RETRACTED ARTICLE: Activation of LncRNA TINCR by H3K27 acetylation promotes Trastuzumab resistance and epithelial-mesenchymal transition by targeting MicroRNA-125b in breast Cancer

The study included 60 patients with HER-2+ breast cancer (female/male: 60/0, range of age (median, years): 27–63 (45)) who underwent surgical resection followed by trastuzumab treatment at Hainan General Hospital, The Fifth People’s Hospital of Chongqing, The Frist Affiliated Hospital of Chongqing Medical University and The First Affiliated Hospital of Zhengzhou University between Jan 2010 and Jun 2013. The diagnosis of recruited patients was pathologically confirmed, and primary cancer tissues were collected before performing trastuzumab treatment. The obtained upon resection tissue samples were immediately snap-frozen in liquid nitrogen and then stored at − 80 °C until further use. This study was approved by Research Scientific Ethics Committee of Hainan General Hospital, The Fifth People’s Hospital of Chongqing, The Frist Affiliated Hospital of Chongqing Medical University and The First Affiliated Hospital of Zhengzhou University. All participants signed informed consent prior to using the tissues for scientific research.

The human HER-2+ breast cancer cell lines SKBR-3, BT474 and human normal breast epithelial cell line MCF-10A were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). SKBR-3 and BT474 cells were cultured in Dulbecco’s modified Eagle (DMEM, Gibco, Carlsbad, CA) medium with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA). Normal breast epithelial MCF-10A cells were grown in DMEM/F-12 medium (HyClone) containing 10% FBS, 100 ng/ml cholera toxin (Sigma-Aldrich, St Louis, MO, USA), 5 μg/ml hydrocortisone (Sigma-Aldrich) and 10 μg/ml insulin (Sigma-Aldrich). The cultures were incubated at 37 °C in 5% CO₂. Trastuzumab (Herceptin) was purchased from Roche (Shanghai, China) and dissolved in enclosed sterile water.

The trastuzumab-resistant cell lines were established according to the method as previously reported [25]. Briefly, 5 × 10⁶ SKBR-3 or BT474 cells were injected subcutaneously into the flanks of nude mice. When the volume of xenograft reached 200 mm³, mice were intraperitoneally injected either with trastuzumab (3 mg/kg) or PBS once every two days for two weeks followed by another two weeks without the drug treatment (one course). Altogether, the mice received four courses of trastuzumab treatment and breast cancer cells were isolated from xenografts after completion of four courses of treatment and confirmed for resistance to trastuzumab.

Total RNA was extracted from trastuzumab-resistant cells and parental cells by using the RNeasy plus mini kit (Qiagen, Waltham, MA) according to the manufacturer’s protocol. LncRNAs were sequenced and microarray was performed using Agilent human lncRNA microarray V.2.0 platform (GPL18109). The data were analyzed by GeneSpring 12.6 software (Agilent) and the raw signals were log transformed and normalized using the Percentile shift normalization method, the value was set at 75th percentile. cDNA was fragmented (Bioruptor, Diagenode) to an average size of 250 bp to build the cDNA library. Data processing and statistical analysis for RNA-sequencing data were performed and heat maps were generated.

The synthetic oligonucleotides used for silencing TINCR (sh-TINCR) and oligonucleotides for overexpression of TINCR (Lv-TINCR) were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Negative control sh-NC or Lv-NC were also obtained from Sangon Biotech Co. Ltd. The above synthetic oligonucleotides were cloned into lentiviral vector to guarantee stable infection. Ribo™ Biotech (Guangzhou, China) synthesized the overexpression plasmid containing Snail-1 coding sequences (p-Snail-1) and mimics of miR-125b and anti-miR-125b. Cells were transduced with the above vectors by using TransFast transfection reagent (Promega; Madison, WI, USA) according to the manufacturer’s protocol. A total of 5 × 10⁵ cells were seeded into each well of a 6-well plate and transfected with respective oligoribonucleotides (final concentration 100 nM) upon reaching 70 to 80% confluence. The altered expression of target genes was measured after 24 h of transfection. The cells were then subjected to RNA/protein extraction and further functional assays.

