expression and GAPDH for cytoplasmic RNA expression. The primers are shown in Table 1.

| Luciferase assay
The putative binding sites of C/EBPβ and TFF3 were obtained by the JASPAR (http://jaspar.gener eg.net/). The pmirGLO-based luciferase reporter plasmids (Promega, Madison, WI, USA) containing wild-type TFF3, wild-type TFF3 truncated the putative C/EBPβ binding sites or TFF3 mutated at the putative C/EBPβ binding sites were designed.
HEK293T cells (Shanghai Beinuo Biotech Ltd., Shanghai, China) were seeded into 6-well plates in triplicate and cotransfected with welldesigned pmirGLO-based reporter plasmids and plasmids carrying C/ EBPβ gene using the dual-luciferase reporter assay system (D0010, Beijing Solarbio Science & Technology Co., Ltd, China). Relative luciferase activity was normalized to that of Firefly luciferase.

| Biotinylated RNA pull-down assays
RNA-protein interactions were examined using the Pierce™ Magnetic RNA-Protein Pull-Down kit (20164, Thermo Fisher Scientific, Waltham, MA, USA). The protein was extracted from the captured RNA-protein complexes for Western blot analysis. Cell lysates (10 μL) were served as an Input.

| RNA immunoprecipitation (RIP)
Enrichment of LINC00160 in the C/EBPβ promoter region was further evaluated using the EZ-Magna RIP Kit (Millipore, Milan, Italy) according to the manufacturer's instructions. Immunoprecipitated RNA and total RNA from the whole cell lysates (input controls) were extracted for RT-qPCR analysis.

| Cell survival assays
Cell survival after drug treatment was examined using a CCK-8 assay as described previously. 21 Absorbance was read at 490 nm using a

| Clonogenic assay
Clonogenic assays were performed as previously described. 23

| Hoechst 33 342 staining
The chromatin dye Hoechst 33258 was used to observe chromosomal condensation and morphological changes in the nucleus with the fluorescence microscopy (BX50-FLA; Olympus, Tokyo, Japan). Viable cells showed normal nuclear size with uniform fluorescence. Apoptotic cells displayed condensed nuclei or nuclear condensations.

| Migration and invasion assays
Cell migration and invasion assays were carried out using Transwell assays (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.

| Flow cytometric analysis
An apoptosis assay was performed using the FITC Annexin V apoptosis detection kit (KeyGen, Nanjing, Jiangsu, China) following the protocol.

| Statistical analysis
Representative experiments were repeated at least three times, and data are expressed as mean ± standard deviation (s.d.). Differences between normally distributed values of two groups were evaluated using Student's t test, and those of multiple groups were evaluated using one-way or two-way analysis of variance (ANOVA). Pearson correlation coefficients were employed to assess the correlation between the expression of LINC00160 and TFF3. A two-tailed probability value less than 0.05 was set as the level of significance. All statistics were performed using SPSS 21.0 (IBM Corp. Armonk, NY, USA).

| Up-regulated LINC00160 was associated with paclitaxel resistance in BC 13
Three pairs of BC and adjacent normal tissue were used to determine the differentially expressed lncRNAs via microarray analysis.
A total of 44 lncRNAs were found to have differential expression between BC and adjacent normal tissue ( Figure 1A). The top 5 lncR-NAs with most differential expression (LINC00160, LINC00558, LINC00260, LINC00597 and LINC01278) were further validated via RT-qPCR, which identified that LINC00160 held the greatest differential expression among all lncRNAs in 47 BC patients ( Figure 1B).
The patients were allocated into responders (to paclitaxel) or nonresponders according to their sensitivity to paclitaxel treatment.
RT-qPCR identified that LINC00160 expression was much higher in the tissue from non-responders (to paclitaxel) than in those from responders ( Figure 1C). Kaplan-Meier survival curves revealed a reduced OS in BC patients with high expression of LINC00160 compared with those with low expression ( Figure 1D)

| LINC00160 knockdown reduced cell migration and invasion of drug-resistant cells
The Transwell assay and flow cytometry results found that

| LINC00160 is a nuclear lncRNA
We firstly searched LncATLAS database for subcellular localization of LINC00160, and the database showed LINC00160 is nucleus-sublocalized ( Figure 4A). FISH experiments with probes targeting LINC00160 were performed to validate the subcellular localization of LINC00160 in MCF-7 and BT474 cells. As shown in Figure 4B, MCF-7 and BT474 cells were stained with probes targeting LINC00160 (red stain), and the nuclei were stained with DAPI (blue stain). The merged image showed LINC00160 was nucleussublocalized in MCF-7 and BT474 cells. In addition, we characterized the nuclear and cytoplasmic expression of LINC00160 by fractionation of nuclear/cytoplasmic RNA and found LINC00160 in the nucleus ( Figure 4C).

