Background
Triple-negative breast cancer (TNBC) is a kind of female malignancy with high mortality [
1]. Its occurrence is owing to lacking estrogen receptor and progesterone receptor, as well as elevated human epidermal growth factor receptor 2 level [
2]. Even thought, immunotherapy [
3,
4], radiotherapy [
5] and others act as presently main therapeutic strategies for patients with TNBC, there are still great challenges for treating TNBC due to poor prognosis and heterogeneous character [
6]. In past few decades, molecules like messenger RNAs (mRNAs) of protein-coding genes have been applied as promising biomarkers for TNBC treatment [
7‐
9]. In the current study, we planned to figure out the role of USP6NL in TNBC.
The level of protein-coding genes is considered to have close association with the development of multiple tumors and diseases [
10‐
12]. For example, strong expression levels of COL1A1 and COL1A2 was suggested to have close relation to low overall survival rate of patients with gastric cancer [
13]. Up-regulation of TCF21 depressed cell proliferation and EMT in breast cancer [
14]. As for the subject ubiquitin specific peptidase 6 N-terminal like (USP6NL) mentioned in this study, this gene encoded a type of GTPase-activating protein which has been reported in past few years to participate in several cancers. For instance, sun el at. suggested that USP6NL was overexpressed in colorectal cancer (CRC) cells to facilitate CRC progression [
15]; USP6NL up-regulation caused glycolysis addiction in breast cancer [
16]. However, the role and molecular mechanism of USP6NL in TNBC have not yet been reported or explored.
In present study, we intended to explore the function and upstream mechanism of USP6NL in TNBC.
Methods
Cell culture
Human TNBC cells lines (AU565, MDA-MB-453, MDA-MB-436, MDA-MB-468 and MDA-MB-231) and human normal mammary epithelial cell line (MCF-10A) were all procured from the ATCC company (Rockville, Maryland). All these TNBC cell lines are derived from metastatic tumors (metastatic site: pleural effusion or pericardial effusion). The DMEM medium (Invitrogen, Carlsbad, CA) supplementing with 10% FBS was employed for cultivating MCF-10A, AU565 and MDA-MB-453 cells. Besides, L-15 medium with 10% FBS was adopted to culture MDA-MB-436, MDA-MB-468 and MDA-MB-231 cells. All these cells were grown in 5% CO2 at 37 °C.
Real-time quantitative PCR (RT-qPCR)
Total RNA was separated through adopting Trizol reagent (Invitrogen), followed by the synthesis of the first cDNA template in accordance with the protocol of suppliers (Takara Bio Inc., Shiga, Japan). Following, quantitative PCR was implemented with the SYBR R Premix Ex TaqTM II (Takara), with the result analyzed via 2-ΔΔCt method. ACTB or U6 served as the control. Three bio-repeats were conducted for this experiment.
Plasmid transfection
The shRNAs targeting LINC00689 or USP6NL, as well as the corresponding negative controls, were devised and composed by Genepharma Company (Shanghai, China). In addition, pcDNA3.1 vectors (Invitrogen) covering full-length cDNA sequence of LINC00689 or USP6NL were adopted to enhance LINC00689 or USP6NL expression, with empty vector as the negative control. The miR-142-3p mimics and NC mimics, as well as miR-142-3p inhibitor and NC inhibitor, were obtained from RiboBio (Guangzhou, China), with the final transfection concentration at 50 nM. Above plasmids were appropriately transfected into MDA-MB-468 and MDA-MB-231 cells for 48 h by utilizing Lipofectamine 2000 (Invitrogen).
Western blot
After cells lysis via RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor, total protein was acquired and the concentration was then examined with a Bio-Rad DC Protein Assay Kit (Yuwei Biotechnology, Guangzhou, China). Subsequently, protein separation was achieved by SDS-PAGE, followed by protein-transferring to PVDF membranes. Thereafter, the membranes were subjected to sealing with 5% BSA and overnight incubation at 4 °C with primary antibodies against USP6NL and β-actin. After being captured by secondary antibodies, the proteins were visualized by using chemiluminescence detection system.