Total RNA was extracted from breast cancer tissues or cells by using the RNeasy plus mini kit (Qiagen) according to the manufacturer’s protocol. RT and qPCR kits were used to evaluate the expression of target RNAs. RT (20 μl) reactions were performed using the PrimeScript® RT reagent kit (Takara, Dalian, China) and incubated for 30 min at 37 °C followed by 5 s at 85 °C. For qPCR, 2 μl of diluted RT product was mixed with 23 μl reaction buffer provided by Takara (Takara Inc., Dalian, China) to a final volume of 25 μl. All reactions were carried out using an Eppendorf Mastercycler EP Gradient S (Eppendorf, Germany) under the following conditions: 95 °C for 30 s followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. The internal expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for the normalization of detected RNAs using the comparative 2^-ΔΔCq method. The primer sequences for qPCR are presented in Additional file 1: Table S1.

Cells were permeabilized with 0.3% Triton X-100 (Beyotime, Shanghai, China) for 15 min after being fixed with 4% paraformaldehyde. The cells were blocked by using goat serum followed by incubation with anti-Ki67 antibody (1:100, ab15580, Abcam, Cambridge, MA) overnight at 4 °C. Subsequently, the slides were incubated with anti-rabbit Alexa Fluor 488 (Jackson Immunoresearch, West Grove, PA, USA) for 1 h at room temperature. DAPI was used for nuclear counterstaining. The slides were observed under a fluorescence microscope (DMI4000B, Leica).

The altered cell viability after treatment with trastuzumab or (and) sh-TINCR was assayed using the MTT Kit (Dojindo, Rockville, MD, USA). Cells were seeded onto 96-well plates at a density of 3000 cells/well and cultured in 200 μL cell culture medium. Ten microlitre MTT (5 mg/mL, pH = 7.4, pre-pared with PBS) was added to culture the cells for 2 h. Afterthe medium was turned away, the precipitate was made soluble in 100 μL DMSO. An enzyme-linked immunosorbent plate reader was utilized to determine the absorbance of each well.

Cell migration ability was evaluated by performing wound-healing assay. Cells were seeded onto six-well plates at a density of 500,000 cells/well. Twelve hours after treatment with trastuzumab or transduction with respective vectors, the layer of cells was scratched to form wounds by using a sterile 20-μl pipette tip; the non-adherent cells were washed away with culture medium and then the cells were further incubated for 48 h and photographed to identify the gap area. Cell invasive ability was evaluated using the transwell invasion assay with Boyden chambers (BD Biosciences) that had 8 μm pore size membranes with Matrigel. Cells in serum-free media were placed in the upper chamber of an insert. Medium containing 10% FBS was added to the lower chamber. After 12 h of incubation, the cells that had invaded through the membrane were stained with methanol and 0.1% crystal violet and imaged using an inverted microscope (Leica, DM IRBE).

Immunohistochemical staining was performed on 4 μm-thick TMA slides as previously described [23]. Anti-HER-2 antibody (1:100, cat. no. ab16901, Abcam, Cambridge, MA) and anti-Snail-1 antibody (1:100, cat. no. ab53519, Abcam) were used to detect their protein level in xenografts tissues. Images were visualized using a Nikon ECLIPSE Ti (Tokyo, Japan) microscope system and processed using Nikon software.

The PARIS™ kit (Ambion, Austin, TX) was used for the nucleo-cytoplasmic separation experiment. Briefly, 5 × 10⁶ cells were re-suspended in 0.6 ml resuspension buffer and incubated for 15 min followed by homogenization. After centrifugation at 400×g for 15 min, the cytoplasmic fraction was obtained in the supernatant. The pellet was then resuspended in 0.3 ml PBS, 0.3 ml nuclear isolation buffer, and 0.3 ml RNase-free H₂O, followed by 20 min incubation on ice. The pellet was the nuclear fraction after centrifugation. TINCR expression was determined by qPCR with GAPDH as cytoplasmic control and U1 as nuclear control. The primers used are shown in Additional file 1: Table S1.

Sangon Biotech synthesized the specific TINCR probe. Briefly, the cells were fixed in 1 ml of 4% formaldehyde for 10 min at room temperature, washed twice with 1× PBS and permeabilized with 70% EtOH in two-chamber dishes. The probes (0.3–0.6 μM final concentration) were hybridized in 10% dextran sulfate (Sigma, cat. no. D8906), 10% formamide and 2× SSC at 37 °C overnight followed by thorough washing. Imaging was performed immediately using a fluorescence microscope (DMI4000B, Leica).

