Long Noncoding-RNA Component of Mitochondrial RNA Processing Endoribonuclease Promotes Carcinogenesis in Triple-Negative Breast Cancer Cells via the Competing Endogenous RNA Mechanism

Abstract

Purpose: Triple-negative breast cancer (TNBC) is a subtype of breast cancer. Increasing evidence supports that dysregulation of long noncoding RNAs (lncRNAs) plays a vital role in cancer progression. RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a lncRNA, is characterized as a tumor-propeller in some cancers, but its mechanism in TNBC remains poorly understood. This study aimed to determine whether and how RMRP functions in TNBC.

Methods: Cell proliferation was determined by cell counting kit-8 (CCK-8) and colony formation assays and cell apoptosis by flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Cell migration and invasion were determined by transwell assays. RNA-binding protein immunoprecipitation (RIP), luciferase reporter, and RNA pulldown assays were implemented to assess the interaction of RMRP with other molecules in TNBC cells.

Results: RMRP expression was elevated in TNBC cells. RMRP knockdown repressed cell proliferation, migration, and invasion, but induced apoptosis in TNBC. In addition, RMRP was found to target microRNA-766-5p (miR-766-5p) in TNBC cells. Silencing miR-766-5p enhanced cell viability and decreased apoptosis, whereas miR-766-5p overexpression had opposite effects. Furthermore, miR-766-5p was found to bind to yes-associated protein 1 (YAP1). Moreover, miR-766-5p inhibition reversed the repressive effect of RMRP knockdown on the malignant progression of TNBC.

Conclusion: The present study manifested that RMRP promotes the growth, migration, and invasion of TNBC cells via the miR-766-5p/YAP1 axis. These findings provide novel perspectives for TNBC treatment.

Keywords: Breast neoplasms; MicroRNAs; RNA, untranslated; Triple negative breast neoplasms.

Figure 1. Inhibition of cell proliferation, migration, and invasion and promotion of cell apoptosis in TNBC by RMRP knockdown. (A) Relative expression of RMRP in TNBC (MDA-MB-231, MDA-MB-436, and BT-549) and MCF-10A cell lines was detected by qPCR. (B) RMRP expression was verified by qPCR in TNBC cells transfected with sh-RMRP#1/2/3 versus sh-NC. (C) CCK-8 assay detected TNBC cell viability following the transfection with sh-RMRP#1/2/3 versus sh-NC. (D) Quantification of colonies of TNBC cells transfected with sh-RMRP#1/2/3 versus sh-NC. (E) Ratio of TUNEL-positive TNBC cells in sh-RMRP#1/2/3 groups versus the sh-NC group. (F) Flow cytometry analysis was implemented to analyze apoptosis of TNBC cells in sh-RMRP#1/2/3 groups versus the sh-NC group (left panel); flow cytometry analysis was conducted within 24 hours (right panel). (G) The migrated and invaded TNBC cells in sh-RMRP#1/2/3 groups relative to the sh-NC group in the Transwell system. Data were analyzed with 1-way analysis of variance and Dunnett's test.

GAPDH = glyceraldehyde 3-phosphate dehydrogenase; TNBC = triple-negative breast cancer; sh-NC = negative control of short hairpin RNA; OD = optical density; RMRP = RNA component of mitochondrial RNA processing endoribonuclease; CCK-8 = cell counting kit-8; TUNEL = terminal deoxynucleotidyl transferase-mediated nick end labeling; qPCR = quantitative polymerase chain reaction; N.S. = no significance. ^*p < 0.05, ^†p < 0.01.

Figure 2. MiR-766-5p as a downstream target of RMRP. (A) Subcellular fractionation demonstrated the distribution of RMRP in TNBC cells (Student's t-test). GAPDH and U6 served as cytoplasmic and nuclear references, respectively. (B) Subcellular localization of RMRP in TNBC cells was detected by fluorescence in situ hybridization. Fluorescent stain, ×1,000. (C) Seven potential miRNAs were screened using starBase. (D) RNA pulldown assay was performed followed by qPCR (1-way ANOVA and Tukey's test). Bio-RMRP was used to pull down candidate miRNAs. (E) RIP assay was performed with IgG or SNRNP70 as NC or positive control, respectively. Enrichment of miR-766-5p in AGO2 RIP products relative to IgG control was analyzed by qPCR (Student's t-test). (F) The sequence of RMRP-WT and the sequence of RMRP-Mut are shown. (G) The relative luciferase activities in TNBC cells co-transfected with RMRP-WT or RMRP-Mut and miR-766-5p mimics or NC mimics were detected by luciferase reporter assays (2-way ANOVA and Tukey's test).

