Long non-coding RNA RP11-70C1.3 confers chemoresistance of breast cancer cells through miR-6736-3p/NRP-1 axis

Chemoresistance remains a major obstacle for improving the clinical outcome of patients with breast cancer. Recently, long non-coding RNAs (lncRNAs) have been implicated in breast cancer chemoresistance. However, the function and underlying mechanism are still largely unknown. Using lncRNA microarray, we identified 122 upregulated and 475 downregulated lncRNAs that might be related to the breast cancer chemoresistance. Among them, RP11-70C1.3 was one of the most highly expressed lncRNAs. In breast cancer patients, high RP11-70C1.3 expression predicted poor prognosis. Knockdown of RP11-70C1.3 inhibited the multidrug resistance of breast cancer cells in vitro and in vivo. Further investigations revealed that RP11-70C1.3 functioned as a competing endogenous RNA for miR-6736-3p to increase NRP-1 expression. Notably, the rescue experiments showed that both miR-6736-3p inhibitor and NRP-1 overexpression could partly reverse the suppressive influence of RP11-70C1.3 knockdown on breast cancer chemoresistance. In conclusion, our study indicated that lncRNA RP11-70C1.3 regulated NRP-1 expression by sponging miR-6736-3p to confer chemoresistance of breast cancer cells. RP11-70C1.3 might be a potential therapeutic target to enhance the clinical efficacy of chemotherapy in breast cancer.


INTRODUCTION
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women worldwide. In 2020, about 2.3 million breast cancer cases were newly established, which surpassed lung cancer as the most commonly diagnosed cancer [1]. Breast cancer is heterogeneous and can be classified into different subtypes depending on the expression status of the hormone receptors [2]. The main treatment strategies include surgery, radiation, and chemotherapy [3]. However, patients with long-term treatment of chemotherapy frequently develop chemoresistance. Nevertheless, the molecular mechanisms responsible for the poor response to chemotherapy remain poorly understood, which have become one of the major obstacles to successful treatment.
NRP-1 encodes a transmembrane glycoprotein neuropilin-1, which contains a large N-terminal extracellular domain that can bind many ligands and various types of coreceptors [4]. Previously, we reported that NRP-1 was frequently upregulated in breast cancer and functioned as an oncogene to accelerate tumorigenesis and progression by promoting proliferation, metastasis, and stemness [5][6][7][8]. Recent studies revealed that NRP-1 could promote breast cancer cells resistance to ADM and PTX resistance through activation of ITGB3/FAK/NF-kB p65 axis and downregulation of BCRP/ ABCG2 [9][10][11]. However, a more detailed role of NRP-1 upregulation in breast cancer pathogenesis has not been fully elucidated.
Recent advances in whole genome and transcriptome sequencing technologies have identified long non-coding RNAs (lncRNAs) as a newly subgroup of non-coding RNAs [12]. LncRNAs have been shown to regulate oncogenes or tumor suppressor genes at both post-transcriptional and transcriptional level [13]. Intriguingly, lncRNAs play important roles in various biological processes, including drug resistance [14]. For example, lncRNA DILA1 promoted tamoxifen resistance of breast cancer cells through stabilizing Cyclin D1 protein [15]. Moreover, lncRNA HCP5 facilitated triple-negative breast cancer cell resistance to cisplatin by regulating PTEN expression [16]. LncRNA SNHG14 contributed to trastuzumab resistance and PABPC1 expression through regulating H3K27 acetylation in the promoter of PABPC1 [17]. Thus, www.bjbms.org plasmids using lipofectamine RNAiMAX (Invitrogen). Cells were harvested 24 or 48 h after transfection for further study.

Establishment of chemoresistant breast cancer cell lines
Adriamycin (ADM)-resistant cells MB231/ADM and paclitaxel (PTX)-resistant cells MCF-7/PTX were constructed by treatment of MCF-7 cells or MDA-MB-231 with stepwise increasing concentrations of ADM or PTX until they acquired resistance to 5 µM ADM or 20 µM PTX (Sigma), respectively. The resistant cells were routinely maintained in the presence of 5 µM ADM or 20 µM PTX every other day and are removed before the experiments being performed.

Colony formation assay
Cells were seeded in 6-well plates at a density of 500 cells per well. After 2 weeks, the cells were washed 2 times with PBS, fixed with methanol for 30 min, and stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 15 min. The number of visible colonies was counted under a microscope.

