LncRNA CBR3-AS1 regulates of breast cancer drug sensitivity as a competing endogenous RNA through the JNK1/MEK4-mediated MAPK signal pathway

GSE20685 was downloaded from the GEO dataset (http://​www.​ncbi.​nlm.​nih.​gov/​geo/​). The whole genome expression profiles and clinicopathological information of human cancer were downloaded from The Cancer Genome Atlas (TCGA) (https://​tcga-data.​nci.​nih.​gov/​). The expression of lncRNAs in the cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE) database (http://​www.​broadinstitute.​org/​ccle). The IC₅₀ values of drug in multiple cell lines were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) database. All transcripts were normalized by log₂ transformation. The Diana lncbase (http://​carolina.​imis.​athena-innovation.​gr/​diana_​tools) was used to predict the binding of miRNAs and lncRNAs. The bioinformatics analysis tool miRdb (http://​www.​miRdb.​org/​) was used to predict the binding of miRNA and mRNA 3′UTR. The RNAhybrid website (https://​bibiserv.​cebitec.​uni-bielefeld.​de/​rnahybrid/​) was used to predict the binding sites of miRNA and lncRNA. The bioinformatics website lncLocator (http://​www.​csbio.​sjtu.​edu.​cn/​cgi-bin/​lncLocator.​py) was used to predict the subcellular localization of lncRNAs.

The correlation between genes was evaluated through the Pearson correlation test. The differences between breast cancer tumor and normal samples were evaluated by unpaired t-test. The log-rank test was used to test the difference in survival rate among different groups of patients.

MCF-7, T47D, MDA-MB-231 and HEK-293 T cell lines were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China), and MCF-7/ADR cells were purchased from the Zhen Shanghai and Shanghai Industrial Co., Ltd. (Shanghai, China). The cells were cultured in L-15 medium, DMEM or RPMI 1640 medium (HyClone, USA) and cultured in a humidified incubator at 37 °C. Except for cells cultured in L-15 medium, all cells were incubated in an environment containing 5% carbon dioxide.

Microarray analysis was performed by Shanghai OE Biotech (China) with the microarray Agilent Human lncRNA 4*180 K (Design ID: 076500). The raw microarray data were submitted to the GEO database (accession number: GSE155478). The lncRNAs were identified by the Gencode database (https://​www.​gencodegenes.​org). The p-value and fold change value of the t-test were used to screen differential lncRNAs. The screening criteria were up-regulated or down-regulated log₂FC ≥ 2.0 and FDR ≤ 0.05.

Data on CBR3-AS1 expression were obtained from breast cancer patients with TCGA. Taking the median CBR3-AS1 expression as the cut-off point, breast cancer cases were divided into two groups. The index of gene sequencing was set as “Pearson”, the curve of the top set was set to 150, and all other parameters were set at the default values.

The cells were seeded on a 6-well plate and incubated for 24 h. The cells reached 70–80% confluence. Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA) transfection reagent (5 μl) was dissolved in 125 μl of serum-free medium. Two micrograms of DNA was diluted with 125 μl of serum-free medium to prepare the DNA (Hanheng, China) premix, and then 10 μl of p3000 reagent was added and mixed well. After incubation for 5 min, the DNA solution was added to the cells. After 4 h, the medium was replaced with fresh medium containing 10% serum. The final transfection concentration of miRNA mimics (RiboBio, Guangzhou, China) was 50 μM, and the final concentration of siRNA (RiboBio, China) transfection was 50 μM. In order not to decompose RNA, p3000 was not used in the transfection process, and the other steps were equivalent to DNA transfection.

In the animal experiments, the plasmid was stably transfected into cells as described above. Then, 1 mg/L puromycin was added to the culture for 1 month, and stably transfected cell lines were selected for the experiment.