RMRP = RNA component of mitochondrial RNA processing endoribonuclease; DAPI = 4′,6-diamidino-2-phenylindole; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; Bio = biotinylated; NC = negative control; Anti-IgG = antibody against immunoglobulin G; Anti-AGO2 = antibody against argonaute 2; Anti-SNRNP70 = antibody against small nuclear ribonucleoprotein U1 subunit 70; WT = wild-type; Mut = mutant; RMRP-WT = RMRP with WT miR-766-5p binding sites; RMRP-Mut = RMRP with Mut miR-766-5p site; TNBC = triple-negative breast cancer; miRNA = microRNA; ANOVA = analysis of variance; RIP = RNA-binding protein immunoprecipitation. ^*p < 0.05, ^†p < 0.01.

Figure 3. Inhibition of TNBC cell proliferation, migration, and invasion and promotion of cell apoptosis by miR-766-5p. (A) Quantitative polymerase chain reaction data of miR-766-5p in TNBC cells transfected with the miR-766-5p inhibitor versus NC inhibitor or miR-766-5p mimics versus NC mimics (1-way analysis of variance and Tukey's test). (B-D) CCK-8 and colony formation assays were carried out to analyze the effect of miR-766-5p overexpression and knockdown on cell proliferation (Student's t-test). (E, F) The apoptosis of TNBC cells was analyzed via TUNEL assay and flow cytometry when miR-766-5p was overexpressed or silenced in TNBC cells (Student's t-test). (G, H) Evaluation of TNBC cell migratory and invasive capacities by Transwell and Matrigel assays in the miR-766-5p inhibitor group versus the NC inhibitor group or the miR-766-5p mimics group versus the NC mimics group (Student's t-test).

TNBC = triple-negative breast cancer; NC = negative control; OD = optical density; CCK-8 = cell counting kit-8; TUNEL = terminal deoxynucleotidyl transferase-mediated nick end labeling; N.S.: not significant. ^*p < 0.01.

Figure 4. Upregulation of YAP1 in TNBC cells by RMRP via miR-766-5p. (A) Target mRNAs processing binding sites of miR-766-5p were predicted using starBase. (B) YAP1 was predicted using DAVID 6.8. (C) qPCR was performed to determine the expression of YAP1 in cells transfected with miR-766-5p mimics versus NC mimics (Student's t-test). (D) Western blot analysis was employed to examine the protein expression of YAP1 in TNBC cells in the miR-766-5p mimics group versus the NC mimics group. (E) RIP assay and qPCR determined the enrichment of RMRP, miR-766-5p, and YAP1 in the binding complex with AGO2 compared to that with IgG (Student's t-test). IgG or small nuclear ribonucleoprotein U1 subunit 70 were used as negative control or positive control, respectively. (F) The sequence of binding sites in YAP1-3′UTR for miR-766-5p was predicted. (G) YAP1-WT/Mut or the empty reporter vector pmirGLO were co-transfected with miR-766-5p mimics or NC mimics into TNBC cells (2-way ANOVA and Tukey's test) and luciferase activities of each group were measured. (H, I) mRNA and protein expression of YAP1 in the indicated TNBC cells with indicated transfections were analyzed by qPCR (1-way ANOVA and Tukey's test) or western blot analysis.

NC = negative control; YAP1 = yes-associated protein 1; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; Anti-IgG = antibody against immunoglobulin G; Anti-AGO2 = antibody against argonaute 2; RMRP = RNA component of mitochondrial RNA processing endoribonuclease; ANOVA = analysis of variance; qPCR = quantitative polymerase chain reaction; YAP1-WT = WT YAP1-3′UTR reporter vectors; YAP1-Mut = Mut YAP1-3′UTR reporter vectors. ^*p < 0.01.

Figure 5. Aggravation of proliferative, migratory, invasive capacities and suppression of apoptosis of triple-negative breast cancer cells by RMRP via miR-766-5p. (A) The decreased cell proliferation caused by sh-RMRP#2 was increased by the miR-766-5p inhibitor. (B) The decreasing number of colonies induced by RMRP knockdown was restored by miR-766-5p inhibition. (C) The increasing number of TUNEL-positive cells induced by RMRP knockdown markedly decreased following miR-766-5p inhibition. (D) Flow cytometry analysis was conducted to analyze cell apoptosis after the indicated transfection. (E) Transwell assays were performed to analyze cell migration and invasion capacities after RMRP knockdown and/or miR-766-5p inhibition.

OD = optical density; sh-NC = negative control of short hairpin RNA; RMRP = RNA component of mitochondrial RNA processing endoribonuclease; NC = negative control; TUNEL = terminal deoxynucleotidyl transferase-mediated nick end labeling; N.S.: not significant. ^*p < 0.01 was based on 1-way analysis of variance and Tukey's test.