An EZ-Magna ChIP kit (Millipore) was used for the ChIP assay according to the manufacturer’s protocol. Briefly, cells were treated with formaldehyde and incubated for 10 min to generate DNA–protein cross-links. Cell lysates were then sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with H3K27 antibody (Abcam, cat. no. ab4729), CBP antibody (Abcam, cat. no. ab2832) or the negative control IgG antibody (EMD Millipore, cat. no. 12–371). RNA was recovered and analyzed by qPCR.

RIPA buffer (Sigma Aldrich, Cambridge, MA) was used to lyse the cells to obtain total protein lysates. Protein concentration was measured using the BCA method (Sigma Aldrich). The quantified protein (25 μg) was transferred onto polyvinylidene fluoride (PVDF) membranes following SDS-PAGE gel electrophoresis. Then, the membrane was blocked with 5% nonfat dry milk in tri-buffered saline plus Tween (TBS-T) buffer for 2 h at room temperature and incubated with respective primary antibodies (1:1000 dilution) at 4 °C overnight, followed by Horseradish peroxidase-conjugated (HRP) secondary antibody (1:5000, Abcam, cat. no. ab7090) at room temperature for 1 h. The following primary antibodies were used: anti-HER-2 antibody (Abcam, cat. no. ab227383), anti-E-cadherin antibody (Abcam, cat. no. ab186533), anti-Snail-1 antibody (Abcam, cat. no. ab8614), anti-N-cadherin antibody (Abcam, cat. no. ab182651), anti-vimentin antibody (Abcam, cat. no. ab8805), anti-β-catenin antibody (Abcam, cat. no. ab8932), anti-GAPDH antibody (Invitrogen, cat. no. PA1–987).

Ten male BALB/c nude mice (19–22 g, 6 weeks old) were obtained from the Animal Center of Chinese Academy of Science (Shanghai, China). They were randomly divided into two groups of five each and housed three per cage in pathogen-free conditions at 28 °C, 50% humidity and were housed in a specific sterile environment suitable and regularly observed. The experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Hainan General Hospital. SKBR-3-TR cells (1 × 10⁷) that were stably transduced with sh-TINCR or sh-NC were subcutaneously injected into the flanks. The mice were housed for 25 days, then the formed tumors were stripped and the tumor mass was measured.

Experimental lung metastases were induced by injections of single-cell suspension (2 × 10⁶ cells in 100 μl) into the mouse lateral tail vein. Cells were stably transduced with sh-TINCR or sh-NC, and all cell injections were administered in a total volume of 500 μL PBS containing 0.1% BSA over a 60 s duration [26]. Five weeks later, prior to in vivo imaging, the mice were anaesthetized with phenobarbital sodium and the established lung metastases images were observed by LB983 NIGHTOWL II system (Berthold Technologies GmbH, Calmbacher, Germany).

Trastuzumab-resistant breast cancer cell lines were established by grafting SKBR-3 and BT474 cells into nude mice followed by cycles of trastuzumab treatment (3 mg/kg, intraperitoneal injection) as described in Methods (Fig. 1a). Breast cancer xenografts that received the fourth course of trastuzumab treatment displayed poor response to trastuzumab therapy. Cells were isolated from these resistant xenografts and named SKBR-3-TR and BT474-TR, respectively. We then validated the trastuzumab-resistant feature of these two sub-lines. As shown in Fig. 1b, the cell viability of both trastuzumab-resistant cell lines was much higher than their respective parental cell lines after treatment with trastuzumab at 3 μg/ml for 48 h. Furthermore, the IC₅₀ values of trastuzumab were much higher in trastuzumab-resistant cell sub-lines when compared to the respective parental ones (Fig. 1c). A different morphological feature was identified between the trastuzumab-resistant cells and parental cells (Fig. 1d). The resistant cells showed increased intercellular separation and formation of pseudopodia, suggesting that these cells may undergo EMT. Moreover, we used TUNEL assay to assess apoptosis after treatment with trastuzumab (3 μg/ml for 48 h). We observed a dramatic decrease in TUNEL intensity in trastuzumab-resistant cells when compared to the respective parental cells (Fig. 1e). It is well-known that chemoresistant cells treated by chemotherapeutics (e.g. oxaliplatin, 5-fluorouracil, etc.) undergo EMT; however, whether this phenomenon applies to cells treated with monoclonal antibody (e.g. trastuzumab) is still unknown. We investigated the migratory and invasive abilities of these cells and demonstrated that both trastuzumab-resistant cell lines showed enhanced migratory and invasive capacities (Fig. 1f, g). In addition, the epithelial proteins (E-cadherin and β-catenin) were downregulated whereas mesenchymal proteins (vimentin, N-cadherin) were upregulated in trastuzumab-resistant cells when compared to non-resistant cells (Fig. 1h). This suggests that trastuzumab resistance induces EMT of breast cancer cells.

