Overexpression of long noncoding RNA LINC01296 indicates an unfavorable prognosis and promotes tumorigenesis in breast cancer

Accepted Manuscript Overexpression of long noncoding RNA LINC01296 indicates an unfavorable prognosis and promotes tumorigenesis in breast cancer

Min Jiang, Yu Xiao, Deshui Liu, Na Luo, Qi Gao, Yueyao Guan PII: DOI: Reference:

S0378-1119(18)30773-X doi:10.1016/j.gene.2018.07.004 GENE 43045

To appear in:

Gene

Received date: Revised date: Accepted date:

26 April 2018 20 June 2018 1 July 2018

Please cite this article as: Min Jiang, Yu Xiao, Deshui Liu, Na Luo, Qi Gao, Yueyao Guan , Overexpression of long noncoding RNA LINC01296 indicates an unfavorable prognosis and promotes tumorigenesis in breast cancer. Gene (2018), doi:10.1016/ j.gene.2018.07.004

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ACCEPTED MANUSCRIPT Overexpression of long noncoding RNA LINC01296 indicates an unfavorable prognosis and promotes tumorigenesis in breast cancer

Department of Ultrasound, Third Affiliated Hospital of Qiqihar Medical University,

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a

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Min Jiang a,*, Yu Xiao b, Deshui Liu c, Na Luo a, Qi Gao a, Yueyao Guan a

b

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Heilongjiang Province, 161000, China

Department of Physiology, Qiqihar Medical University, Heilongjiang Province,

Department of Oncomolecularbiology, Medical Research Institute of Qiqihar

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c

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161000, China

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* Corresponding author.

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Medical University, Heilongjiang Province, 161000, China

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E-mail address: [email protected] (M. Jiang)

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Abstract: Breast cancer (BC) is one of the most common malignancies in female worldwide. Long non-coding RNAs (lncRNAs) play imperative roles in cancer cell initiation and progression. Recently, aberrantly expressed LINC01296 was observed in several malignancies. To the best of our knowledge, its clinical significance and exact effects on BC is still unclear. In this work, the clinical value of LINC01296 was evaluated in patients with BC. Additionally, cell proliferation, apoptosis, migration and invasion capacities were detected after silencing of LINC01296. Furthermore, the 1 / 26

ACCEPTED MANUSCRIPT xenograft experiment was used to confirm the in vitro results. As a result, LINC01296 is up-regulated in both BC tissue samples and cells. Up-regulated LINC01296 is correlated with larger tumor size, positive lymph node metastasis, and advanced TNM stage of patients with BC. Additionally, Cox regression analysis confirmed

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LINC01296 as an independent prognostic indicator for patients with BC. For the part

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of functional assays, silencing of LINC01296 inhibited BC cell growth in vitro and in

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vivo. Also, cell apoptosis was enhanced after LINC01296 silenced. Moreover, cell migration and invasion potential were both abrogated in the si-LINC01296 groups.

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therapeutic target for patients with BC.

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Collectively, LINC01296 may function as a potential prognostic predictor and

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1. Introduction

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Keywords: breast cancer, lncRNA, LINC01296, prognosis

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Breast cancer (BC) is one of the most common cancers in female worldwide [1]. With the existence of intrinsic resistance, BC is quite resistant to currently available therapeutic approaches [2]. Despite recent advances in multimodality therapies, the prognosis of BC remains unfavorable [3]. Additionally, BC treatment is also challenged by its distinct pathogenesis, which is a main cause of failures in radiotherapy [4]. Thus, better exploring molecular mechanisms underlying BC development and progression will be helpful to exploit effective prognostic/diagnostic 2 / 26

ACCEPTED MANUSCRIPT biomarker and therapeutic target for this malignancy. Long non-coding RNAs (lncRNAs) is a family of non-coding RNAs (ncRNAs) family with length > 200nt. LncRNAs are involved in various physiological and pathological processes by functioning as enhancers, scaffolds, decoys, and guides in

facilitate

cholangiocarcinoma

cell

progression

via

scaffolding

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could

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gene regulation [5-8]. For instance, a recent report demonstrated that SPRY4-IT1

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EZH2/LSD1/DNMT1 and sponging miR-101-3p [9]. Up to now, several lncRNAs have been identified as key regulators in the initiation and development of BC [10-12].

