lncRNA PCNAP1 predicts poor prognosis in breast cancer and promotes cancer metastasis via miR‑340‑5p‑dependent upregulation of SOX4

Abstract

The high metastatic rate of breast cancer is the significant cause of its poor prognosis. The long noncoding RNA (lncRNA) proliferating cell nuclear antigen pseudogene 1 (PCNAP1) plays important roles in the initiation and progression of cancers; however, its regulatory function and molecular mechanism in breast cancer metastasis remains unknown. Therefore, we investigated the roles of lncRNA PCNAP1 in breast cancer metastasis by modulating the microRNA (miR)‑340‑5p/SOX4 axis using quantitative real‑time PCR, in vivo mouse models, nucleo‑cytoplasmic separation, western blot analysis, scratch assays, Transwell assays, luciferase reporter assays and MS2‑RIP, in vitro and in vivo. lncRNA PCNAP1 was found to be upregulated in human breast cancer tissues, and high lncRNA PCNAP1 levels predicted poor overall survival. Function assays showed that knockdown of lncRNA PCNAP1 suppressed the migration and invasion of breast cancer cells in vitro and in vivo. Mechanistically, lncRNA PCNAP1 functioned as a competing endogenous (ce)RNA for miR‑340‑5p to facilitate the expression of its target gene SRY‑box transcription factor 4 (SOX4), promoting migration and invasion of breast cancer cells. Overall, we found that lncRNA PCNAP1 predicted a poor prognosis in breast cancer and promoted cancer metastasis via miR‑340‑5p‑dependent upregulation of SOX4 expression. These results suggest that lncRNA PCNAP1 has potential as an alternative therapeutic target to suppress breast cancer metastasis.

Keywords: lncRNA PCNAP1; miRNA‑340‑5p; SOX4; breast cancer; cancer metastasis.

Figure 1.

lncRNA PCNAP1 is upregulated in human BC tissues, and high lncRNA PCNAP1 levels predict poor overall survival. (A) RT-qPCR results showed that PCNAP1 was significantly increased in BC tissues compared with that in adjacent normal tissues. (B) Tumor samples from patients with metastasis displayed increased lncRNA PCNAP1 levels compared with those in patients without metastasis. (C) Samples were divided into low (below the median, n=35) and high (above the median, n=35) lncRNA PCNAP1 expression groups according to the mean level of lncRNA PCNAP1. (D and E) An increased level of lncRNA PCNAP1 was significantly associated with poor overall survival (HR 3.24; 95% CI 1.26–8.49; P=0.014). Results are representative of three independent experiments. Data are shown as mean ± SD. **P<0.01, ***P<0.001. BC, breast cancer; lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1.

Figure 2.

Knockdown of lncRNA PCNAP1 suppresses the migration and invasion of BC cells in vitro and in vivo. (A and B) Loss-of-function experiments showed that lncRNA PCNAP1 expression levels were markedly decreased following transfection with siPCNAP1#a and siPCNAP1#b compared with scrambled controls (siCtrl) in BC cells (by ~80 and ~70% in MDA-MB-231 and MCF7 cells, respectively). (C and D) Knockdown of lncRNA PCNAP1 had no significant effect on BC cell viability in MDA-MB-231 and MCF7 cells, as confirmed by CCK-8 assay results. (E-H) Loss of lncRNA PCNAP1 significantly decreased the speed of wound closure in BC cells (by ~64 and ~82% in MDA-MB-231 and MCF7 cells, respectively). (I-K) Transwell invasion assays showed that lncRNA PCNAP1 inhibition significantly reduced invasion. (L) Cells with lncRNA PCNAP1 knockdown (siPCNAP1#a and siPCNAP1#b) showed observably increased protein levels of epithelial marker E-cadherin, and decreased levels of mesenchymal marker N-cadherin. (M and N) Injection of siCtrl cells led to lung metastasis in BALB/c nude mice; these metastases were inhibited in nude mice injected with siPCNAP1-transfected MDA-MB-231 cells. (O) Lung metasasis tumor volume of nude mice. Results are representative of three independent experiments. (P) Changes in nude mouse body weight. Data are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, compared with the siCtrl group. BC, breast cancer; lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1.

