Long noncoding RNA PPP1R14B-AS1 imitates microRNA-134-3p to facilitate breast cancer progression by upregulating LIM and SH3 protein 1

Abstract

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.

Keywords: Precision therapy; Therapeutic target; ceRNA theory; miRNA sponge.

Figure 1. PPP1R14B-AS1 is overexpressed in breast cancer. (A) PPP1R14B-AS1 ranks 16^th among overexpressed lncRNAs in BRCA samples from TCGA dataset. (B) The TCGA database was analyzed to examine PPP1R14B-AS1 expression in BRCA tissue samples. (C) qRT–PCR results showing expression levels of PPP1R14B-AS1 in breast cancer tissue samples from our own cohort. (D) qRT–PCR results showing PPP1R14B-AS1 expression in different breast cancer cell lines. (E) The correlation between PPP1R14B-AS1 levels and survival rates of patients with breast cancer. ***p < 0.001 (n = 3).

Figure 2. PPP1R14B-AS1 silencing inhibits the malignant progression of breast cancer cells in vitro. (A) qRT–PCR results confirming the transfection efficiency of si-PPP1R14B-AS1. (B, C) CCK-8 and colony formation assays results revealing the change in the proliferation of breast cancer cells after si-PPP1R14B-AS1 transfection. (D, E) Effects of PPP1R14B-AS1 silencing on the migratory and invasive abilities of breast cancer cells. 100× magnification. **p < 0.01 and ***p < 0.001 (n = 3).

Figure 3. PPP1R14B-AS1 acts as an miR-134-3p sponge. (A) Relative distribution of PPP1R14B-AS1 in nuclear and cytosolic fractions of breast cancer cells was detected via subcellular localization analysis. (B) qRT–PCR results showing the levels of the seven candidate molecules in breast cancer cells after PPP1R14B-AS1 depletion. (C) The predicted miR-134-3p binding site within PPP1R14B-AS1. Mutated binding sequences are underlined. (D) Luciferase reporter assay results showing the luciferase activity of breast cancer cells upon cotransfection with miR-134-3p mimic or NC mimic and PPP1R14B-AS1-wt or PPP1R14B-AS1-mut. (E) The relationship between PPP1R14B-AS1 and miR-134-3p in breast cancer cells as assessed by the RIP assay. ***p < 0.001 (n = 3).

Figure 4. miR-134-3p suppresses the malignancy of breast cancer cells. (A) The efficiency of miR-134-3p mimic transfection into breast cancer cells was investigated. (B, C) The proliferation of miR-134-3p-overexpressing breast cancer cells was evaluated. (D) The migration and invasion of miR-134-3p-overexpressing breast cancer cells. 100× magnification. **p < 0.01 and ***p < 0.001 (n = 3).

Figure 5. Verification of LASP1 as a direct target of miR-134-3p. (A) The predicted miR-134-3p binding sequences within the 3′-UTR of LASP1. (B) A luciferase reporter assay showing the binding interaction between miR-134-3p and LASP1. (C, D) LASP1 expression in breast cancer cells was measured when miR-134-3p was overexpressed. ***p < 0.001 (n = 3).

Figure 6. PPP1R14B-AS1 indirectly regulated LASP1 expression by functioning as a ceRNA of miR-134-3p. (A, B) Breast cancer cells were transfected with si-PPP1R14B-AS1 in parallel with anti-miR-134-3p, followed by the measurement of LASP1 levels. (C) The relationship between PPP1R14B-AS1, miR-134-3p and LASP1 in breast cancer cells as assessed by the RIP assay. ***p < 0.001 (n = 3).

Figure 7. Anti-miR-134-3p or reintroduction of LASP1 counteracts the influence of si-PPP1R14B-AS1 on breast cancer cell proliferation. (A) miR-134-3p levels measured in breast cancer cells upon anti-miR-134-3p transfection. (B) LASP1 levels measured in breast cancer cells upon pcDNA3.1-LASP1 transfection. (C) Breast cancer cells were further treated with si-NC, si-PPP1R14B-AS1, si-PPP1R14B-AS1 + anti-NC, si-PPP1R14B-AS1 + anti-miR-134-3p, si-PPP1R14B-AS1 + pcDNA3.1, or si-PPP1R14B-AS1 + pcDNA3.1-LASP1. Cell proliferation change was determined after transfection. **p < 0.01 and ***p < 0.001 (n = 3).

Figure 8. The regulatory effect of si-PPP1R14B-AS1 on the colony formation and motility of breast cancer cells was reversed by lowering miR-134-3p or overexpressing LASP1 levels. (A) PPP1R14B-AS1-depleted breast cancer cells were further cotransfected with anti-miR-134-3p or pcDNA3.1-LASP1. After transfection, colony formation assay was implemented to assess colony formative ability. (B, C) Breast cancer cells were transfected with si-NC, si-PPP1R14B-AS1, si-PPP1R14B-AS1 + anti-NC, si-PPP1R14B-AS1 + anti-miR-134-3p, si-PPP1R14B-AS1 + pcDNA3.1, or si-PPP1R14B-AS1 + pcDNA3.1-LASP1. Cell migration and invasion was detected utilizing Transwell migration and invasion experiments. ***p < 0.001 (n = 3).

Figure 9. PPP1R14B-AS1 knockdown hinders tumor growth in vivo. (A) Images of xenograft tumors. (B) Tumor volume was recorded every 5 days to examine the growth of the tumors. (C) The weight of xenograft tumors. (D, E) PPP1R14B-AS1 and miR-134-3p levels in xenograft tumors. (F) The protein level of LASP1 in xenograft tumors. ***p < 0.001 (n = 3).