Drug resistance assay
The breast cancer cells (5 × 10 3 per well) were seeded in 96-well plates. After cellular adhesion, cells were incubated with freshly prepared anticancer drugs (Sigma) including ADM, PTX, cisplatin (DDP), cyclophosphamide (CPM), fluorouracil (5-Fu), vincristine (VCR), and methotrexate (MTX) for 48 h. Subsequently, 10 µL CCK-8 solution (Beyotime) was added to each well. After 1 h of incubation at 37℃, the optical density (OD) at 450 nm was measured under a microplate reader (Thermo Fisher Scientific). The half-maximal inhibitory concentration (IC 50 ) value of anticancer drugs was analyzed understanding the expression profile and function of individual lncRNA will help to clarify the mechanisms underlying breast cancer chemoresistance.
To identify the candidate lncRNAs responsible for breast cancer chemoresistance, we first conducted an lncRNA microarray profiling analysis in three chemotherapy-resistant and three chemotherapy-sensitive breast cancer tissues. A total of 597 lncRNAs were differentially expressed, including 122 upregulated and 475 downregulated. We further identified one of the most upregulated lncRNAs RP11-70C1.3, located in chromosome 3:42876736-42893917, as a prognostic biomarker associated with a worse overall survival. The effects of RP11-70C1.3 on breast cancer cell chemoresistance were evaluated by loss-of-function analysis in vitro and in vivo. Moreover, we explored the mechanism underlying the biological role of RP11-70C1.3 in which RP11-70C1.3 functioned as a competing endogenous RNA (ceRNA) of miR-6736-3p to upregulate NRP-1 expression. Taken together, these results provide new insights into the key role of the lncRNA RP11-70C1.3 in breast cancer chemoresistance.

Clinical specimens
Thirty-two chemotherapy-resistant breast cancer tissues and 28 chemotherapy-sensitive breast cancer tissues were collected from diagnosed patients who underwent surgical resection or biopsy between April 2008 and March 2013 at the Second Affiliated Hospital of Xuzhou Medical University. All enrolled patients received preoperative chemotherapy (36% adjuvant anthracycline-based therapy, 44% anthracyclines in combination with taxanes, and 20% other chemotherapy agents). All patients were signed with informed consent and the project protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xuzhou Medical University. RNA pull-down assay Biotinylated at 3' ends of miR-6736-3p (Bio-miR-6736-3p) and miR-NC (Bio-miR-NC) were synthesized by RiboBio and transfected into MB231/ADM and MCF-7/PTX cells at a concentration of 50 nM. After 48 h incubation, cells were harvested and lysed with a lysis buffer. The freshly prepared cellular lysates containing Biotinylated miRNAs were incubated with streptavidin magnetic beads (Invitrogen) for 1 h at room temperature. After washing, the NRP-1 expression was measured by qRT-PCR assay.

Microarray analysis
The total RNA from three chemotherapy-resistant breast cancer tissues and three chemotherapy-sensitive breast cancer tissues was extracted by Trizol reagent (Invitrogen) and used to double-stranded cDNA. cDNA was synthesized, labeled, and hybridized to microarray (Arraystar 8*60K Human LncRNA Array V5.0, Aksomics Inc., Shanghai, China). The data were extracted and analyzed using GeneSpring software V11 (Agilent Technologies, Santa Clara, CA, USA). The threshold for differentially expressed lncRNAs was set as a fold change >= 2.0 and p <0.05.

Apoptosis assay
The Annexin V-FITC/Propidium-Iodide (PI) Apoptosis Detection kit (Invitrogen) was utilized to determine cell apoptosis. Briefly, cells were harvested after transfection for 48 h and washed with phosphate-buffered solution twice. Then, these cells were stained with Annexin V-FITC and PI for 15 min at room temperature, and then immediately analyzed with BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Mice experiments
All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Xuzhou Medical University. MB231/ADM or MCF-7/ PTX cells (2 × 10 6 ) stably expressing with sh-RP11-70C1.3 or sh-NC were subcutaneously injected into right flank area of BALB/c nude mice (Vital River, Beijing, China). Animals were randomly grouped (n = 6 per group). After 7 days, mice were treated with or without 5 mg/kg ADM or 20 mg/kg PTX through intraperitoneal injections twice a week until the end of the study. Tumor volume was calculated by the following formula: Volume = (length × width 2 ) × 0.5. After 5 weeks, the mice were killed and the tumor weight was recorded.

Immunohistochemical staining
Tumor tissues were fixed in 10% formaldehyde in PBS, embedded in paraffin, and cut into 4 µm thick slices. Then, the slices were deparaffinized with xylene, rehydrated, and microwaved for antigen retrieval with a citrate buffer using routine methods. The tissue sections were blocked with 1% bovine serum albumin buffer for 20 min at room temperature, incubated with the primary antibodies against Ki-67 (Cell Signaling Technology, 1:50) overnight at 4℃, and then treated with biotin-labeled secondary antibodies for 30 min at room temperature, followed by staining with diaminobenzidine, counterstaining with 10% Mayer' s hematoxylin, dehydration, and mounting.

Luciferase reporter assay
The wide type (wt) or mutated (mut) response elements of miR-6736-3p in the RP11-70C1.3 and 3'UTR of NRP-1 were synthesized and cloned into pMIR-REPORT plasmid downstream of luciferase reporter gene. Then, wt-or mut-reporters and miR-6736-3p mimics or negative control were cotransfected into breast cancer cells. After 48 transfection, luciferase activities were assessed by a luciferase reporter assay system (Promega) according to the manufacturer' s instruction.

