Introduction

Breast cancer (BC) is one of the most serious public health issues that physicians and different professionals are concerned about it and interested to cure (Harris et al. 1992). The statistics indicate that one out of eight women will be suffered from BC in their lifetime (Berns et al. 2007). One of the major obstacles in varying breast cancer prognosis and therapy is genetic and epigenetic modifications in cell signaling pathways, which are accompanied by significant molecular and cellular heterogeneity (Baselga and Swain 2009; Hynes and MacDonald 2009). Despite recent diagnostic and therapeutic advances, the molecular mechanisms of BC development are still poorly understood. Therefore, the study and discovery of the molecular basis involved in the progression of BC are so important in using advanced therapeutic strategies in the prevention, diagnosis, prognosis, and treatment of this malignancy.

The PI3K/AKT/mTOR or PAM pathway is mostly activated in breast cancer and plays an important driver of proliferation in this cancer (Lee et al. 2015). The pathway hyper-activation has mostly occurred in the cells that are responding to growth factor receptor engagement, such as IGF1R, where attaching to ligand converts PIP2 to PIP3. This modification, by recruiting PDK1, phosphorylates and triggers AKT protein and subsequently leads to activation of the downstream signaling cascade (Drury et al. 2011). The researches show that any disruption and mutation in the pathway promote neoplastic transformation by increasing enzymatic activity and enhancing downstream signaling elements (Baselga 2011).

Owing to recent advances in genome sequencing technologies, long non-coding RNAs (RNA transcripts longer than 200 nucleotides in length) have emerged as critical regulators in almost all aspects of biology (Yang et al. 2014; Li et al. 2015; Han et al. 2017; Dang et al. 2015; Vance et al. 2014; Lu et al. 2017; Jiang et al. 2015). Accumulating evidence indicates lncRNAs are engaged in practically every level of gene expression regulation, as well as a range of disease conditions (Blythe et al. 2016).

Several publications have appeared in recent years documenting various well-known lncRNA.

that play critical roles in signaling pathways and BC development. For instance, H19 (Blythe et al. 2016; Lottin et al. 2002; Hibi et al. 1996; Roozendaal et al. 1998), SRA (Cooper et al. 2009; Colley and Leedman 2011), HOTAIR (Gupta et al. 2010; Sørensen et al. 2013; Lu et al. 2012), and LncRNA-Smad7 (Arase et al. 2014) are oncogenic and GAS5 (Mourtada-Maarabouni et al. 2009; Özgür et al. 2013; Piao and Ma 2012; Zhang et al. 2013) and XIST (Sirchia et al. 2005; Sirchia et al. 2009; Richardson et al. 2006) have tumor suppressive roles in BC.

In recent investigations, LINC02381 performs distinct roles in various cancer. In cervical cancer, overexpression of LINC02381 resulted in more viability of cancer cells and increased metastasis (Chen et al. 2020). The other studies in colorectal and gastric cancer revealed that LINC02381 expression was reduced in tumor tissues, and LINC02381 overexpression in cancer cells promoted apoptosis and decreased cell proliferation, viability, migration, and invasion (Jafarzadeh et al. 2020; Jafarzadeh and Soltani 2020).

In the present study, we intend to dissect the expression pattern of LINC02381 in breast cancer tissues and cell lines and investigate the functional effects of LINC02381 in breast cancer cells. To this aim, the effect of LINC02381 overexpression on breast cancer-originated cells was examined through different cellular and molecular techniques. Collectively, the results indicated that LINC02381 by modulating IGFR1/PI3K/AKT pathway affects breast cancer cell phenotypes.

Materials and methods

Bioinformatics analysis

The RNA-seq data of lncRNA expression related to breast cancer were obtained from the starBase v3.0 (http://starBase.sysu.edu.cn). Also, other RNA-seq data of breast cancer tissues were retrieved from the Sequence Read Archive (SRA, SRP218935) database (https:// www. ncbi. nlm. nih. gov/ sra) using the online SRAToolkit software. Then, the adapters were trimmed with Trim Galore software, and the reads were aligned to the reference genome with HISAT2. The reads were assembled and counted using the HTseq-count program. Finally, the differential expression of lncRNAs was determined using the DEseq2 package. Additionally, the microarray data of lncRNA expression in breast cancer was retrieved from GEO (GSE9574/3744/41422/32474/1299) (https://www.ncbi.nlm.nih.gov/geo/) databases. The Kaplan–Meier survival analysis was retrieved from the Kaplan–Meier plotter (https://kmplot.com). The BRCA data were downloaded from the TCGA database and negatively correlated genes with LINC02381 expression were extracted by using R software (version 4.0.2). Biological pathway analyses were performed by DAVID (https://david.ncifcrf.gov) and Enrichr (https://maayanlab.cloud).

