Large quantities of long noncoding RNAs (lncRNAs) have been verified to exert vital functions in the process of breast cancer (BC). lncRNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was reported to suppress the development of several cancers. However, its detailed function in BC remained unclear. In the current study, HAND2-AS1 was discovered to be low expressed in BC cell lines, and overexpression of HAND2-AS1 could repress proliferation, migration, and invasion but facilitate apoptosis in BC cells. Moreover, HAND2-AS1 was found to act as a sponge of miR-3118 which was detected to be upregulated in BC cell lines. miR-3118 depletion could constrict the progression of BC. HAND-AS1 hindered the course of BC by reducing the expression of miR-3118. Besides, PHLPP2 was treated as a downstream target of miR-3118 under the selection of RNA pull-down assays. HAND2-AS1 inhibited the process of BC by enhancing expression of PHLPP2. In summary, our study testified that HAND2-AS1 suppressed BC growth by targeting the miR-3118/PHLPP2 axis, indicating that HAND2-AS1 could be regarded as a potential target for BC treatment.
Breast cancer (BC) is a common cancer with high morbidity among females globally [
Substantial long noncoding RNAs (lncRNAs) were reported to bring about vital effects on the progression of cancers [
Numerous essays acclaimed that the competing endogenous RNA (ceRNA) regulatory system played huge roles in regulating the mechanism of cancers [
miRNAs were a class of RNAs which were around 18-22 nucleotides in length without the capacity of coding proteins [
The main purpose of our current study was to explore the role of HAND2-AS1 in BC cells.
To investigate the role of HAND2-AS1 in BC, we used RT-qPCR assays to determine the expression of HAND2-AS1 in BC cell lines (MCF-7, MDA-MB-231, SK-BR-3, and MDA-MB-453) and normal mammary epithelial cell (MCF-10A). The results showcased that HAND2-AS1 was at a low level of expression in BC cell lines in comparison with MCF-10A (Figure
Overexpression of HAND2-AS1 repressed proliferation, migration, and invasion in BC cell. (a) RT-qPCR assays were constructed to evaluate expression of HAND2-AS1 in BC cell lines (MCF-7, MDA-MB-231, SK-BR-3, and MDA-MB-453) and normal mammary epithelial cell (MCF-10A). (b) RT-qPCR assays were performed to appraise the expression of HAND2-AS1 in cells transfected with pcDNA3.1/HAND2-AS1. (c, d) CCK8 and colony formation assays were conducted to appraise cell proliferation in cells transfected with pcDNA3.1/HAND2-AS1. (e) Flow cytometry analysis was performed to assess the apoptosis rate. (f, g) Transwell assays were carried out to assess the migration and invasion capacity. (h) Western blot was used to measure E-cadherin, N-cadherin, MMP2, Vimentin, and slug proteins.
To analyze the role of HAND2-AS1 in the ceRNA regulatory system, we performed FISH assays, and the outcomes displayed that HAND2-AS1 accumulated in cytoplasm (Figure
miR-3118 depletion inhibited BC proliferation while facilitating apoptosis in BC. (a) FISH assays were constructed to judge the subcellular localization of HAND2-AS1. (b) pcDNA3.1/HAND2-AS1 were transfected into cells, and the expression of miRNAs was detected in MCF-7 and SK-BR-3 cells. (c) The binding sequences between HAND2-AS1 and miR-3118 were projected by bioinformatics. (d) RNA pull-down assays were carried out to illustrate the combination between miR-3118 and HAND2-AS1. (e) miR-3118 mimics were transfected into cells, and the expression of miR-3118 was tested by RT-qPCR assays. (f) Luciferase reporter assays were conducted to demonstrate the combination between HAND2-AS1 and miR-3118. (g) The expression of miR-3118 was examined in BC cell lines by RT-qPCR assays. (h) miR-3118 expression was tested in cells transfected with miR-3118 inhibitor. (i, j) CCK8 and colony formation assays were applied to detect cell proliferation ability. (k) Flow cytometry analysis was conducted to probe the apoptosis rate. (l, m) Transwell assays were performed to estimate capacities of migration and invasion. (n) Western blot assays were conducted to measure associated proteins of the EMT process.