Cells were lysed on ice in RIPA buffer (Beyotime, China). The lysate was centrifuged at 15000×g at 4 °C for 15 min. SDS polyacrylamide gel electrophoresis was used to isolate proteins before they were transferred onto PVDF membranes. The membrane was blocked in 5% skim milk and incubated with the primary antibody in TBST at 4 °C overnight. Then, the cells were incubated with a horseradish peroxidase-labelled secondary antibody at a concentration of 1:10000 for 1 h. Finally, chemiluminescence was carried out using a Microchemi 4.2 system (DNR Bioimaging System, Israel). The details of all antibodies used are shown in Table S1.

HEK-293 T cells were transfected with MEK4–3′UTR-WT, MEK4–3′UTR-MU, JNK1–3′UTR-WT and JNK1–3′UTR-MU plasmids. At the same time, the four groups of cells were transfected with either NC mimic or miR-25-3p mimic to form eight experimental groups. Finally, each group of cells was transfected with the Renilla luciferase plasmid. After 36 h, the cells were harvested for luciferase analysis using a dual luciferase reporting and detection system (Promega, USA). The results were normalized against Renilla luciferase activity.

TRIzol (Invitrogen, USA) was used to extract total RNA from cultured cells. For mRNA and lncRNAs, a ReverTra Ace qPCR RT Kit (Toyobo, Japan) was used, and 200 ng of total RNA was used to synthesize cDNA in a 10 μl reaction volume. For miRNAs, the Bulge-Loop™ miRNA qRT-PCR Starter Kit (RiboBio, China) was used, and 1 μg of total RNA was used to synthesize cDNA in a 10 μl reaction volume. qRT-PCR of mRNA and lncRNAs was performed using SYBR Green Real-time PCR Master Mix (Toyobo, Japan) in a 12.5 μl reaction volume. The qRT-PCR of miRNAs used Bulge-LoopTM miRNA qRT-PCR Starter Kit (Ribobio, China) in a 20 μl reaction volume. The primers used are listed in Table S2.

MCF-7/ADR and MCF-7 cells (1 × 10³/well) in the logarithmic growth phase were resuspended and seeded into a 60 mm cell culture dish. For the drug treatments, 100 nM ADR was added to MCF-7 cells and 30 μM ADR was added to MCF-7/ADR cells for 48 h. Afterward, the medium in the culture was replaced with normal medium. Two weeks later, the cells were washed with PBS twice, fixed with methanol, and stained with 0.1% crystal violet (Beyotime, China) for 30 min. The concentration of JNK1 inhibitor (SP600125, MedChemExpress, USA) used in the cell experiment was 40 nM.

MCF-7/ADR and MCF-7 cells that were transiently transfected were seeded into 96-well plates at 3 × 10³ cells per well. After 24 h, the cells were treated with ADR (Sigma Chemical Co, St. Louis, MO) for 48 h. Next, cells were incubated with 10 μl of CCK-8 reagent (Dojindo, Japan) for 1 h before the absorbance was measured at 450 nm on a spectrophotometer.

According to the method of our previous articles [22], a cumulative ADR measurement assay was performed. Briefly, the cells were exposed to 5 μM ADR for 2 h and then washed with PBS. Next, flow cytometry was used to measure ADR fluorescence to determine the intracellular ADR level.

MCF-7/ADR and MCF-7 cell apoptosis after ADR treatment was evaluated using the Annexin-V APC/7AAD Iodide Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Cells were then analysed by MACSQuant (Miltenyi Biotec, Germany).

The negative control plasmid (GFP alone) or OE-CBR3-AS1 plasmid were transfected into MCF-7 cells, which were then treated with puromycin to obtain stably expressing cell lines. All BALB/c mice (4 weeks old) were purchased from Shanghai Laboratory Animal Centre (Shanghai, China). MCF-7/control and MCF-7/CBR3-AS1 cells (1 × 10⁷ cells/100 μl/nude mice) were subcutaneously injected into the axillary region of nude mice. When the subcutaneous tumors grew to 200–250 mm³ (16 days later), nude mice in the treatment group were intraperitoneally injected with ADR (2 mg/kg) and/or JNK1 inhibitor (15 mg/kg; MedChemExpress, USA) once every 3 days. All nude mice were treated 6 times. The diameter of the transplanted tumor was measured with digital calipers every week. The tumor volume was calculated as follows: volume = 0.5 × width² × length. All nude mice were humanly sacrificed at the end of the experiment, and the tumor tissues were collected for further study by qRT-PCR, IHC and western blotting.