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For instance, HOTAIR exerts oncogenic properties in MCF7 cells by affecting

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P53/Akt/JNK pathway [13].

LINC01296 is an intergenic lncRNA and mapped to 14q11.2. It was found

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up-regulated and functions as an oncogene.in several malignancies. Qiu et al. found

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that LINC01296 might act as a potential prognostic predictor for colorectal cancer patients [14]. Additionally, Wu et al. reported that up-regulated LINC01296 is closely

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correlated with unfavorable prognosis for the patients with prostate cancer and

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facilitates cancer cell growth, migration, and invasion [15]. Moreover, LINC01296 could facilitate cholangiocarcinoma cell proliferation and progression via sponging miR-5095 [16]. LINC01296 could also aggravate gastric cancer cell progression via miR-122/MMP-9 axis [17]. Although emerging evidence has identified LINC01296 as a crucial factor in cancer initiation and development, its expression profile, clinical significance and biological functions in BC remains unknown.

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ACCEPTED MANUSCRIPT 2. Materials and methods

2.1 Clinical tissue collection

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55 paired BC tissues and matched adjacent non-tumorous tissue samples were

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collected from the Third Affiliated Hospital of Qiqihar Medical University between

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March 2011 and February 2013. The present study was carried out with the approval of the Ethics Review Committee of Qiqihar Medical University and all the recruited

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BC patients signed informed consent before participating in the study. The tumor

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tissues and matched adjacent non-tumorous tissue samples were immediately stored in

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liquid nitrogen until used.

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2.2 Cell culture and transfection

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MCF7, MDA-MB-231, SKBR3, BT-20, T47D, MDA-MB-436, and normal

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mammary epithelial cells MCF10A were commercially acquired from American Type Cell Culture (ATCC, Manassas, VA, USA). RPMI-1640 or Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) was used for cell culture. All the cells used in the study were cultured in a 37°C cell chamber with 5% CO2. siRNAs specifically targeting LINC01296 and si-NC were purchased from GenePharma (Shanghai, China). Lipofectamine 3000 (Thermo Fisher Scientific, USA) was used for the transfection followed by the 4 / 26

ACCEPTED MANUSCRIPT protocols of manufacturer. At 48h after transfection, the knockdown studies were performed.

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2.3 RNA isolation and RT-qPCR analysis

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Total RNA was isolated from tissues or cultured cells using Trizol reagent

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(Thermo Fisher Scientific, Waltham, MA, USA) according to the directions of manufacturer. RNA integrity number (RIN) was measured by Agilent 2100 and

RNA

6000

LabChip

kit

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Bioanalyser

(Agilent

Technologies).

For

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reverse-transcription polymerase chain reaction, RNA was reverse-transcribed to cDNA by using random primers (10μM) and a reverse-transcription kit obtained from

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Takara (Kyoto, Japan). RT-qPCR was carried out on an ABI StepOnePlus (Thermo

LINC01296:

F,

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Fisher Scientific) using Power SYBR Green (Takara). The primers used in the study: 5’-

AAGTGGCACCAGCCTCACT

-3’;

R,

5’-

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CGGCCAAGTTCTTTACCATC -3’. GAPDH: F, 5’- GGGAGCCAAAAGGGTCAT

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-3’; R, 5’- GAGTCCTTCCACGATACCAA -3’. Standard curves were constructed before the quantification of gene expression. Amplification efficiencies = 10 -1.