To identify the potential lncRNA that may influence trastuzumab resistance in breast cancer cells, we performed microarray analysis by using trastuzumab-resistant cells and respective parental cells. According to the microarray data, we identified 187 transcripts that were upregulated with more than 2-fold change in SKBR-3-TR cells in contrast to SKBR-3 cells while 66 transcripts were downregulated by more than 2-fold. Moreover, we identified 224 transcripts that were upregulated with more than 2-fold change and 78 transcripts that were downregulated by 2-fold in BT474-TR cells in contrast to BT474-TR cells (Fig. 2a). Among these upregulated lncRNAs, we focused on TINCR, which has been previously reported to be linked to HER-2 expression and breast cancer tumorigenesis [24]. We validated the expression of TINCR in trastuzumab-resistant cells by using qPCR. As shown in Fig. 2b, TINCR is significantly upregulated in SKBR-3-TR and BT474-TR cells than in SKBR-3 and BT474 cells, respectively. To further validate the upregulation of TINCR, we included 30 tissue samples from HER-2+ breast cancer patients exhibiting poor response to trastuzumab therapy and another 30 tissue samples from HER-2+ breast cancer patients responding to trastuzumab therapy. As expected, TINCR expression was increased in trastuzumab-resistant patients compared to trastuzumab-responsive patients (Fig. 2c).

We then investigated the biological function of TINCR by constructing two kinds of shRNAs and cloned them in lentiviral vectors. We observed that both shRNAs induced significant downregulation of TINCR in SKBR-3-TR and BT474-TR cells (Fig. 3a). The MTT assay demonstrated that TINCR silencing significantly promotes trastuzumab-induced inhibition of cell viability (Fig. 3b). As trastuzumab mainly affects HER-2-regulated cell proliferation, we detected the cell proliferation protein Ki67 in these cells. Immunofluorescence assay revealed that TINCR knockdown decreased Ki67 expression level in both trastuzumab-resistant cell lines (Fig. 3c). In addition, TINCR knockdown damaged the acquired cell migration and invasion properties of trastuzumab-resistant cells as evidenced by wound healing and the matrigel-transwell assay (Fig. 3d, e). Western blot analysis of EMT proteins revealed that TINCR knockdown suppressed EMT in trastuzumab-resistant cells (Fig. 3f).

Furthermore, we overexpressed TINCR in parental SKBR-3 and BT474 cells by transduction of lentiviral vector containing TINCR sequence (Lv-TINCR) (Fig. 3g). We treated the cells with trastuzumab for 48 h at a concentration of 3 μg/ml and found that the cells transduced with Lv-TINCR decreased the trastuzumab-induced cell cytotoxicity compared to that of Lv-NC-transduced cells (Fig. 3h). Interestingly, we found that enhanced TINCR expression showed minimal effect on cell migration, invasion, and EMT of SKBR-3 and BT474 cells (data not shown), indicating that TINCR is a critical regulator of trastuzumab resistance and resistance-induced EMT process.

As TINCR is associated with trastuzumab resistance, we hypothesized that TINCR may regulate HER-2 expression. To confirm this hypothesis, we measured the expression level of HER-2 in cells by performing immunofluorescence assay. As shown in Fig. 4a, HER-2 expression was increased in both the trastuzumab-resistant cell lines. To further validate the upregulated HER-2 expression that was induced by TINCR, we detected the expression level of HER-2 in SKBR-3-TR and BT474-TR cells upon TINCR knockdown. HER-2 expression was significantly inhibited at both transcript and protein levels in these cells (Fig. 4b). Similarly, overexpression of TINCR in SKBR-3 and BT474 cells increased HER-2 expression (Fig. 4c). Our data suggests that TINCR induces trastuzumab resistance by increasing HER-2 expression.