Tissue samples

Fifteen breast paired tissue samples were obtained from patients who underwent breast surgery in the Khatam-al-Anbya Hospital and confirmed by pathological diagnosis. The age, gender, IHC staining result, proliferation rate, histological grade, lymphovascular invasion, and other clinical data were collected and the data are shown in Table 1. This research involves collecting human tissues without any experimentation on human subjects. All patients in this study have signed informed consent forms and pathological samples were approved by the Ethical Committee of the Tarbiat Modares University (code number: IR.MODARES.REC.1400.116). Fresh tissues were quickly stored in liquid nitrogen.

Table 1 The clinical characteristics of the BC patients
Full size table

Cell lines and cell culture

Breast cancer cell lines SKBR3, MCF7, and MDA-MB-231 were cultured in HDMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin (HyClone, USA). Normal breast cell line MCF10A was cultured in DMEM medium (Gibco, USA) containing 5% horse serum (Gibco, USA), 10 μg/ml insulin (Gibco, USA), 20 ng/ml epidermal growth factor (EGF) (Invitrogen, USA), 100 ng/ml cholera toxin (CT) (Calbiochem, USA), and 0.5 μg/ml hydrocortisone (HC) (Sigma, USA). All the cells were incubated at 37 °C with 5% CO2. The cell bank of the Pasteur Institute (Tehran, Iran) provides all the cell lines.

Plasmid construction and transfection

The full-length of LINC02381 cDNA were cloned into pcDNA3.1 (Invitrogen, USA). The cells were seeded one day before transfection. Six hours before transfection, the medium was changed to an incomplete medium. The cells were washed with PBS (Phosphate Buffered Saline, pH = 7.4) and then transiently transfected with LINC02381-overexpressing or corresponding empty vectors into breast cancer cells using TurboFect Reagent (Thermo Scientific, USA) according to the manufacturer’s instructions.

RNA extraction, cDNA synthesis, and RT-qPCR

Total RNA was isolated using Trizol reagent (Invitrogen, USA). RNA integrity, purity, and concentration were detected by using agarose gel electrophoresis and Nano-drop, and each paired sample was adjusted to the same concentration. The extracted RNA was reverse-transcribed into cDNA by a reverse transcription kit (Fermentase, Canada) using oligo-dT and random hexamer primers. Transcript levels were measured by quantitative reverse transcription PCR using SYBR green (SMOBIO, Taiwan) and StepOne Real-Time PCR (Applied Biosystems, USA). Primers used in the study are shown in Table 2. β-actin and GAPDH were used as internal controls for expression normalization. Expression levels and relative RNA abundances were calculated by the standard 2−ΔCt and 2−ΔΔCt methods, respectively.

Table 2 The list of primers for RT-qPCR
Full size table

Protein extraction and western blot

MCF7 and SKBR3 cells were transfected with LINC02381-expressing or control vectors. 48 h after transfection, the cells were lysed and total proteins extracted with lysis buffer (RIPA, Cell Signaling Technology, USA) on ice. The extracted proteins were quantified using spectrophotometer at a 630 nm wavelength. Fifty micrograms of extracted proteins were run with 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) (Thermo Scientific, USA). The PVDF was blocked with TBST-BSA overnight at 4 °C and then incubated with primary antibodies β-actin (Santa Cruz, USA, #sc-47778), AKT (Elabscience, USA, # E-AB-30471), p-AKT (Santa Cruz, USA, # sc-271966), and IGF1R (Abcam, UK, #ab39675) overnight at 4 °C. After washing with TBST (5 min, three times), the PVDF was incubated with m-IgGκBP-HRP (Santa Cruz, USA, # sc-516102) or mouse anti-rabbit (Santa Cruz, United States, #sc-2357) for 1 h at 37 °C. The protein bands were visualized using the ECL detection kit (Pierce, USA). Western blot band quantification was performed using the ImageJ software (version 1.52a).