[− 1/slope]

2.4 Cell proliferation assays

Cell counting kit-8 (CCK-8) and clonogenic assays were induced to detect cell 5 / 26

ACCEPTED MANUSCRIPT growth altered by LINC01296. After 48h transfection, MCF7 and MDA-MB-231 cells were planted into 96-well plates at a concentration of 2000 cells per well. In brief, transfected cells were cultured for consecutive 4 days with complete medium. 10μl of CCK-8 (Dojindo, Japan) was added into the corresponding wells at each

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monitored time. After incubated for about 2h, the absorbance of each well was

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detected at 450nm using a microplate reader (Tecan, Männedorf, Switzerland).

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For clone formation assay, after 48h of transfection, a certain number of MCF7 and MDA-MB-231 cells were collected and inoculated into 6-well plates. Then, the

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cells were allowed to grow in a 37°C cell-culture incubator for another 2w. At last,

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colonies were washed with phosphate-buffered saline (PBS), fixed with formaldehyde, stained with 0.1% crystal violet solution and photographed. The cell colony

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containing more than 50 cells indicated one clone.

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2.5 Cell apoptosis assay

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Flow cytometric analysis was induced to evaluate the apoptosis upon LINC01296 siRNAs transfection in BC cells. In brief, transfected cells at exponential growth phase were trypsinized and re-suspended with 1X Annexin V-FITC binding buffer containing 10mM Hepes/NaOH, pH 7.4, 140mM NaCl, 2.5mM CaCl2. 5μL of Annexin V-FITC and 5μL of propidium iodide (PI, Beyotime, Beijing, China) were added into the tubes and co-incubated for 15min. Then, the treated cells were measured by flow cytometry (FACScan, BD). 6 / 26

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2.6 Relative caspase activity determination

The relative activities of caspase-3/-9 were evaluated by a Caspase-3 Activity

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Kit and Caspase-9 Activity Kit, respectively (Solarbio, Beijing, China).

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2.7 Wound scratch assay

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Transfected MCF7 and MDA-MB-231 cells were planted into a 6-well plate. The

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plate was replaced with the serum-free medium after cell adherence. When 90-100% cells were fused, wounds were made uniformly using a 200μL spearhead

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perpendicular to the bottom of the 6-well plate. Afterwards, PBS was used to clear

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away the floating cells. At 0h and 36h after wound formation, the wound closure area

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was observed and photographed.

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2.8 Transwell assay

Transfected cells were collected, counted, and suspended in serum-free medium. Cell suspension were planted into the upper compartment of Transwell unit (Costar, Washington, DC, USA), while RPMI-1640 medium supplied with FBS was added into the lower compartment. The cells were incubated at 37°C for 24h prior to sweep the cell in the upper chamber. Then, the membranes were fixed with formaldehyde 7 / 26

ACCEPTED MANUSCRIPT and stained with 0.1% crystal violet solution. Five fields of view were randomly selected and photographed under an inverted microscope (Leica, Germany). The number of cells traversed through the membrane were counted. For the invasion assay, what was different from migration assay was that the upper membrane of transwell

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was coated with a layer of Matrigel. The remaining steps were identical to those in

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migration assay.

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2.9 Immunoblotting assay

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The proteins were isolated by RIPA lysis buffer with protease inhibitors (Beyotime) and the concentration of protein was then detected. The same amount of

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proteins were electrophoretically separated on sodium dodecyl sulfate polyacrylamide

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gel (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Afterwards, 5% defatted milk was used to block the membranes prior to incubating

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with primary antibodies at 4°C overnight. Primary antibodies against Bcl-2 (1:2000,

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ab182858); Bax (1:2000, ab32503) and GAPDH (1:10000, ab181602) were obtained from Abcam (Cambridge, MA, USA). At last, the membranes were incubated with HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, USA) before visualized by an enhanced chemiluminescence (Beyotime).

