Long noncoding RNA PPP1R14B antisense RNA 1 (PPP1R14B-AS1) has emerged as a critical modulator of liver cancer and lung adenocarcinoma progression. However, the functional importance and biological relevance of PPP1R14B-AS1 in breast cancer remain unclear. Therefore, this study was designed to detect PPP1R14B-AS1 levels in breast cancer cells using qRT–PCR and elucidate the influence of PPP1R14B-AS1 on aggressive phenotypes. Furthermore, molecular events mediating the action of PPP1R14B-AS1 were characterized in detail. Functional experiments addressed the impacts of PPP1R14B-AS1 knockdown on breast cancer cells. In this study, PPP1R14B-AS1 was found to be overexpressed in breast cancer, exhibiting a close correlation with poor patient prognosis. Results also showed that breast cancer cell proliferation and motility were suppressed when PPP1R14B-AS1 was silenced. Mechanistically, PPP1R14B-AS1 acted as a competing endogenous RNA for microRNA-134-3p (miR-134-3p) in breast cancer cells. PPP1R14B-AS1 also increased LIM and SH3 protein 1 (LASP1) levels by imitating miR-134-3p in breast cancer cells. Rescue experiments further corroborated that the knockdown of miR-134-3p or an increase in LASP1 restored the aggressive malignant characteristics of breast cancer cells that were weakened by PPP1R14B-AS1 depletion. In summary, PPP1R14B-AS1 facilitated the oncogenicity of breast cancer cells by controlling the miR-134-3p/LASP1 axis. We believe that our findings may contribute to the development of precision therapy techniques in the field of breast cancer treatment.
Breast cancer ranks as the most frequently diagnosed human cancer and the leading cause of death among women worldwide [
Long noncoding RNAs (lncRNAs) are characterized as a group of RNA transcripts containing over 200 nucleotides [
MicroRNAs (miRNAs) are short single-stranded noncoding RNA transcripts typically composed of 17–22 nucleotides [
Alternatively, PPP1R14B-AS1 has emerged as a critical controller of liver cancer and lung adenocarcinoma progression [
The Ethics Committee of The First Hospital of Jilin University approved our research. First, breast cancer tissues and adjacent healthy tissues were obtained from 41 patients in our hospital with breast cancer. The inclusion criteria were as follows: (i) patients diagnosed with breast cancer; (ii) those previously treated with radiotherapy, chemotherapy, or other types of anticancer treatments; and (iii) those who agreed to participate in the research. The exclusion criteria were as follows: (i) patients receiving radiotherapy or chemotherapy, (ii) those with other types of cancer, and (iii) those who refused to participate in the research. RNALater™ RNA Stabilization Reagent (Beyotime; Shanhgai, China) was used, and all clinical samples were stored in liquid nitrogen until further use. Additionally, all participants signed informed consent forms before they were enrolled in the study.
A normal human immortalized breast epithelial cell line MCF-10A (ATCC, Rockville, MD, USA) was cultured in MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM (Lonza/Clonetics Corporation, Walkersville, MD, USA) that was supplemented with 100 ng/ml cholera toxin. The Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) is the provider of all breast cancer cell lines. Cell lines BT-549 was maintained in Roswell Park Memorial Institute Medium 1640 containing 10% fetal bovine serum (FBS), while 10% FBS-supplemented L-15 medium (all from Gibco, Grand Island, NY, USA) was used for the culture of cell lines MDA-MB-468 and MDA-MB-231. Minimum Essential Medium (Gibco) was added with 1% Glutamax, 1% non-essential amino acids, 1% sodium pyruvate 100 mM solution for culturing MCF-7 cells. All cells were cultured at 37°C in a humidified incubator under 5% CO2.
For gene silencing studies, small interfering RNA (siRNA) against PPP1R14B-AS1 (si-PPP1R14B-AS1) and negative control siRNA (si-NC) were bought from GenePharma (Shanghai, China). Then, for high mobility group box 3 (LASP1) overexpression plasmid generation, LASP1 sequences were inserted into the pcDNA3.1 + vector, yielding the pcDNA3.1-LASP1 plasmid. MiR-134-3p mimic, NC mimic, miR-134-3p inhibitor (anti-miR-134-3p), and NC inhibitor (anti-NC) were all designed and synthesized by RiboBio (Guangzhou, China). Furthermore, lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for transfection experiments.
Total RNA was isolated using TRIzol (Invitrogen). The reverse transcription step was performed using miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen, Beijing, China) to determine miRNA expression. Next, the miRcute miRNA qPCR Detection Kit (Tiangen) was employed for the quantification of miRNA.
For detecting PPP1R14B-AS1 and LASP1 levels, the PrimeScript™ RT reagent kit and TB Green® Premix Ex Taq™ II (both from Takara, Dalian, China) were employed for reverse transcription and PCR amplification. Furthermore, U6 and GAPDH were used to normalize miRNA and mRNA (lncRNA) expression, respectively. Finally, all data were analyzed using the 2−ΔΔCt method.