To find out how HAND2-AS1 regulated miR-3118 in BC, we conducted rescue assays. MCF-7 and SK-BR-3 cells were transfected with pcDNA3.1/HAND2-AS1 and miR-3188 mimics. The cell proliferation assessed by CCK8 and colony formation assays was declined by pcDNA3.1/HAND2-AS1, but miR-3118 mimics could rescue the results induced by HAND2-AS1 overexpression (Figures
HAND2-AS1 restricted the process of BC by inhibiting miR-3118. (a, b) CCK8 and colony formation assays were performed to measure cell proliferation ability in cells transfected with pcDNA3.1/HAND2-AS1 and miR-3118 mimics. (c) The rate of flow cytometry analysis was set up to estimate the apoptosis rate. (d, e) Transwell assays were conducted to determine abilities of cell migration and invasion. (f) Western blot was carried out to evaluate the relevant proteins of the EMT process.
Then, we searched the starBase and found that several mRNAs had possibilities to bind to miR-3118. RNA pull-down assays were applied, and the results manifested that biotinylated miR-3118-WT only could make PHLPP2 abundant while no changes could be seen in other mRNAs (Figure
PHLPP2 was a downstream target of miR-3118. (a) RNA pull-down assays were carried out to verify which mRNA could bind to miR-3118. (b) The binding sites between miR-3118 and PHLPP2 were predicted by bioinformatics. (c) PHLPP2 expression and protein were tested in cells transfected with miR-3118 inhibitor by RT-qPCR and western blot. (d) RIP assays were performed to certify that HAND2-AS1, miR-3118, and PHLPP2 coexisted in RNA-induced silencing complexes (RISCs). (e) Luciferase reporter assays were conducted to testify the competing relationship between HAND2-AS1 and PHLPP2.
To investigate how HAND2-AS1 mediated PHLPP2 in BC, we constructed rescue assays. Firstly, we examined the expression of HAND2-AS1 in cells transfected with sh-PHLPP2. The results revealed that PHLPP2 expression was cut down by sh-PHLPPP2 (Figure
HAND2-AS1 hindered the course of BC through elevating PHLPP2 expression. (a) PHLPP2 expression was evaluated in cells transfected with pcDNA3.1/PHLPP2. (b, c) CCK8 and colony formation assays were carried out to estimate cell proliferation in cells transfected with pcDNA3.1/PHLPP2 and sh-PHLPP2. (d) The apoptosis rate was measured in flow cytometry analysis. (e, f) Transwell assays were conducted to evaluate capacity of migration and invasion. (g) Western blot was performed to measure relevant proteins of the EMT process.
The effects of HAND2-AS1 on BC growth were verified in vivo. MCF-7 cells were transfected into pcDNA3.1/HAND2-AS1 and pcDNA3.1 vector. And the cells were injected into nude mice. After culture for 28 days, the process of tumor growth was recorded. The tumors transfected with pcDNA3.1/HAND2-AS1 grew slower than that with pcDNA3.1 vector (Figure
Overexpression of HAND2-AS1 constricted the growth of BC in vivo. (a–c) Tumor growth curve, volume, and weight were recorded and calculated in cells transfected with pcDNA3.1 and pcDNA3.1/HAND2-AS1. (d) Immunohistochemistry was conducted to examine the expression of Ki67 in cells transfected with pcDNA3.1 and pcDNA3.1/HAND2-AS1.
In this study, we mainly analyzed HAND2-AS1 and discovered that it is downregulated in BC cell lines, which was in accordance with previous reports [
Previous studies demonstrated that lncRNAs sponged miRNAs to regulate mRNA expression and thus the progression of cancers in the ceRNA regulatory system [
PHLPP2 was unveiled as a tumor suppressor in multiple cancers acting as an inhibitor of AKT and inducing apoptosis of cancer cells [
To sum up, our studies confirmed that HAND2-AS1 could inhibit BC proliferation, migration, and invasion and induce apoptosis as a ceRNA of miR-3188 to elevate PHLPP2 expression, which poses a potential that HAND2-AS1/miR-3188/PHLPP2 might be a therapeutic axis for breast cancer in the future.