2.10 In vivo study and immunohistochemistry (IHC) assay

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ACCEPTED MANUSCRIPT Animal study was approved and reviewed by the Animal Care and Use Committee of the Third Affiliated Hospital of Qiqihar Medical University. Eight BALB/c nude mice (female, 6-week-old, Vital River, Beijing, China) were raised under pathogen-free conditions. For tumor propagation analysis, MCF7 tumor cells

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were injected into either side of the posterior flank of nude mice. Tumor volume was

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calculated followed by the formula: Volume=0.5×longitudinal diameter×latitudinal

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diameter2. All the mice were sacrificed at 18d after inoculation. The tumors were removed from mice and weighed. Then, they were made into formalin-fixed

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paraffin-embedded blocks. The slices were cut at a thickness of 5μm before incubated

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with anti-Ki67 polyclonal antibody (Abcam, Cambridge, MA, USA). Additionally,

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2.11 Statistical analysis

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total RNA from tumors was extracted to detect the relative expression of LINC01296.

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The statistical analyses of this study were calculated by using GraphPad Prism

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5.01 (GraphPad software, La Jolla, CA, USA) and SPSS 19.0 (Chicago, IL, USA). The data are presented as mean±standard deviation from three independent repeated trials and analyzed using Student’s t-test. The links of LINC01296 expression and clinicopathologic characteristics was analyzed by Fisher’s exact test. The survival rate was analyzed using Kaplan-Meier method and Log-rank test. The factors affecting prognosis were analyzed via Cox regression analysis. A p-value less than 0.05 was considered significant. 9 / 26

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3. Results

3.1 LINC01296 is highly expressed in BC tissues and cells and predicts unfavorable

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prognosis

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To explore the expression profile of LINC01296 in BC, RT-qPCR was used to detect the levels of LINC01296 in 55 paired cancerous tissues and para-cancerous

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tissue samples. As Fig. 1A showed, compared with normal tissues (MCF10A), the

further

analysis

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expression of LINC01296 was strikingly increased in BC tissues (p<0.01). After of the relationship

between

LINC01296

expression

and

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clinicopathological features of BC, it was found that enhanced expression of

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LINC01296 was tightly associated with larger tumor size (p=0.038), positive lymph node metastasis (p=0.022) and more advanced TNM stage (p=0.032) of BC, but had

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no significant correlations with other clinical features (Table 1). Further survival

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analysis showed that the prognosis of patients in low LINC01296 expression group was markedly superior to that of patients in high LINC01296 expression group (p=0.002, Fig. 1B). Cox regression analyses indicated that overexpressed LINC01296 was an independent prognostic factor for the patients with BC (p=0.017, Table 2). In vitro experiments also indicated that the transcription of LINC01296 in normal breast epithelial MCF10A cells was strikingly lower than that in BC cells. Moreover, the expression levels of LINC01296 in MCF7 and MDA-MB-231 cells were higher than 10 / 26

ACCEPTED MANUSCRIPT other cells (Fig. 1C).

3.2 Silencing of LINC01296 attenuates cell growth and facilitates apoptosis in BC

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cells

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To identify the functions of LINC01296 exerts in BC cells, we transfected

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si-LINC01296-1/-2 into MCF7 and MDA-MB-231 cells to knockdown the expression of LINC01296. RT-qPCR results indicated that both si-LINC012961-1 and

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si-LINC012961-2 had a good inhibition efficiency relative to si-NC group (Fig. 2A).

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Thus, they were chosen for the subsequent study. CCK-8 assay documented that silencing of LINC01296 could suppress the proliferation of BC cells (Fig. 2B).

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Clonogenic analysis demonstrated that the colony numbers were dramatically reduced

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in the LINC01296 silenced group (Fig. 2C). Results of flow cytometry showed that the proportion of apoptotic cells in knockdown groups was markedly boosted

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compared with that in the si-NC group (Fig. 2D). Additionally, relative activities of

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caspase-3 and caspase-9 were increased in the LINC01296 knockdown groups (Fig. 2E). Furthermore, Western blotting analysis indicated a higher expression of Bax after silencing of LINC01296, while Bcl-2 expression was reduced (Fig. 2F). Ultimately, the findings above indicated that silencing of LINC01296 could inhibit cell proliferation and promote apoptosis in BC cells.