The assay was carried out according to the method mentioned in a previous study [23]. We collected breast cancer tissues from patients from the First Affiliated Hospital of China Medical University. The ISH probes were ordered from BOSTER Biological Technology Co., Ltd. (USA). For the IHC assays, we collected breast cancer tissues from different groups of subcutaneous. The details of all antibodies used are shown in Table S1.

MCF-7 cell lysates were incubated with CBR3-AS1 or control probe (Sangon, China) conjugated to streptavidin agarose resin beads (Thermo Fisher Scientific, USA) to generate probe-bound Dynabeads. After the samples were washed with buffer, DNase I (20 U) was added, and purified RNA was collected. The purified RNA was analysed by qRT-PCR. The whole experimental process and the buffer formulation were described by Hsu et al. [24].

To identify whether the differential expression of lncRNAs can be induced during the course of breast cancer drug resistance, we investigated the expression of lncRNAs in MCF-7 cells and drug-resistant MCF-7/ADR breast cancer cells. LncRNAs with log₂FC > 1 and an FDR (false discovery rate) < 0.05 were selected from the microarray data. The results showed that, compared with MCF-7 cells, MCF-7/ADR cells had 949 up-regulated lncRNAs and 915 downregulated lncRNAs (Fig. 1a-b). The top 20 lncRNAs with the most obvious up-regulation and downregulation in MCF-7/ADR cells are listed in Fig. 1c. Moreover, in the three groups of MCF-7/ADR cells and MCF-7 cells, differential expression of lncRNAs was used for principal component analysis (PCA). The results showed that the differential expression of lncRNAs could effectively distinguish MCF-7/ADR and MCF-7 cells (Fig. S1 A). We further selected five lncRNAs (DSCR8, TP53TG1, CBR3-AS1, LINC01006, HAGLROS) that were not previously reported to be related to the drug sensitivity of breast cancer and performed qRT-PCR to verify their expression levels (Fig. 1d). Among them, CBR3-AS1 showed the highest expression in MCF-7/ADR cells. To confirm the relationship between the expression of the five lncRNAs and the sensitivity of breast cancer cells to ADR, we observed the expression patterns of lncRNAs in various breast cancer cells from the CCLE database and then obtained the IC₅₀ values of ADR in these breast cancer cells from the GDSC database. The results showed that the expression of CBR3-AS1 was positively correlated with the resistance of breast cancer cells to ADR (p < 0.05, r > 0.3) (Fig. 1e, Fig. S1B). To verify this result, we performed CCK-8 experiments on four breast cancer cell lines. The IC₅₀ value of ADR in each cell line was calculated (Fig. 1f). The qRT-PCR results showed that CBR3-AS1 was highly expressed in breast cancer cell lines with high IC₅₀ values but showed reduced expression in cell lines with a low IC50 value (Fig. 1g). In addition, we obtained the mRNA expression profile and clinical information of breast cancer patients from the GSE20685 dataset in the GEO dataset and used the Kaplan-Meier plotter website to calculate whether the survival of patients with breast cancer with different expression patterns of the five lncRNAs was different. The results showed that in breast cancer patients receiving chemotherapy, the prognosis of patients with high expression of CBR3-AS1 was worse (Fig. S1C). We also found that in the TCGA database, CBR3-AS1 was up-regulated in breast cancer tissues compared with adjacent normal tissues (Fig. 1h). Gene set enrichment analysis (GSEA) showed that CBR3-AS1 was enriched in the ABC transporter signal transduction pathway (Fig. 1i). In addition, the expression of CBR3-AS1 was significantly correlated with the mRNA expression of p-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) (Fig. 1j, k). The log-rank test of the OS curve of breast cancer patients in the TCGA also indicated that compared with low CBR3-AS1 expression, high CBR3-AS1 expression was significantly correlated with worse OS (Fig. 1l). We evaluated the expression of CBR3-AS1 via ISH in 96 breast cancer samples from the First Affiliated Hospital of China Medical University. High and low expression is shown in Fig. 1m. There was a significant correlation between CBR3-AS1 level and both tumor size and pathological staging (Table 1). The log-rank test of the overall survival curve of these breast cancer patients showed that elevated expression of CBR3-AS1 was significantly related to the worse OS of breast cancer (Fig. 1n).