| LINC00160 recruits TF C/EBPβ into the promoter region of TFF3
We performed a computer-based prediction in the LncMAP database (http://bio-bigda ta.hrbmu.edu.cn/LncMA P/), in which C/EBPβ is a F I G U R E 1 Up-regulated LINC00160 is associated with chemotherapeutic drug resistance in BC. A, Microarray analysis was performed to determine differentially expressed lncRNAs between BC and adjacent normal tissue. B, RT-qPCR were performed to determine LINC00160, LINC00558, LINC00260, LINC00597 and LINC01278 expression in 47 BC patients C, LINC00160 expression in paclitaxelresistant tumour tissue (n = 22) and in paclitaxel-sensitive tumour tissue (n = 25) determined using RT-qPCR. Unpaired t test was used for data analysis. D, Kaplan-Meier survival curves were used to reflect the OS of BC patients. E ~ F, Viability of the parental MCF-7, MCF-7/ Tax, parental BT474 and BT474/Dox cells detected using CCK-8 assays. Two-way ANOVA was used to determine statistical significance. G, LINC00160 expression in MCF10A cells, parental drug-sensitive MCF-7 and BT474 cells, and in MCF-7-Tax and BT474/Dox cells determined via RT-qPCR. One-way ANOVA was used to determine statistical significance. Data are expressed as mean ± s.d., representative of three independent experiments. **P < .01 putative TF regulated by LINC00160, and TFF3 is the downstream target of C/EBPβ ( Figure 5A). We conducted RNA pull-down assay and RIP assay to confirm the interaction between LINC00160 and C/EBPβ in both MCF-7 and BT474 cells ( Figure 5B-C). As expected, we found more C/EBPβ proteins were pulled down by biotinylated RNA fragments of LINC00160. More enrichment of LINC00160 on the C/EBPβ promoter region was detected by RIP-qPCR assay.
Next, the focus of experiments shifted to identifying whether TFF3 is a downstream target gene of C/EBPβ. We first performed a computer-based prediction in the JASPAR website and obtained three binding sites of C/EBPβ and TFF3 ( Figure 5D). The results of luciferase activity assays showed that the site 2 was responsible for C/ in MCF-7 and BT474 cells. More TFF3 was immunoprecipitated in the presence of C/EBPβ antibody, and increased amplification products of TFF3 were measured in the site 2 ( Figure 5G). Subsequently, we found TFF3 was up-regulated in BC tissue compared with adjacent normal tissue. In 47 BC tissue, the relative expression of TFF3 was found to positively correlate with the expression of LINC00160 ( Figure 5H). In addition, TFF3 expression was increased in BC cells compared to MCF10A cells. As expected, TFF3 expression was upregulated in MCF-7/Tax and BT474/Dox cells relative to the parental MCF-7 and BT474 cells ( Figure 5I). In LINC00160-depleted MCF-7/ Tax and BT474/Dox cells, the expression of TFF3 was found to be decreased ( Figure 5J) (all P < .05).  Figure 6D).

| LINC00160 knockdown reduces paclitaxel resistance of MCF-7/Tax cells and doxorubicin resistance of BT474/Dox cells in vivo
To assess the impact of LINC00160 on paclitaxel and doxorubicin resistance in vivo, we applied a xenograft model in nude mice. In panels A and C, two-way ANOVA was used to determine statistical significance of quantification of immunostaining, whereas in panel B one-way ANOVA was used. *P < .05 vs si-NC group were smaller than those in controls ( Figure 7A~B). It was shown that LINC00160-siRNA tumours had less anti-TFF3 immunostaining and anti-Ki67 immunostaining than scramble siRNA tumours ( Figure 7C).

| D ISCUSS I ON
BC is a frequently diagnosed fatal cancer and the leading cause of cancer-related death among women. 25 has been proposed as an independent risk factor contributing to lymphovascular invasion and lymph node metastasis in BC. 38 TFF3 renders high sensitivity and specificity to differentiate metastatic BC from the non-metastatic one. 15 In addition, TFF3 has also been identified as a sensitive indicator predicting the response of BC cells to endocrine therapy. 39 It has been suggested that the transcriptional regulation of TFF3 could be realized through promoter binding sites of C/EBPβ. 40 Partially consistent with our results, Albergaria Taken together, this study provided evidence that overexpressed LINC00160 up-regulated TFF3 expression through C/EBPβ, whereby potentiating the resistance of MCF-7 cells to paclitaxel and BT474 cells to doxorubicin. In microarray analysis, 44 lncRNAs were found to have differential expression between BC and adjacent normal tissue ( Figure 1A). In the future, we will study the other top 4 ln-cRNAs with most differential expression (LINC00558, LINC00260,

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.