3.3 Down-regulation of LINC01296 attenuates metastatic properties of BC cells 11 / 26

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The effects of LINC01296 on the migratory and invasive capacities of BC cells were further analyzed. First, wound scratch assay was used to detect the migration capacities of MCF7 and MDA-MB-231 cells. Results showed that the wound closure

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areas in LINC01296 knockdown groups were remarkably smaller than that in the

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si-NC group at 36h after wound creation (Fig. 3A). Afterwards, transwell migration

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assay was performed and obtained a similar result (Fig. 3B). For the cell invasion assay, transfected cells that traversed through the Matrigel and membrane in the

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LINC01296 depletion groups were decreased relative to si-NC groups (Fig. 3C). The

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findings above demonstrated that knockdown of LINC01296 in BC cells could

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significantly attenuate cell metastatic properties.

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3.4 Decreased LINC01296 inhibits xenograft growth

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In vivo study was performed to further confirm the oncogenic properties of

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LINC01296 exerts in BC. As Fig. 4A and B indicated, silenced LINC01296 could dramatically suppress tumor growth relative to shCtrl group. Additionally, tumor weight was also decreased in the LINC01296 knockdown group (Fig. 4C). Meanwhile, IHC assay showed a lower expression of Ki67 in shLINC01296 group (Fig. 4D). RT-qPCR also showed a lower transcription of LINC01296 in tumors formed from shLINC01296 group (Fig. 4E). Collectively, the animal study further validated that LINC01296 knockdown significantly inhibited BC tumor growth. 12 / 26

ACCEPTED MANUSCRIPT

4. Discussion

BC is the leading cause of cancer-related deaths in women worldwide [18]. The

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initiation and progression of BC is a complicated process. Moreover, the pathological

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classification based on estrogen and progestrone receptor status has become the basis

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for the treatment of this fatal disease [19,20]. Recently, the crucial roles of lncRNAs were widely identified in various diseases [21,22], including cancer [23]. In general,

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lncRNAs regulate downstream genes expression by interacting with proteins or acting

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as ceRNAs for miRNAs [9]. For instance, lncRNA FTH1P3 could promote oral cancer cell progression via sponging miR-224-5p to regulate fizzled 5 expression [24].

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Sun et al. reported that HOXA11-AS facilitates gastric cancer cell growth and

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invasion by scaffolding the RNA binding proteins PRC2, LSD1 and DNMT1 [25]. For BC, several lncRNAs have been identified to participate in tumor progression

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[26,27]. In this work, we focused on a novel cancer-associated lncRNA, LINC01296,

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which has been proved up-regulated in several cancers and functions as an oncogene in tumorigenesis and progression. Wang et al. reported that LINC01296 harbors miR-21a to promote colon cancer proliferation and invasion [28]. Moreover, prostate cancer patients with enhanced expression of LINC01296 had a dismal prognosis proved by a recent study [15]. These findings prompted us to explore the clinical relevance and functional role of LINC01296 in BC. In line with our expectation, LINC01296 transcription is strikingly up-regulated in BC tissues relative to their 13 / 26

ACCEPTED MANUSCRIPT counterparts. Additionally, this up-regulation is significantly correlated with aggressive phenotypes of BC including larger tumor size (p=0.038), positive lymph node invasion (p=0.022) and more advanced TNM stage (p=0.032). Additionally, we found that the patients with overexpressed LINC01296 in tumor tissues had a worse

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5-year survival rate relative to the patients with decreased transcription of LINC01296.

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Moreover, LINC01296 may regard as an independent prognostic predictor for BC

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patients after surgery. These results indicate that LINC01296 might exert oncogenic properties in BC cells.

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After validating the clinical value of LINC01296 in BC patients, a series of

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experiments were performed to assess the biological activities of LINC01296 played in BC cells. Loss-of-function assays were performed on MCF7 and MDA-MB-231

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cells, which have the highest expression of LINC01296 compared with MCF10A.