Cell cycle analysis

To investigate the effect of LINC02381 overexpression on the cell cycle, MCF7 and SKBR3 cells were harvested 36 h after transfection and centrifuged at 300 g. After washing with PBS thrice; the cells were fixed with precooled 70% ethanol overnight at 4 °C. Subsequently, the cells were washed and then stained with propidium iodide (PI) staining buffer and incubated at 37 °C for 30 min away from light. Eventually, the quantification of cell cycle distribution was accomplished with FACS Flow cytometry (BD Biosciences, USA). The cell population in Sub-G1, G1, S, and G2/M phases were counted and compared. Results were analyzed by Flowing software (version 2.5.1; Turku Centre for Biotechnology, Finland).

Colony formation assay

MCF7 and SKBR3 cells were transfected with pcDNA 3.1 vector with or without LINC02381 sequence in six-well plates with a density of 1000 cells/well and cultured at 37 C for 14 days in neomycin (Invitrogen, USA) supplemented medium. The colonies were counted by staining with 1% crystal violet and photographed. The colonies were quantified using the ImageJ software (version 1.52a).

MTT assay

Cell viability analysis was measured by MTT assay. A total of 10,000 cells/well were seeded in 96-well plates and then transfected with LINC02381 overexpressing plasmid or mock control vectors. 0, 24, 48, and 72 h after transfection, 20 µl MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (5 mg/ml in PBS, Sigma-Aldrich, USA) was added and the cells incubated for 4 h. Then, 200 µl DMSO (Dimethyl sulfoxide, Sigma, USA) was added to dissolve formazan crystals. Finally, the absorbance at 570 nm was measured using an ELISA reader (Biotech, Santa Cruz, USA).

Apoptosis assay

Cell apoptosis was assessed by Annexin V-FITC/PI staining kit (Roche, Germany) according to the manufacturer’s instruction. Cells were seeded in 12 well plates. 36 h after transfection, cells were treated with trypsin digestion and then washed with cold PBS, followed by staining with Annexin V-FITC and propidium iodide (PI) at 25 °C. Finally, flow cytometry is done by using a FACS flow cytometer (BD Biosciences, USA) to detect the rate of apoptotic cells. Results were analyzed by the FlowJo software (version10, TreeStar, USA).

Caspase activity assay

Twenty-four and 48 h after transfection, the MCF7 and SKBR3 transfected cells were lysed using lysis buffer. The extracted protein concentration was determined and equal amount of protein was used for caspase-3/7 activity test using the Caspase-Glo® 3/7 assay kit (Promega, USA), according to the manufacturer’s protocols. The fluorescence signals were measured using the Cytation 3™ microplate reader (BioTek, USA).

Acridine orange/ethidium bromide dual staining

Acridine orange/ethidium bromide (AO/EB) dual staining was carried out to detect the morphological evidence of apoptosis. Thirty-six hours after transfection with LINC02381 and mock vectors, MCF7 and SKBR3 cells were stained with 1 ml of acridine orange (10 mM) and ethidium bromide (10 mM) dye mix in PBS. Then, the images were photographed under fluorescent microscopy (Olympus, Japan).

Hoechst33342/PI dual staining

Hoechst 33,342 (H33342) and propidium iodide (PI) double staining were used to measure apoptosis and cell death rate. Transfected cells were stained with H33342 (1 mg/ml) and PI (1 mg/ml), 36 h after LINC02381 overexpression or mock vectors transfection. Cell imaging was performed by fluorescent microscopy (Olympus, Japan), and the percentage of cell death and apoptotic cells was calculated.

Wound-healing assay

To assess the effect of LINC02381 overexpression on cell migration, the scratch test was performed. The cells were seeded in a 24-well plate and cultured until 80% confluent. After transfection, a straight wound line was made by scratching with a sterile yellow pipette tip. The cell migration was photographed at 0, 24, 48, and 72 h after transfection, and the images were analyzed with the ImageJ software (version 1.52a).