MDA-MB-468 and MDA-MB-231 cells were gathered and then planted in 96-well plate (500 cells/well). After fortnight, cells were subjected to fixation through 4% paraformaldehyde and staining via 0.1% crystal violet. In the end, the number of colonies was counted manually. The experiment was repeated no less than three times.
TUNEL assay
After fixation, MDA-MB-468 and MDA-MB-231 cells were rinse through precooled PBS. Afterward, cells were subjected to permeation as least 15 min. In accordance with the protocol pf suppliers, TUNEL assay Kit (Beyotime, Shanghai, China) was adopted to measure apoptotic cells. Then DAPI was utilized to stain the nuclei. In the end, the apoptotic cells were captured through fluorescence microscopy (Olympus). This assay was implemented with three repeats.
Flow cytometry analysis
Firstly, the transfected MDA-MB-468 and MDA-MB-231 cells were gathered and rinsed via precooled PBS. Then Annexin V-FITC/PI Apoptosis kit bought from BD Biosciences (San Jose, CA) was utilized to estimate the apoptosis rate of 2 × 105 cells. In detail, cells were separately treated with FITC or PI for 15 min, followed by analysis through flow cytometry (BD Biosciences). The experiment was repeated no fewer than three times.
JC-1 assay
The transfected MDA-MB-468 and MDA-MB-231 cells were subjected to cultivation in 96-well black microplate. After a night, cells were centrifuged and then the culture medium was removed. Following, cells were processed with JC-1 staining for half an hour. In the end, fluorescence microscope (Olympus) was adopted to observe and analyze the stained cells. This experiment was carried out for at least three times.
Transwell assay
With regard to migration assay, cells in serum-free medium were put in the upper transwell chambers without Matrigel. As for invasion assay, such cells were added into chambers pre-laid with Matrigel. After that, the culture medium with 20% FBS was added in bottom chamber. After cultivating for 24 h, the residual cells in upper chamber were eliminated, while cells in the lower chamber were separately fixed and stained with methanol and crystal violet. In the end, a microscopy (Olympus) was utilized to compute the number of migrated and invaded cells. Three bio-repeats were carried out for this assay.
Immunofluorescence (IF)
A 6-well plate was utilized to cultivate TNBC cells at the consistence of 2.5 × 104 cells per well. One day later, cells were fixed at room temperature (RT) and then permeabilized by 0.1% Triton X-100. Following blockaded with 5% BSA, cells were treated with the primary antibodies against E-cadherin and N-cadherin at 4 °C. After a night, secondary antibodies (Abcam) were supplemented and incubated cells at RT for half an hour. Finally, DAPI staining was performed and cells were observed under fluorescence microscopy (Olympus). The experiment was repeated no fewer than three times.
Subcellular fraction
MDA-MB-468 and MDA-MB-231 cells (1 × 106) rinsed by precooled PBS were gathered for incubation with cell fractionation buffer and cell disruption buffer based on the PARIS™ Kit (Invitrogen). After centrifugation, LINC00689 content in the cytoplasmic or nuclear fraction was evaluated through RT-qPCR. The cytoplasmic control was ACTB and the nuclear control was U6 served. This experiment was repeated no less than three times.
FISH assay
The devised specific RNA-FISH probe for LINC00689 was acquired from Ribobio. Based on the protocol of suppliers, cells were air-dried and cultivated with LINC00689-probe in hybridization buffer. After Hoechst staining, fluorescence microscopy was utilized to observe cells. This assay was performed with at least three repeats.
RNA immunoprecipitation (RIP)
Firstly, cell lysates obtained via RIP lysis buffer were cultivated with human Ago2 antibody (Millipore) and magnetic beads in RIP buffer. The normal IgG antibody (Millipore) was thought as the negative control. Finally, RT-qPCR was adopted for analyzing RNA in the whole precipitates. The experiment was conducted for no fewer than three times.