Nuclear and cytosolic RNAs were isolated from breast cancer cells using a cytoplasmic and nuclear RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada). qRT–PCR was then used to determine the relative abundance of PPP1R14B-AS1 in nuclear and cytosolic RNAs.
Transfected cells were inoculated into 96-well plates at a density of 2 × 103 cells per well. Each group contained five replicate wells. After the cells were cultivated for different time periods, 10 µl of the CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added, and the cells were incubated at 37°C for an additional 2 h. Subsequently, a microplate reader was used to determine the absorbance at 450-nm wavelength. Next, the average absorbance value for each group of replicate wells was analyzed.
For colony formation assay, 2 ml culture medium containing 500 cells was added into 6-well plates. Subsequent to 2 weeks cultivation at 37°C with 5% CO2, the newly formed colonies were fixed with 4% paraformaldehyde, followed by staining with 0.1% crystal violet. After extensive washing, the number of colonies was counted applying an inverted light microscope (Olympus, Tokyo, Japan).
Transwell chambers (8-μm pore size) precoated with Matrigel® (BD Biosciences, Franklin Lakes, NJ, USA) were used to detect cell invasive capacity. Briefly, a cell suspension was produced using a serum-free culture medium. Then, while 600 µl of 10% FBS-containing culture medium was seeded into the lower compartments, the upper chambers were inoculated with 200 μl of cell suspension containing 5 × 104 cells. Subsequently, the transferred cells were cultivated at 37°C for the whole day. After removing the noninvading cells, 4% paraformaldehyde and 0.1% crystal violet were used to immobilize and stain the invading cells. Next, after extensive washing, the invading cells were viewed and counted under an inverted light microscope. The migration experiment was performed in accordance with the abovementioned experimental procedures, except that Matrigel® was not used.
BALB/C female nude mice aged 4–6 weeks (SLAC Laboratory Animal, Co., Ltd., Shanghai, China) were employed for developing xenograft tumors. First, mice were injected subcutaneously with 1 × 106 MCF-7 cells with stable sh-PPP1R14B-AS1 or sh-NC expression. Each group had three nude mice. Then, tumor width (W) and length (L) were recorded every 5 days, and the acquired data was used for calculating tumor volume using the following formula: V = 0.5 × L × W2. On day 30, all mice were euthanized using the cervical dislocation method. Next, the xenograft tumors were resected from nude mice, weighed, and stored until further analysis.
PPP1R14B-AS1 expression in breast invasive carcinoma from TCGA samples was analzyed utilizing UALCAN (
GenePharma amplified PPP1R14B-AS1 and LASP1 fragments carrying the wild-type (wt) miR-134-3p binding sequences and inserted them into a psiCHECK™-2 vector (Promega, Madison, WI, USA), generating the PPP1R14B-AS1-wt and LASP1-wt reporter plasmids. Subsequently, the PPP1R14B-AS1-mutant (PPP1R14B-AS1-mut) and LASP1-mut reporter plasmids were yielded in the same way. Then, reporter plasmids and miR-134-3p mimic or NC mimic were introduced into breast cancer cells, followed by a 48-h incubation at 37°C. Finally, the activity of the reporter was assayed using a Dual-Luciferase Reporter Assay System (Promega).
Breast cancer cells were collected and lysed in an equal volume of RIP buffer. The assay was performed using a Millipore Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Merck-Millipore, Bedford, MA, USA). First, a 100 µl aliquot of cell lysate was added to 900 μL of RIP immunoprecipitation buffer, followed by further cultivation overnight at 4°C with magnetic beads conjugated with human anti-Ago2 or control anti-IgG antibodies (Merck-Millipore). After digestion with proteinase K, the immunoprecipitated RNA was further analyzed using qRT–PCR.
RIPA reagent (Sigma, St. Louis, MO, USA) was employed for total protein isolation. First, after quantification using bicinchoninic acid protein assay kit (Beyotime), proteins were electrophoresed using a 10% SDS–PAGE electrophoresis protocol. Then, separated proteins were transferred to polyvinylidene difluoride membranes before blocking them with 5% nonfat dried milk. Thereafter, primary antibodies against LASP1 (ab156872; Abcam, Cambridge, UK) and GAPDH (ab204481; Abcam) were added, and the solution was incubated overnight at 4°C. Next, tris-buffered saline with Tween buffer was used to clean the membranes and then HRP-conjugated secondary antibody (ab205718; Abcam) was applied and incubated at room temperature for 2 h. Finally, protein signals were detected using an ECL Western blotting Substrate Kit (Abcam).