Statistical analysis

All statistical analyses were accomplished using GraphPad Prism 9.0. The student's t-test was used to measure differences between the two groups. One-way analysis of variance (ANOVA) was used to compare differences among three groups or more. Error bars are presented as mean + / − SEM (standard error of the mean) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Results

Bioinformatics and RT-qPCR analysis suggest that LINC02381 is downregulated in breast cancer

A volcano plot of the differential expression genes (DEGs) showed LINC02381 as a top ten significantly downregulated lncRNA in breast invasion carcinoma compared with normal ones (p-value = 1.08e-9, fold change = 0.49876, tumor = 76, and normal = 19, Fig. 1A), adopted from RNA-seq data (SRP218935). In addition, bioinformatics analysis of TCGA data indicated significant differential expression of LINC02381 in 1104 breast cancer tissues compared to 113 normal ones (p-value = 7.8e-9, Fig. 1B). Kaplan–Meier curve is used to analyze the overall survival time of the BC patients and to compare two groups with different LINC02381 expression. The related curves represented that the patients with higher expression in LINC02381 have more survival probability than the other patients with lower expression (HR = 0.68, log rank p = 8.9e-07, low expression = 883 & high expression = 881, Fig. 1C).

Fig. 1
figure 1

Expression pattern of LINC02381 in different human cell lines and breast tissues. A Volcano plot analysis represents the difference between the normal and tumor data, adopted from SRP218935. B Differential expression of LINC02381 in breast tumors and matched normal tissues from starBase v3.0 database. C Kaplan–Meier survival analysis of breast cancer patients from Kaplan–Meier plotter with different LINC02381 expression. D Normalized LINC02381 expression in 14 breast cancer and 15 normal breast tissues from the GSE9574. E Normalized expression of LINC02381 in normal breast tissues, non-basal-like cancer, and basal-like cancer from the GSE3744 dataset. F Normalized expression of LINC02381 in normal breast samples, breast cancer patients with ductal carcinoma in situ, and invasive ductal carcinoma from the GSE41422 dataset. G The LINC0381 expression in human breast cancer tissues compared to their adjacent normal pairs was detected by RT-qPCR. N = 15 for each group. H Shows the mean fold change for the data of part G. I Normalized expression of LINC02381 in breast cancer cell lines from the GSE32474/1299 datasets. J Expression of LINC02381 in aggressive and normal breast cancer cell lines was detected by RT-qPCR. Data represent as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

Analysis of multiple lncRNA expression profile from microarray datasets has demonstrated that LINC02381 was significantly down-regulated in 14 breast cancer tissues compared to 15 normal breast samples (GSE9574, p-value < 0.0001, Fig. 1D). Also, lower expression of LINC02381 was observed in breast basal-like cancer (p-value = 0.0005) and non-basal-like cancer tissues (p-value = 0.0021) versus normal breast tissues (GSE3744, Fig. 1E). Furthermore, the decreased expression level of LINC02381 was observed in invasive ductal carcinoma (p-value = 0.01) and ductal carcinoma in situ (p-value = 0.04) compared to healthy breast controls (GSE21422, Fig. 1F).

To verify the suppression of LINC02381 in breast cancer specimens, the LINC02381 expression level was measured by RT-qPCR. Results showed that LINC02381 expression is downregulated (mean fold change = 0.21) in tumor specimens compared to their adjacent normal pairs (N = 15; p-value < 0.001) (Fig. 1G and H).

Bioinformatics analysis of LINC02381 expression in GSE32474/1299 dataset showed significantly lower levels of LINC02381 in breast cancer cell lines compared to normal breast epithelium (Fig. 1I). In accordance with bioinformatics data, RT-qPCR results indicated the significant downregulation of LINC02381 expression in breast cancer cell lines SKBR3, MCF7, and MDA-MB-231 relative to normal breast MCF10A cells (Fig. 1J).

LINC02381 regulates IGF1R/PI3K/AKT signaling pathway in breast cancer cell lines

To examine the effect of LINC02381 on IGFIR and PI3K signaling pathway, this lncRNA was overexpressed in both MCF7 and SKBR3 cell lines (Fig. 2A). Then, at the transcript and protein levels, IGF1R and AKT (total AKT and phospho-AKT) expressions were investigated. Following the overexpression of LINC02381 in both cell lines, RT-qPCR indicated that IGF1R has been significantly downregulated while total AKT (AKT1, AKT2, and AKT3) transcript levels were not significantly changed (Fig. 2B). Consistent with the transcript levels, IGF1R protein level was remarkably reduced and total AKT protein level was not significantly changed. However, p-AKT level was significantly reduced in both cell lines (Fig. 2C). Overall, these results presented that LINC02381 overexpression attenuated PI3K signaling activity.