RNA pull down assay
In accordance with the protocol of suppliers, Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA) was taken for determining the interaction between molecules. The prepared cell lysates were blended with Bio-USP6NL-WT/Mut, Bio-LINC00689-WT/Mut or Bio-NC, followed by being mixed with streptavidin-linked magnetic beads. In the end, RT-qPCR was utilized to analyze RNAs in the pulled down complexes. The experiment had no less than three bio-repeats.
Luciferase reporter assay
The LINC00689 or USP6NL 3’UTR fragments which included wild-type or mutated miR-142-3p binding sites were separately inserted to the pmirGLO luciferase vector (Promega, Madison, WI). Following, above reporters were subjected to co-transfection into indicated TNBC cells with miR-142-3p mimics or NC mimics. Two days later, Luciferase Reporter Assay System (Promega) was utilized for analysis of the luciferase activity. This assay was repeated for no fewer than three times.
Statistical analysis
All the experiments were bio-repeated no less than three times. Data were represented by means of mean ± SD. Moreover, PRISM 6 (GraphPad, San Diego, CA) software was employed for data analysis with one-way ANOVA or Student’s t-test. The p-value below 0.05 was considered to be statistically significant.
Discussion
TNBC, one subtype of breast cancer, has been paid increasing attention due to high occurrence and mortality. Prevalently, targeted therapy has become a hot topic in diverse cancers, including TNBC. In this regard, promising molecules suitable for such therapy have caught researchers’ eyes in recent years, especially protein-coding genes. For instance, MCP-1 was suggested to play a key role in TNBC by affecting tumor invasiveness and metastasis [
17]. Strong expression of CDCA7 was indicated to have correlations with metastatic relapse status and shorted the survival time of TNBC patients [
18]. Similarly, RAB1A knockdown suppressed cell proliferation and EMT in TNBC [
19]. Presently, we unveiled that the expression of USP6NL was enhanced in TNBC cells, and the elevated expression of USP6NL contributed to cell proliferation, migration and invasion as well as EMT in TNBC cells. Thus, we deduced that USP6NL served a cancer-facilitating part in TNBC, suggesting it as an effective and useful biomarker for TNBC treatment.
Since the mRNAs of protein-coding genes could be recognized and silenced by miRNAs, we then made use of bioinformatics tools to find out probable miRNAs upstream of USP6NL. Herein, miR-142-3p was finally proved to target USP6NL in TNBC. Moreover, we disclosed that miR-142-3p up-regulation had similar effects like silencing USP6NL on TNBC cellular behaviors. In other word, miR-142-3p worked as a cancer-repressor in TNBC. Previously, miR-142-3p has also been indicated to participate in cancer development by a similar manner. For instance, in colorectal cancer, miR-142-3p inhibited CDK4 to restrain cell growth [
20]. Besides, miR-142-3p also regulated the progression of cervical cancer and gastric cancer [
21,
22].
Nowadays, more and more evidence disclosed that long non-coding RNAs (lncRNAs) are also implicated in TNBC development. For example, lncRNA BORG accelerated the survival and chemoresistance of TNBC cells [
23]. LncRNA PVT1 mediated TNBC progression via KLF5/beta-catenin signaling [
24]. Generally speaking, lncRNAs are able to be a ceRNA to regulate mRNA expression by competitively combining with miRNAs [
25,
26]. As for LINC00689, Liu et al. explained that LINC00689 might affect glioma progression by regulating miR-338-3p/PKM2 pathways [
27]. In current study, LINC00689 was recognized as the upstream of miR-142-3p and worked as a ceRNA to boost USP6NL expression by sequestering miR-142-3p. In the end, rescue assays proofed that inhibited miR-142-3p or up-regulated USP6NL could absolutely reverse the suppression of depleted LINC00689 on TNBC cell proliferation, migration/invasion and EMT.
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