To identify the effect of CBR3-AS1 on the drug sensitivity of breast cancer cells, we overexpressed CBR3-AS1 in MCF-7 cells with low expression of CBR3-AS1 and silenced CBR3-AS1 in MCF-7/ADR cells with high expression of CBR3-AS1. The overexpression or silencing efficiency of CBR3-AS1 was evaluated by qRT-PCR. After transfection of the CBR3-AS1 overexpression plasmid, CBR3-AS1 levels were significantly increased compared with those in the control group (Fig. S2A). Upon completion of si-CBR3-AS1 siRNA transfection, CBR3-AS1 expression was significantly downregulated compared with that in the si-control group (Fig. S2B). The results showed that in cells overexpressing CBR3-AS1, the protein expression levels of P-gp increased significantly (Fig. 2a). After CBR3-AS1 was silenced, the protein expression levels of P-gp decreased significantly (Fig. 2b). Then, we used flow cytometry to detect drug accumulation in cells. The overexpression of CBR3-AS1 significantly reduced the accumulation of ADR in cells (Fig. 2c), while silencing of CBR3-AS1 significantly increased the accumulation of ADR in cells (Fig. 2d). Next, we used the CCK-8 cytotoxicity test to detect changes in the drug sensitivity of cells. After overexpressing CBR3-AS1, cells exhibited a decrease in drug sensitivity (Fig. 2e, Table 2). However, after CBR3-AS1 was silenced, the cells’ drug sensitivity was significantly increased (Fig. 2f). We performed CCK-8 cytotoxicity experiments with other anticancer drugs in cells overexpressing CBR3-AS1. The results showed that after overexpressing CBR3-AS1, MCF-7 cells showed decreased sensitivity to paclitaxel decreased, while their sensitivity to cisplatin did not change (Fig. S2C, D). The above results showed that the effects of CBR3-AS1 on drug sensitivity are not specific to ADR. Colony formation experiments showed that colony-forming ability of cells was significantly enhanced by overexpression of CBR3-AS1 after adding ADR (Fig. 2g). However, compared with the control cells, si-CBR3-AS1 cells showed reduced colony-forming ability (Fig. 2h). At the same time, the IC50(48 h) concentration of ADR was used to induce apoptosis, and flow cytometry showed that CBR3-AS1 overexpression significantly reduced apoptosis (Fig. 2i).