Functional experiments were repeated three times independently (obtained data are shown as the mean ± standard deviation). A normality test was performed utilizing Shapiro–Wilk normality test. The overall survival of the patients with breast cancer was estimated using Kaplan–Meier analysis and compared using the log-rank test. All statistical evaluations were also conducted using the Student’s
First, utilizing TCGA database, we found that PPP1R14B-AS1 ranks 16th among overexpressed lncRNAs in breast invasive carcinoma (BRCA;
Therefore, to evaluate the effect of PPP1R14B-AS1 on breast cancer cells, MDA-MB-468 and MCF-7 cell lines were chosen for functional assays and transfected with si-PPP1R14B-AS1 to lower PPP1R14B-AS1 expression. Then, qRT–PCR was used to verify the successful knockdown of PPP1R14B-AS1 by si-PPP1R14B-AS1 (
To decipher the molecular events by which PPP1R14B-AS1 exerts its cancer-promoting activity, subcellular localization analysis was performed to test the location of PPP1R14B-AS1 in breast cancer cells. Results proved that PPP1R14B-AS1 was abundant in the nucleus and cytoplasm, with the latter harboring a larger portion (
hsa-miR-8063 | hsa-miR-6715b-5p | hsa-miR-4269 |
hsa-miR-4774-3p | hsa-miR-3135b | hsa-miR-3168 |
hsa-miR-12119 | hsa-miR-765 | hsa-miR-4303 |
hsa-miR-6811-5p | hsa-miR-4318 | hsa-miR-11181-3p |
hsa-miR-4519 | hsa-miR-134-3p | hsa-miR-6511b-5p |
The predicted binding site between miR-134-3p and PPP1R14B-AS1 is presented in
Since miR-134-3p was downregulated in breast cancer cells, its detailed effects on breast cancer cell behavior were also examined. The upregulation of miR-134-3p by miR-134-3p mimic (
Using bioinformatics analysis, LASP1 was predicted as a candidate molecule that regulates miR-134-3p (
Since we identified a relationship between miR-134-3p; its upstream regulator, PPP1R14B-AS1; and its downstream target, LASP1, the following experiments were performed to uncover the regulatory associations between PPP1R14B-AS1, miR-134-3p, and LASP1 in breast cancer cells. The absence of PPP1R14B-AS1 led to the downregulation of LASP1 levels in breast cancer cells. However, such regulatory activity was counteracted by the cotransduction of anti-miR-134-3p (
The contribution of the miR-134-3p/LASP1 axis toward the oncogenic roles of PPP1R14B-AS1 in breast cancer cells was evaluated using rescue experiments. Anti-miR-134-3p decreased miR-134-3p levels (
Finally, a subcutaneous tumor was generated in nude mice to investigate the carcinogenic roles of PPP1R14B-AS1 in breast cancer cells. Notably, the growth of xenograft tumors formed by MCF-7 cells stably expressing sh-PPP1R14B-AS1 was hindered compared to the growth of those formed by MCF-7 cells expressing sh-NC (
It has already been established that lncRNAs are important regulators and control complex cellular behavior in breast cancer [
High PPP1R14B-AS1 expression has been reported in patients with liver cancer and lung adenocarcinoma [
Importantly, our research further elucidated the mechanisms underlying the aggravation of breast cancer malignancy by PPP1R14B-AS1. LncRNAs perform their biological functions by working together with different molecules. Furthermore, cytosolic lncRNAs can competitively sequester miRNAs through miRNA response elements, which decreases the availability of miRNAs and indirectly increases the levels of target mRNAs, thus functioning as a ceRNA [
The use of a bioinformatics database revealed that miR-134-3p contains complementary binding sequences for PPP1R14B-AS1. Prominently, luciferase reporter and RIP assays demonstrated the direct binding between PPP1R14B-AS1 and miR-134-3p in breast cancer cells. Moreover, results provided sufficient evidence to identify LASP1 as a direct target of miR-134-3p. Furthermore, PPP1R14B-AS1 positively regulated LASP1 in breast cancer cells by imitating miR-134-3p. Similarly, PPP1R14B-AS1, miR-134-3p, and LASP1 existed in the same RNA-induced silencing complex. In brief, PPP1R14B-AS1 can imitate miR-134-3p and antagonize the inhibitory effect of miR-134-3p on LASP1 in breast cancer cells, consequently constructing a novel PPP1R14B-AS1/miR-134-3p/LASP1 ceRNA pathway.
Aberrant expression of miR-134-3p was reported in several human cancer types [
The considerable functional repertoire of lncRNAs during cancer onset and progression supports the opportunities for their therapeutic targeting [
To summarize, our study highlights the importance of the interplay among PPP1R14B-AS1, miR-134-3p, and LASP1 in promoting the oncogenicity of breast cancer. Mechanistically, PPP1R14B-AS1 operates as a miR-134-3p sponge and relieves regulatory effect of miR-134-3p on LASP1 in breast cancer cells. Our observations may lead to the development of novel precision therapy techniques in the breast cancer treatment field.
Study conception and design: Limin Zhou and Tao Liu; data collection: Xin Guan and Yi Dong; analysis and interpretation of results: Limin Zhou and Lianbo Zhang; draft manuscript preparation: Limin Zhou and Tao Liu. All authors reviewed the results and approved the final version of the manuscript.
The Ethics Committee of The First Hospital of Jilin University approved our research.
Data available with the communication author and can be provided upon reasonable request.
The authors received no specific funding for this study.
The authors declare that they have no conflicts of interest to report regarding the present study.