LncRNA in cytoplasm could compete for MREs with the driver genes closely related to cancer occurrence and development as competing endogenous RNAs (ceRNAs), which would weaken the inhibition of miRNA upon target genes, indirectly regulate the expression level of target genes. To elucidate the regulatory mechanism of TINCR, we identified the subcellular location of TINCR in breast cancer cells. qPCR analysis of nuclear and cytoplasmic lncRNA showed that TINCR was mainly detected in the cytoplasm of SKBR-3-TR and BT474-TR cells (Fig. 5a). By performing fluorescence in situ hybridization of TINCR, we found that TINCR in breast cancer cells was mainly located in the cytoplasm (Fig. 5b). MiRNAs are present in the form of miRNA ribonucleoprotein complexes (miRNPs) which contains AGO2, a core component of the RNA-induced silencing complex [27]. Hence, we performed RIP assay with Ago2 to verify whether TINCR exerts its function through sponging with miRNAs. Clearly, both TINCR and HER-2 were enriched with Ago2 (Fig. 5c). In addition, TINCR knockdown led to decreased enrichment of Ago2 and TINCR but showed an increased recruitment of Ago2 to HER-2 transcript (Fig. 5d), suggesting that there is a competition between TINCR and HER-2 genes for the silencing effects induced by Ago2-based miRNAs.

Subsequently, we sought to identify the miRNA that is responsible for sponging TINCR and HER-2. Interestingly, the microRNA miR-125b was predicted to target both HER-2 and TINCR according to miRcode (http://mircode.org/) (Fig. 5e). TINCR knockdown increased miR-125b expression (Fig. 5f). Moreover, co-transfection of anti-miR-125b abrogated the downregulated the expression of HER-2 that was induced by TINCR knockdown (Fig. 5g). Luciferase reporter assay showed that enhanced expression of miR-125b significantly suppressed the luciferase activity in both TINCR and HER-2 wild type reporters but was relatively unaffected in presence of mutant HER-2 reporter (Fig. 5h-j). RIP assay revealed that TINCR silencing promoted the enrichment of miR-125b bound to HER-2 (Fig. 5k). Collectively, our data indicates TINCR as a molecular sponge for miR-125b to modulate HER-2 expression.

We further investigated the underlying mechanism by which TINCR mediates the EMT of trastuzumab-resistant cells. As we identified miR-125b as a sponge target of TINCR, we were interested to see whether other downstream mRNA targets of miR-125b are involved in the EMT process. Among the potential targets, we focused on Snail-1, which is well-known as a crucial regulator of EMT [28]. Bioinformatics analysis using miRcode indicated a potential binding site of miR-125b at the 3′UTR of Snail-1 (NM_005985.3, position: 1245–1251) (Fig. 6a). Overexpression of miR-125b significantly suppressed Snail-1 expression in SKBR-3-TR and BT474-TR cells (Fig. 6b). In contrast, anti-miR-125b reversed the TINCR knockdown-induced inhibition of Snail-1 expression (Fig. 6c). Furthermore, co-expression of p-Snail-1 dramatically rescued Snail-1 expression that was -downregulated by sh-TINCR (Fig. 6d), as well as the migratory and invasive abilities of SKBR-3-TR and BT474-TR cells (Fig. 6e-f). Western blot analysis showed that enhanced Snail-1 expression dramatically abrogated the sh-TINCR-induced suppression of EMT in both trastuzumab-resistant cell lines (Fig. 6g). To conclude, our data indicates that Snail-1 is a functional target of TINCR/miR-125b in the regulation of trastuzumab resistance-induced EMT.

Recent studies have shown that aberrant expression of lncRNAs is attributed to acetylation mediated-transcriptional activation [29]. To further understand the reason for increased expression of TINCR in trastuzumab-resistant cells, we explored the probable mechanisms using genome bioinformatics analysis (http://genome.ucsc.edu/), and found that the promoter region of TINCR had high enrichment of H3K27ac (Fig. 7a). To verify the existence of histone acetylation, we used ChIP experiment. As shown in Fig. 7b, there is H3K27ac enrichment at the promoter region of TINCR gene in both trastuzumab-resistant cells and parental cells. Moreover, the enriched intensity of H3K27ac was dramatically higher in trastuzumab-resistant cells compared to the respective parental cells. We also performed ChIP assay using five tissues from trastuzumab-resistant patients and five tissues from trastuzumab-responsive patients, and observed TINCR expression to be aberrantly upregulated in resistant tissues compared to responsive tissues. As expected, the resistant tissues showed an increased enrichment level when compared to that in responsive tissues (Fig. 7c). Moreover, treatment with histone acetyltransferase (HAT) inhibitor C646 significantly decreased the expression level of TINCR (Fig. 7d).