The bioinformatics analysis tool Diana lncbase revealed that lncRNA CBR3-AS1 targeted miRNAs (Fig. 3a, Fig. S3A). Research has shown that activation of miR-25-3p can overcome chemotherapy resistance [25]. qRT-PCR analysis showed that miR-25-3p expression was downregulated in MCF-7/ADR cells (Fig. 3b). The bioinformatics analysis tool miRdb was used to discover the 3′UTR binding site for miR-25-3p with JNK1/MEK4 in the MAPK pathway (Table S3). CBR3-AS1 showed a significant negative correlation with miR-25-3p and positive correlation with JNK1/MEK4 mRNA expression in the TCGA breast cancer data (Fig. 3c). To determine whether JNK1 and MEK4 are possible targets for miR-25-3p, a luciferase reporter gene assay was conducted. We constructed luciferase reporter vectors Luc-JNK1 and Luc-MEK4 containing the 3′UTRs of JNK1 and MEK4 (Fig. 3d). Overexpression of miR-25-3p significantly reduced the luc-jnk1 luciferase activity in MCF-7 cells (Fig. 3e), indicating that JNK1 and MEK4 are direct targets of miR-25-3p. The RNAhybrid website predicted that CBR3-AS1 has a binding site with miR-25-3p (Fig. 3f). RNA pulldown assays showed that CBR3-AS1 could directly bind to miR-25-3p in breast cancer cells (Fig. 3g). Since the function of lncRNAs depends on their subcellular distribution, we examined the subcellular localization of CBR3-AS1. The bioinformatics website lncLocator predicted that CBR3-AS1 was expressed in both the nucleus and cytoplasm (Fig. S3B). The results were verified by qRT-PCR (Fig. 3h). qRT-PCR analysis and western blot analysis showed that the overexpression of CBR3-AS1 increased the mRNA and protein expression of JNK1 and MEK4 in MCF-7 cells after ADR was added (Fig. 3i, j), while silencing CBR3-AS1 reduced the mRNA and protein expression of JNK1 and MEK4 in MCF-7/ADR cells (Fig. 3k, l). Moreover, overexpression of miR-25-3p decreased JNK1 and MEK4 expression in MCF-7/ADR cells (Fig. S3C, D), while silencing miR-25-3p expression may increase JNK1 and mek4 expression in MCF-7/ADR cells (Fig. S3E, F). We next used ISH and IHC to examine 96 breast cancer tissues, sliced into serial sections (Fig. 3m). Consistent with the results in breast cancer cells, a negative correlation was confirmed to exist between CBR3-AS1 and miR-25-3p within this cohort (Fig. 3n), the expression of CBR3-AS1 was positively correlated with JNK1/MEK4 (Fig. S3G) and miR-25-3p was negatively correlated with JNK1/MEK4 (Fig. S3H). In conclusion, CBR3-AS1 regulates the expression of MEK4/JNK1 through miR-25-3p.

To determine whether miR-25-3p and JNK1 are involved in CBR3-AS1-mediated chemotherapy resistance in breast cancer cells, a CCK-8 assay was conducted with MCF-7 cells, and the results showed that overexpression of miR-25-3p or inhibition of JNK1 reversed the decrease in ADR sensitivity caused by overexpression of CBR3-AS1 (Fig. 4a). Colony-forming experiments confirmed that overexpression of miR-25-3p or inhibition of JNK1 reversed the increased cell proliferation by overexpression of CBR3-AS1 in cells treated with ADR (Fig. 4b). Apoptosis experiments confirmed that overexpression of CBR3-AS1 led to a decrease in the number of apoptotic cells, which was reversed by overexpressing miR-25-3p or inhibiting JNK1 (Fig. 4c). qRT-PCR analysis and western blot analysis showed that overexpression of miR-25-3p reversed the activation of JNK1 and MEK4 caused by overexpression of CBR3-AS1 (Fig. 4d-e).

To determine whether the overexpression of CBR3-AS1 can reduce the sensitivity of tumors to ADR in vivo and whether this effect is achieved by regulating JNK1, MCF-7 cells stably transfected with CBR3-AS1-cDNA or NC-cDNA were subcutaneously injected into nude mice. A schematic of the experiment is shown in Fig. 5a. After 34 days, the expression level of CBR3-AS1 in subcutaneous tumor tissue was detected. The expression level of CBR3-AS1 in the transfected group was still significantly higher than that in the control group, confirming the construction of the model (Fig. 5b). Next, the expression level of miR-25-3p in the tumor was detected, and it was found that the expression level of miR-25-3p in the OE-CBR3-AS1 group was significantly reduced (Fig. 5c). In vivo imaging in mice showed that overexpression of CBR3-AS1 resisted the inhibitory effect of ADR on tumor cells, which was reversed by inhibition of JNK1 (Fig. 5d). Furthermore, the effect of CBR3-AS1 on increasing tumor size and weight could be reversed by a JNK1 inhibitor (Fig. 5e-g). Western blot analysis showed that overexpression of CBR3-AS1 increased the expression of JNK1 and MEK4 (Fig. 5h). JNK1 and MEK4 expression was also detected by IHC (immunohistochemistry) (Fig. 5i). These results showed that CBR3-AS1 promotes ADR resistance in breast cancer through JNK1/MEK4 in vivo.