Fig. 2
figure 2

LINC02381 regulates PI3K signaling pathway in breast cancer cell lines. A Shows successful overexpression of LINC02381 following the transfection of MCF7 and SKBR3 cells, using recombinant vector. B Represents RT-qPCR results against IGF1R, AKT1, AKT2, and AKT3 genes, following the overexpression of LINC02381 in MCF7 and SKBR3 cells. C Shows western blotting analysis against IGF1R and AKT and p-AKT protein levels, following the overexpression of LINC02381 in MCF7 and SKBR3 cells. While total-AKT protein level was not changed, p-AKT level and IGF1R protein level were reduced at this condition. GAPDH and β-actin were used as the internal controls in RT-qPCR. β-actin was used as the loading control in western blot. Data represent as mean ± SEM. *p < 0.05, **p < 1 0.01, ***p < 0.001

Full size image

LINC02381 overexpression suppresses breast cancer cell proliferation and viability

Enrichr and DAVID databases indicated the most significant negative correlation between LINC02381 and the cell cycle associated genes (odds ratio = 19.58, combined score = 751.8, adjusted p-value = 2.706e-15, Fig. 3A). The anti-proliferative effect of LINC02381 on MCF7 and SKBR3 cells was investigated through cell cycle analysis. PI flow cytometry results indicated that LINC02381 overexpression was followed by a significant increase of the sub-G1 cell proportion and a decrease in both S and G2/M phase population in both cell lines (Fig. 3B and C). To verify the effect of LINC02381 overexpression on the cell cycle inhibition, expression levels of c-Myc, Cyclin D1, Cyclin D2, P21, and P53 genes, as cell cycle regulators, were investigated. RT-qPCR results indicated that LINC02381 suppression effect on c-Myc expression and induction effect on P21 gene expression in both cell lines. Also, Cyclin D2 relative expression level was decreased in MCF7 and P53 relative expression was increased in SKBR3 cells (Fig. 3D). The data was consistently confirmed by colony formation assay in which a significant reduction in colony formation rate was observed in LINC02381-overexpressing breast cancer cells, compared to mock-transfected cells (Fig. 3E and F). Additionally, cell proliferation and viability of transfected cell lines were negatively affected following the LINC02381 overexpression, detected by MTT assay (Fig. 3G).

Fig. 3
figure 3

LINC02381 overexpression effects on cell cycle progression of MCF7 & SKBR3 cells. A Gene enrichment analysis (TCGA data) shows negatively correlated genes with LINC02381. B and C Histograms and bar plot analysis of cell cycle in MCF7 and SKBR3 that were transfected by either LINC02381 overexpressing and mock vectors, 36 h after transfection. D The c-Myc, cyclin D1, cyclin D2, P21, and P53 expression status in transfected MCF7 and SKBR3 cells with LINC02381 overexpressing and mock vectors. E and F The images of colony formation assay (E) and their quantitative analysis (F), after transfection of MCF7 and SKBR3 cells with LINC02381 or mock vectors. G The results of the MTT assay to determine the viability of MCF7 and SKBR3 cells, following the overexpression of LINC02381. Data represent as mean ± SEM. *p < 0.05, **p < 0.01

Full size image

LINC02381 overexpression promotes apoptosis in breast cancer cell lines

The Annexin-V/PI assay was performed to examine the anti-tumor effect of LINC02381 on breast cancer cells. The flow cytometric analysis verified that apoptotic cell proportion was significantly increased in both MCF7 and SKBR3 cells following the LINC02381 overexpression compared to the control group (Fig. 4A and B). The necrosis rate was also significantly increased in both transfected cell lines. Consistent with flow cytometry data, overexpression of LINC02381 enhanced caspase 3/7 activity level at 24 and 48 h post-transfection in both tested cell lines (Fig. 4C). Moreover, LINC02381 overexpression inhibited the relative expression level of Bcl2 and Bcl-xl anti-apoptotic genes and significantly increased the relative expression level of BAX apoptotic gene (Fig. 4D) Additionally, acridine orange/ethidium bromide and hoechst33342/propidium iodide staining showed overexpression of LINC02381 remarkably increased cell apoptosis and death rate in both cell lines compared to mock transfected cells (Fig. 4E and F). Taken together, these results represented that LINC02381 overexpression promoted cell apoptosis and death in both MCF7 and SKBR3 cell lines.