It is well known that CREB-binding protein (CBP) is essential for chromatin acetylation, we then performed ChIP assay and confirmed the enrichment of CBP at the TINCR promoter (Fig. 7e). In addition, knockdown of CBP using specific siRNA (Fig. 7f) dramatically decreased the enrichment of H3K27ac at the TINCR promoter region (Fig. 7g), thereby downregulating TINCR (Fig. 7h). Together, our results strongly indicate that the upregulated levels of TINCR are due to histone acetylation at the TINCR promoter region mediated by CBP enzyme.

To validate the functional role of TINCR in vitro, we established in vivo xenografts in BALB/cnude mice by grafting SKBR-3-TR cells that are stably transduced with sh-TINCR or sh-NC. When tumors were palpable, mice were intraperitoneally injected with 3 mg/kg trastuzumab once every two days for three weeks. The mouse models and tumor xenografts stripped from nude mice are presented in Fig. 8a and b, respectively. The quantitative data showed that TINCR-silenced mice exhibited significantly less tumor growth than did mice transduced with sh-NC (Fig. 8c). In addition, both the luciferase flux count of lung metastases and visible number of metastases on the lung surface were significantly less in sh-TINCR group compared to sh-NC group (Fig. 8d, e). Moreover, qPCR analysis showed that the expression of TINCR, HER-2, and Snail-1 in implanted breast cancer tissues was dramatically decreased, while miR-125b level was significantly increased in the sh-TINCR group in contrast to that in the sh-NC group (Fig. 8f). Immunohistochemical analysis further revealed a significant downregulation of HER-2 and Snail-1 in the tumor tissues of the sh-TINCR group compared with that in the sh-NC group (Fig. 8g). To conclude, our results confirmed the functional role of TINCR in trastuzumab resistance in in vivo xenografts.

Finally, we performed a preliminary study to find the clinical role of TINCR in 60 primary breast cancer tissues from patients receiving trastuzumab treatment (30 responsive and 30 non-responsive cases according to the Immune-related Response Evaluation Criteria In Solid Tumors (irRECIST) [30]). The clinicopathological analysis showed that expression of TINCR correlated with advanced TNM stage, lymph node invasion, and distant metastasis (Additional file 2: Table S2). As TINCR expression was upregulated in non-responsive patients compared to responsive patients (previously shown in Fig. 2c), we performed ROC curve analysis to investigate the predictive value of TINCR in differentiating trastuzumab responsive patients from non-responsive patients. As shown in Fig. 9a, area under the curve (AUC) was 0.833 with the diagnostic sensitivity and specificity reaching 76.7 and 70.0% with the cut-off value of 0.138, respectively. Under this criterion (0.138), we divide the patients into low- and high-TINCR expression groups and found that the number of patients with low TINCR expression was significantly higher in the responsive group than in the non-responsive group (Fig. 9b). Kaplan-Meier analysis showed that patients exhibiting a high level of TINCR were correlated with shorter overall survival time and progression-free survival time (Fig. 9c). In addition, Cox proportional-hazards model based univariate and multivariate analysis for progression-free survival showed that TINCR is an independent prognostic factor (Additional file 3: Table S3). By analyzing TCGA breast cancer dataset with the online database Starbase (http://​starbase.​sysu.​edu.​cn/​panCancer.​php), we identified positive correlations between the RNA expression of TINCR and HER-2, TINCR and Snail-1 in breast cancer, while a negative correlation was found between TINCR and miR-125b expression (Fig. 9d). More importantly, the correlation coefficients were much higher in our 30 non-responding samples (Fig. 9e), which strongly supports the regulatory role of TINCR in trastuzumab resistance.

Collectively, our study demonstrates that the H3K27 acetylation-induced upregulation of TINCR sponged miR-125b, thereby inducing trastuzumab resistance by upregulating HER-2 expression and promoting trastuzumab resistance-induced EMT process by increasing Snail-1 expression (Fig. 9f). Therefore, TINCR might serve as a potential treatment target for breast cancer patients to enhance the benefit of trastuzumab treatment.