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Uncontrolled cell growth is one of the most essential characteristics of cancer. In the current study, silencing of LINC01296 dramatically attenuated cell proliferation and

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clonogenic capacity in the two cells. Subsequent animal study further validated the

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proliferation-promoting role of LINC01296 in BC cells. We next evaluated whether the anti-proliferative effect of si-LINC01296 could be partly attributed to the promotion of cell apoptosis, we detected cell apoptosis alteration after transfection. As we expected, flow cytometric analysis demonstrated that cell apoptosis is boosted after LINC01296 silenced. It is known that mitochondrial pathway is able to activate cell apoptosis and this pathway is tightly correlated with the regulation of apoptosis-related factors including caspase-3 (effector caspase), caspase-9 (initiator 14 / 26

ACCEPTED MANUSCRIPT caspase) and Bcl-2 family [29]. Bcl-2 and Bax are key members in Bcl-2 family which act as on-off action in cell life and death [29-31]. In our study, we found that caspase-3, caspase-9 and Bax expression were enhanced after silencing of LINC01296, whereas Bcl-2 was down-regulated in the LINC01296 decreased groups.

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Ultimately, Bcl-2/caspase-3 pathway might play key roles in si-LINC01296 induced

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cell apoptosis.

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Previously, researchers have found that up-regulated LINC01296 could facilitate prostate cancer cell migration and invasion [15]. Cell migration and invasion are

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crucial prerequisites for tumor cell metastasis. Therefore, we evaluated the alteration

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of migratory and invasive capacities of BC cells induced by LINC01296 knockdown. As a result, we found LINC01296 had a robust metastatic properties promoting role in

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MCF7 and MDA-MB-231 cells, which is consistent with the previous study [15].

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However, there are still some limitations to make an indestructible conclusions in this study. The molecular mechanisms behind these regulatory functions were not

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investigated and more patients should be studied to confirm the clinical significance

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of LINC01296.

In conclusion, we first documented that LINC01296 was up-regulated in BC tissues and cells. Additionally, this overexpresion was closely correlated with unfavorable prognosis of patients with BC. Furthermore, Silencing of LINC01296 abrogated cell proliferation and metastatic properties in MCF7 and MDA-MB-231 cells. Importantly, cell apoptotic rate was increased after silencing of LINC01296 partly via Bcl-2/caspase-3 pathway. Collectively, LINC01296 may be a potential 15 / 26

ACCEPTED MANUSCRIPT prognostic indictor and therapeutic target for BC.

Conflict of Interests

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The authors report no conflicts of interest in this work.

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Acknowledgment

Special thanks to Dr. Liguo Hao for the revision of English usage in the

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manuscript.

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Figure legends

Fig. 1. Relative transcription of LINC01296 in BC tissue samples and cells and its

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clinical relevance. A. Relative transcription of LINC01296 in BC tissue samples and

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non-tumorous tissues evaluated by RT-qPCR. B. Kaplan–Meier survival analysis applied based on postoperative survival time and LINC01296 transcription. C.

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Relative transcription of LINC01296 in BC cells and MCF10A evaluated by

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RT-qPCR. C: The data are shown as the mean ± SD (n=3). *p<0.05, **p<0.01.

Fig. 2. LINC01296 depletion attenuated cell growth and facilitated apoptosis in BC cells. A. Silencing of LINC01296 with LINC01296 siRNAs in MCF7 and MDA-MB-231 cells. B. Cell growth was evaluated by CCK-8 assays in MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC. C. Clone formation assays were performed to detect colony-forming capacities 20 / 26

ACCEPTED MANUSCRIPT of MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC. D. Flow cytometric analyses were carried out to evaluate cell apoptosis in MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC. E. Relative activities of caspase-3 and

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caspase-9 in MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1,

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si-LINC01296-2 and si-NC were read by a microplate reader. F. Bax and Bcl-2

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protein levels in MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC were evaluated by immunoblotting

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assays. A-E: The data are shown as the mean ± SD (n=3). *p<0.05, **p<0.01.

Fig. 3. Silenced LINC01296 abrogated migratory and invasive capacities of BC cells.