Fig. 4
figure 4

LINC02381 overexpression effects on apoptosis in MCF7 and SKBR3 cells. A and B Cell apoptosis assay was performed to determine the apoptosis rate, following the transfection of LINC02381 and mock vectors. A Histograms and B bar plots. C The results of caspase-3/7 activities of MCF7 and SKBR3 cells at 24 and 48 h post-transfection with LINC02381 overexpressing vectors. D Relative expression levels of BAX, Bcl2, and Bcl-xl genes in MCF7 and SKBR3 cells after transfection with LINC02381. E Morphological evidence of apoptosis and cell death using acridine orange (AO)/ethidium bromide (EB) and Hoechst33342/propidium iodide (PI) dual staining. The fluorescent microscopic images of MCF7 and SKBR3 were captured, following the transfection with LINC02381 or mock vectors using different filters: for AO/EB staining, green (the AO-stained cells showed the live cells) and red (the EB-stained cells demonstrated apoptotic or necrotic cells), for Hoechst33342/PI staining, blue (Hoechst-stained nucleus of living cells) and red (PI-stained dead cells) (scale bar = 50 µm). F The quantitative analysis of these MCF7- and SKBR3-stained cells, following LINC02381 overexpression to measure apoptosis and cell death rate. Data represent as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

LINC02381 overexpression inhibits breast cancer cell migration

To investigate the LINC02381 overexpression effect on MCF7 and SKBR3 cell migration, transfected cells were investigated at 0, 24, 48, and 72 h post-scratch, under a microscope. Results indicated that LINC02381 overexpression resulted in a significant reduction in the number of migrated cells, in comparison with controls (Fig. 5A and B). Also, EMT marker expression was investigated in MCF7 and SKBR3 cells after LINC02381 overexpression. CDH1 and Claudin1 (epithelial markers) were upregulated, and ZEB1 (mesenchymal marker) was downregulated in both cell lines, but Twist2 (mesenchymal marker) was significantly decreased just in SKBR3 cells. These results demonstrated that overexpression of LINC02381 affected migration and EMT processes in breast cancer cells (Fig. 5C).

Fig. 5
figure 5

Effects of LINC02381 overexpression on cell migration. A A wound healing assay in MCF7 and SKBR3 cells transfected with either LINC02381 or mock vectors at 0, 24, 48, and 72 h post-scratching (scale bar = 200 μm). B Quantitative analysis of scratch wound closure. C RT-qPCR showing the expression of epithelial and mesenchymal markers (CDH, Claudin1, ZEB1, and Twist2 genes) in MCF7 and SKBR3 cell lines upon LINC02381 transfection, compared to mock vector transfected cells. Data represent as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Full size image