BC cell lines (MDA-MB-231, MCF-7, SK-BR-3, and MDA-MB-453) and human mammary epithelial cell line (MCF10A) were both procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DMEM, 10% fetal bovine serum (FBS), and 100 U/L penicillin/streptomycin were bought from Gibco (Grand Island, NY, USA) for cell culture in 5% CO2 at 37°C. MCF-7 and SK-BR-3 cells in 6-well plates (
Total cellular RNAs were extracted from cell samples via the TRIzol reagent (Invitrogen) to synthesize cDNA with the PrimeScript RT reagent kit (Takara Biotechnology, Tokyo, Japan). qPCR was developed using SYBR Green PCR Master Mix (Invitrogen) on a Bio-Rad IQ5 thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RNA levels of HAND2-AS1, miR-3118, and PHLPP2 in all groups were normalized to U6 or GAPDH using the 2-
BC cells in 96-well plates (
Clonogenic cells of MCF-7 and SK-BR-3 were incubated in 6-well plates (
BC cells were first washed in cold phosphate-buffered saline (PBS). Then, they were resuspended in 100 mL of binding buffer. 5 mL of Annexin V-FITC (Invitrogen) was added for 15 min in the dark at room temperature. Then, 5 mL of propidium iodide (PI; Invitrogen) was added for 15 min, followed by analysis of flow cytometry (BD Biosciences, San Jose, CA, USA). The flow cytometry assays analyzed changes in apoptosis rates in all groups.
Transfected BC cells were collected and incubated in serum-free medium. The 24-well transwell chamber (Costar, MA, USA) with matrigel coating was for invasion or migration assay. BC cells were put into the upper compartment, while the lower compartment was filled with complete culture medium. After 48 h of incubation, invasive and migratory cells were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet for counting. The number of invaded or migrated cells was counted and served as a reference to cell migration or invasion ability in different groups.
The western blotting method was used to observe the relative protein expression in cells of all groups. Protein samples from MCF-7 and SK-BR-3 cells were separated by electrophoresis on 10% SDS polyacrylamide gels and transferred onto PVDF membranes which were then blocked with 5% skimmed milk at room temperature for 2 h. The incubation with primary antibodies against E-cadherin, N-cadherin, MMP2, Vimentin, slug, PHLPP2, and GAPDH, along with the corresponding secondary antibodies (all from Abcam, Cambridge, MA, USA), was performed prior to analysis of the enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
To investigate the localization of HAND1-AS1 in cells, we resorted to the FISH method. For the FISH assay, the HAND2-AS1 RNA probe was produced by RiboBio. After treating MCF-7 and SK-BR-3 cells with Hoechst, stained cells were photographed by a laser scanning confocal microscope (ZEISS, Jena, Germany).
To investigate the interactions between RNAs involved, we performed RNA pull-down assay. The miR-3118-WT, miR-3118-Mut, and NC were biotin-labeled to Bio-miR-3118-WT, Bio-miR-3118-Mut, and Bio-NC by RiboBio, then incubated with cell lysates and Dyna-beads (Invitrogen). After washing, the bound RNAs were assayed by RT-qPCR.
StarBase (
In light of the guidebook of Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA),
6 weeks old of male nude mice were procured from the National Laboratory Animal Center (Beijing, China) and maintained in SPF animal laboratory, with the approval of the Animal Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. MCF-7 cells transfected with pcDNA 3.1/HAND2-AS1 or pcDNA 3.1 were injected to mice subcutaneously for 28 days to form in vivo models of breast cancer, with tumor volume recorded every 4 days. The tumor samples from killed mice were weighed for further analysis.
The tumor tissue samples from in vivo study were fixed by 4% paraformaldehyde and embedded in paraffin. After cutting, the sections of 4
All assays included three biological repeats. Data were processed by Student’s test to compare between two groups, and ANOVA (one-way) was adopted when three or more groups were included in data in GraphPad PRISM 6 (GraphPad, San Diego, CA, USA), with
The data used to support the findings of this study are available from the corresponding authors upon request.
The authors declare that they have no conflicts of interest.
The study was funded by the Key Task Project of Tianjin Health and Family Planning Commission (16KG128), Anticancer Key Technologies R&D Program of Tianjin (12ZCDZSY16200), Natural Science Foundation of Tianjin (18JCYBJC91600), and National Natural Science Foundation of China (81672623).