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A. Wound scratch assays were carried out to evaluate migration capacities of MCF7

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and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC. B. Transwell migration assays were carried out to evaluate the migration

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capacities of MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1,

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si-LINC01296-2 and si-NC. C. Transwell invasion assays were carried out to evaluate the invasion capacities of MCF7 and MDA-MB-231 cells after transfected with si-LINC01296-1, si-LINC01296-2 and si-NC. A-C: The data are shown as the mean ± SD (n=3). **p<0.01.

Fig. 4. Silenced LINC01296 inhibited xenograft tumor growth in vivo. A. Tumors were removed from nude mice at 18 days after inoculation. B. Tumor volume was 21 / 26

ACCEPTED MANUSCRIPT evaluated every 3 days after inoculation. C. Tumor weights were measured. D. Location and relative expression of Ki67 was detected by IHC. E. RT-qPCR was carried out to evaluate the average expression of LINC01296. B, C, E: The data are

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shown as the mean ± SD (n=3). *p<0.05.

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ACCEPTED MANUSCRIPT clinicopathologic characteristics of LINC01296 expression High Low

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p 0.788

15(27.3%) 15(27.3%)

11(20.0%) 14(25.4%) 0.038

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6(10.9%) 24(43.6%)

22(40.0%) 8(14.5%) 12(21.8%) 18(32.7%) 13(23.6%) 17(30.9%)

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0.032

15(27.3%) 10(18.2%)

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9(16.3%) 21(38.2%)

11(20.0%) 14(25.4%)

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5(9.1%) 25(45.5%)

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Table 1 LINC01296 expression and breast cancer patients Clinicopathologic Total No. characteristics Age (years) ≤50 26 > 50 29 Tumor size (cm) ≤2 16 >2 39 TNM stage I + II 24 III 31 Lymph node metastasis Negative 19 Positive 36 Her-2 status Negative 38 Positive 17 ER status Negative 20 Positive 35 PR status Negative 21 Positive 34

0.022

13(23.7%) 12(21.8%) 0.562

16(29.1%) 9(16.4%) 0.585 8(14.6%) 17(30.9%) 0.419 8(14.6%) 17(30.9%)

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Table 2 Univariate and multivariate analysis of prognostic factors for overall survival in breast cancer patients Univariate analysis Multivariate analysis Variables p-val p-val HR 95% CI HR 95% CI ue ue Overall Survival 0.9 0.511-1. 0.89 Age (> 50 vs. ≤50) 59 800 7 1.3 0.663-2. 0.40 Tumor size (> 2 vs. ≤2) 62 801 0 2.1 1.101-4. 0.02 1.6 0.858-3. 0.12 TNM stage (III vs. I + II) 23 096 95 345 9 5 Lymph node metastasis (Positive 1.8 0.939-3. 0.07 vs. Negative) 90 803 4 Her-2 status (Positive vs. 1.6 0.883-3. 0.11 Negative) 88 227 4 0.6 0.349-1. 0.20 ER status (Positive vs. Negative) 61 252 4 0.5 0.314-1. 0.10 PR status (Positive vs. Negative) 91 113 3 LINC01296 expression 2.7 1.375-5. 0.00 2.3 1.163-4. 0.01 (High vs. Low) 02 311 42 715 4 7

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ACCEPTED MANUSCRIPT Abbreviations BC: Breast cancer; CCK-8: cell counting kit-8; DMEM: Dulbecco’s Modified Eagle Medium; FBS: fetal bovine serum; IHC: immunohistochemistry; lncRNAs: long non-coding RNAs; PBS: phosphate-buffered saline; PI: propidium iodide; PVDF:

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polyvinylidene fluoride; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel.

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ACCEPTED MANUSCRIPT Highlights:

LINC01296 is upregulated in BC tissues and cells.

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Knockdown of LINC01296 could inhibit cell growth in vitro and in vivo.

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LINC01296 protects against apoptosis partly via affecting Bcl-2/Caspase-3 pathway.

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LINC01296 expression is associated with tumor size, TNM stage and lymph node

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metastasis in BC patients.

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Patients with high LINC01296 expression had poorer overall survival.

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Figure 1

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