Discussion

Breast cancer is the most common cancer in women and the second-leading cause of cancer worldwide (McPherson et al. 2000). Understating the molecular mechanism underlying breast cancer progression, can lead to the identification of novel diagnostic and prognostic biomarkers. In recent years, according to existing and new evidence, long noncoding RNAs (lncRNAs) play a critical role in many pathologies (Fang and Fullwood 2016; Huarte 2015; Cheetham et al. 2013). Deregulation of lncRNA expression has been observed in several malignancies, particularly breast cancer, where it has been shown to affect the cell proliferation, apoptosis, and migratory abilities (Bin et al. 2018; Li et al. 2016; Weidle et al. 2017). As researches continue to grow, an increasing number of roles that lncRNAs play in breast tissue has been found (Blythe et al. 2016; Colley and Leedman 2011; Arase et al. 2014; Piao and Ma 2012; Sirchia et al. 2009), which may help to understand the molecular pathways associated with the development of breast cancer and find approaches for cancer treatment and prevention. The analysis of the RNA-seq and microarray data adopted from TCGA, SRA, and GEO databases showed the LINC02381 as a highly deregulated lncRNA in breast tissues that was significantly downregulated in breast cancer specimens compared to normal ones. The data were further confirmed by RT-qPCR and the results showed significant downregulation of LINC02381 expression in the patients with breast cancer and cancerous cell lines. The deregulation of this lncRNA was correlated with the overall survival time of breast cancer patients and the ones with higher expression of LINC02381 presented a better prognosis. It has been reported that LINC02381, by sponging different microRNAs including miR-133b (Chen et al. 2020), miR-503-5p (Wang and Zhao 2020), miR-21-5p (Jafarzadeh et al. 2020), miR-27, miR-18, miR-96, and miR-590-5p (Wang and Zhao 2020), could affect different signaling pathways such as PI3K and Wnt. Therefore, it is likely that LINC02381 regulates the PI3K pathway in breast cancer cells by binding to these miRNAs. This observation was consistent with the data reported on the inhibitory effect of this lncRNA on PI3K signaling in colorectal cancer (Jafarzadeh et al. 2020). The PI3K/AKT pathway is one of the deregulated critical pathways in breast cancer, and this deregulation is closely associated with cancer progression and also resistance to therapies (Guerrero-Zotano et al. 2016). It has been reported that IGF1R, as an upstream receptor of PI3K signaling, is hyper-activated in at least 50 percent of breast tumors and plays a crucial role in PI3K activation in breast cancer cells (Farabaugh et al. 2015; Yerushalmi et al. 2012). In the present study, we aimed to investigate the effect of LINC02381 on breast cancer cells’ survival, proliferation, and migration and elucidate its molecular effect on this cancer. Therefore, to investigate the molecular mechanism of LINC02381 action and its relation to IGF1R expression in breast cancer, its overexpression effect on the IGF1R/PI3K/AKT pathway was investigated. Gene set enrichment analysis of the genes correlated with LINC02381 expression revealed that this lncRNA could influence breast cancer cell phenotype by regulating cell cycle progression. Consistently, overexpression of this lncRNA suppressed the mRNA and protein expression of IGFR1 and also protein level of p-AKT as a cell cycle regulator in breast cancer-originated MCF7 and SKBR3 cells. Furthermore, at the cellular level, overexpression of LINC02381 resulted in elevated cell cycle arrest and induction of apoptosis in both transfected cell lines. Overexpression of LINC02381 was also followed by the reduced migratory rate of the transfected cells. Interestingly, our results are consistent with the other reports about tumor suppressor effect of LINC02381 on colorectal and gastric cancer cell progression (Jafarzadeh et al. 2020).

We have already reported that LINC02381 sponges miR-21 in gastric cancer (Jafarzadeh and Soltani 2020). On the other hand, this microRNA is reported to downregulate the von hiple lidau (VHL) gene in pancreatic cancer (Sun et al. 2019) and cervical carcinoma (Cai et al. 2018). Furthermore, knockdown of miR-21 suppressed EMT and PI3K/AKT signal pathway in TPC-1 and BCPAP cells (Zang et al. 2019). VHL protein is known as a negative regulator of IGF1R transcripts in renal carcinoma cells (Yuen et al. 2007). These reports overall are consistent with our RT-qPCR and western analysis results showing LINC02381 overexpression resulted in reduced IGF1R expression. The relation between LINC02381, miR-21, and IGF1R and their overall effects on cell cycle progression via regulating PI3K/AKT signaling pathway are shown in Fig. 6.

Fig. 6
figure 6

Schematic representation of LINC02381 effect on IGF1R/PI3K/AKT signaling pathway. LINC02381 act as a competitive endogenous RNA (CeRNA) by sponging miR-21. LINC02381 and IGF1R inhibitor genes are targets of miR-21. This miRNA, by inhibiting the expression of IGF1R inhibitors, enhances the activation of IGF1R/PI3K/AKT signaling. LINC02381 competitively binds this miRNA, resulting in an increased expression of VHL (IGF1R inhibitor) that prevents PI3K activation. Subsequently, downregulation of the PI3K/AKT signaling results in decreased breast cancer cell proliferation, viability, and migratory ability as well as increasing apoptosis

Full size image

As a conclusion, the data presented here implies the important role of LINC02381 in breast cancer. The data show that LINC02381 is suppressed in the breast cancer specimens and its expression is inversely associated with the patient’s survival. In addition, the results indicate that LINC02381 overexpression impedes breast cancer cells’ survival, proliferation, migration, and metastasis, as well as induces apoptosis. This lncRNA seems to exert its effect on breast cancer at least partly through modulating the IGF1R/P13K/AKT pathway. Further studies are needed to dissect the exact molecular mechanism of LINC02381 in breast cancer. This finding once again emphasizes the potential application of this lncRNA in dealing with cancers like colorectal, gastric, and breast cancer.