Statistical analysis
The mean values ± S.D. were calculated and plotted using GraphPad Prism 9 software (GraphPad Software). Comparisons were analyzed by the one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test or Student' s t-test. In addition, Kaplan-Meier survival curves were plotted, and a log-rank test was performed. Differences were defined as statistically significant for p < 0.05. www.bjbms.org

RESULTS
LncRNA RP11-70C1.3 is ectopically overexpressed in the chemoresistant breast cancer tissues and associated with poor overall survival To explore the roles of lncRNAs in breast cancer chemoresistance, an lncRNA microarray analysis was performed in three chemotherapy-resistant and three chemotherapy-sensitive breast cancer tissues. A total of 122 upregulated and 475 downregulated lncRNAs were identified ( Figure 1A). Among them, lncRNA RP11-70C1.3 was one of the most highly expressed lncRNAs in chemoresistant breast cancer tissues. qRT-PCR results validated that RP11-70C1.3 expression was strikingly increased 6.32-fold compared with chemosensitive breast tissues ( Figure 1B). The expression of RP11-70C1.3 was further confirmed in five breast cancer cell lines and a normal breast epithelial cell line MCF-10A using qRT-PCR. Undoubtedly, compared with MCF-10A cells, RP11-70C1.3 expression was significantly increased in breast cancer cell lines ( Figure 1C). Kaplan-Meier survival analysis and log-rank tests demonstrated that patients with higher expression level of RP11-70C1.3 had a poor overall survival (p = 0.023; Figure 1D). Furthermore, we observed that there was a strong correlation of RP11-70C1.3 expression with distant metastasis and clinical stage in breast cancer patients (Table 1). These results indicate that lncRNA RP11-70C1.3 might be an important regulator of breast cancer chemoresistance.

RP11-70C1.3 knockdown inhibits breast cancer chemoresistance in vivo
To further evaluate the influence of RP11-70C1.3 on breast cancer chemoresistance, in vivo xenograft assay was performed by injecting stably expressing sh-RP11-70C1.3 or sh-NC-resistant cells subcutaneously into right flank area of nude mice and then treated with or without chemotherapeutic agents ( Figure 3A). Our results demonstrated that RP11-70C1.3 knockdown could significantly reduce the tumor growth ( Figure 3B and C) and weight ( Figure 3D and E) in mice treated with chemotherapeutic agents. However, there was no statistical difference of tumor growth and weight between sh-RP11-70C1.3 group and sh-NC group without chemotherapeutic agents' treatment. Immunohistochemical staining demonstrated that Ki-67-positive cells in the sh-RP11-70C1.3 group were lower than that in the sh-NC group ( Figure 3F-H). These findings consolidated the crucial role of RP11-70C1.3 in breast cancer chemoresistance.

DISCUSSION
At present, de novo or acquired resistance of breast cancer cells to chemotherapeutic drugs is particularly relevant to recurrence and metastasis, leading to a poor prognosis [20][21][22]. In this study, we focused on the molecular mechanism of breast cancer chemoresistance, with a particular interest in lncRNAs, which have been broadly implicated in several types of human tumor [23].
Based on our lncRNA microarray result, we identified 597 differentially expressed lncRNAs that might be related to the development of drug resistance in breast cancer. Among them, RP11-70C1.3 was one of the most highly expressed lncRNAs and associated with an unfavorable prognosis. Interestingly, we found that high expression level of RP11-70C1.3 significantly correlated with distant metastasis, indicating that RP11-70C1.3 might also play an important role in regulating breast cancer cell metastasis. Through the loss-of-function assays in vitro and in vivo, we discovered that knockdown of RP11-70C1.3 sensitized breast cancer cells to a series of chemotherapeutic drugs, including PTX, DDP, ADM, CPM, 5-Fu, VCR, and MTX, however, the underlying mechanism was unclear.
In accordance with the ceRNA hypothesis, lncRNA transcripts can function as endogenous decoys for miRNAs through their miRNA binding sites [24][25][26]. Bioinformatics analysis revealed that several miRNAs were candidate downstream targets of RP11-70C1.3. Among them, miR-6736-3p was the most downregulated miRNA after RP11-70C1.3 knockdown. Luciferase reporter assays further validated the direct interaction between RP11-70C1.3 and miR-6736-3p. Moreover, miR-6736-3p was lowly expressed in chemoresistant breast cancer tissues and cell lines, which was different from a recent report of miR-6736-3p upregulating in gastric cancer [27]. However, the biological role of miR-6736-3p in tumorigenesis and development has not been investigated. In addition, miR-6736-3p showed a negative correlation with RP11-70C1.3 in our enrolled 60 breast cancer tissues. Taken together, these findings indicated that miR-6736-3p was a downstream target of RP11-70C1. 3.

CONCLUSION
Our data showed that RP11-70C1.3 promoted chemoresistance of breast cancer cells in vivo and in vitro. Furthermore, we established a novel mechanism that RP11-70C1.3 functioned as a molecular sponge for miR-6736-3p and upregulated its target NRP-1 expression. Overall, RP11-70C1.3 may be a potential therapeutic target for breast cancer treatment.