Figure 3.

miR-340-5p has a target relationship with lncRNA PCNAP1 and is downregulated in BC. (A) lncRNA PCNAP1 at 80.1% was found in the cytoplasmic fraction of MCF7 cells. (B) The first four miRNAs predicted by the TargetScan software to bind to lncRNA PCNAP1 all directly targeted SOX4. (C) Predicted binding sites of miR-340-5p and lncRNA PCNAP1 according to miRcode. (D and E) miR-340-5p mimics inhibited the luciferase activity of lncRNA PCNAP1-WT by ~60% but did not affect the luciferase activity of lncRNA PCNAP1-MUT in MDA-MB-231 and MCF7 cells. **P<0.01, compared with NC. (F and G) Knockdown of lncRNA PCNAP1 significantly increased miR-340-5p expression in both MDA-MB-231 and MCF7 cell lines. **P<0.01, ***P<0.001, compared with the siCtrl group. (H) MS2-RIP followed by miRNA RT-qPCR was used to detect levels of miRNA-340-5p endogenously associated with lncRNA PCNAP1. **P<0.01. (I) miR-340-5p expression was significantly lower in BC tissues compared with that noted in the adjacent non-tumor tissues. **P<0.01. (J) miR-340-5p expression was significantly lower in metastatic BC tissues compared with non-metastatic tissues. ***P<0.001. Results are representative of three independent experiments. Data are shown as mean ± SD. BC, breast cancer; lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1; SOX4, SRY-box transcription factor 4; MUT, mutant; WT, wild-type.

Figure 4.

lncRNA promotes the migration of BC cells by downregulating miR-340-5p. (A and B) Transfection of miR-340-5p mimics significantly upregulated the expression of miR-340-5p in MDA-MB-231 and MCF7 cells compared with the miR-ctrl group, ***P<0.001, compared with the miR-ctrl group. Transfection of miR-340-5p inhibitor obviously downregulated the expression of miR-340-5p compared with the inhibitor NC group, indicating successful transfection. ***P<0.001, compared with the inhibitor NC group. (C and D) Overexpression of miR-340-5p had no significant effect on BC cell viability, as confirmed by CCK-8 assay results. (E-H) Overexpression of miR-340-5p mimics decreased the speed of the wound closure by ~66% in MDA-MB-231 cells and by ~72% in MCF7 cells (both **P<0.01, compared with the miR-ctrl group). (I-K) Transwell invasion assays showed that overexpression of miR-340-5p mimics could inhibit cell invasion in MDA-MB-231 and MCF7 cells compared with the negative control group. **P<0.01, compared with the miR-ctrl group. (L) Overexpression or inhibition of miR-340-5p had no influence on the expression of lncPCNAP1 in MCF7 cells. Results are representative of three independent experiments. Data are shown as mean ± SD. BC, breast cancer; lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1.

Figure 5.

SOX4 has a target relationship with miR-340-5p. (A) TargetScan Human, EV-miRNA, and miRTarBase jointly predicted 111 proteins directly targeted by miR-340-5p, including SOX4. (B) Predicted binding sites between miR-340-5p and SOX4 according to miRcode. (C and D) Luciferase reporter gene assay confirmed the target relationship between miR-340-5p and SOX4 in MDA-MB-231 and MCF7 cells. Results are representative of three independent experiments. Data are shown as mean ± SD. **P<0.01, compared with the NC group. SOX4, SRY-box transcription factor 4.

Figure 6.

lncRNA PCNAP1 promotes BC metastasis by downregulating miR-340-5p, then upregulating SOX4. (A) Immunohistochemistry results showed that SOX4 was significantly increased in BC tissues compared with that in adjacent normal tissues (magnification, ×100). (B) RT-qPCR validation of the immunohistochemistry results. **P<0.01, compared with adjacent tissues. (C) SOX4 expression was increased in metastatic BC tissues compared with non-metastatic tissues. ***P<0.001, compared with non-metastatic tissues. (D) There was a positive correlation between the expression levels of lncRNA PCNAP1 and SOX4 in metastatic tissues. (E-G) Knockdown of PCNAP1 (siPCNAP1#a and siPCNAP1#b) significantly suppressed the protein expression of SOX4 in MDA-MB-231 and MCF7 cells compared with the negative control group (siCtrl). ***P<0.001, compared with the siCtrl group. (H) There was a negative correlation between miR-340-5p and SOX4 expression in metastatic tissues. (I-K) miR-340-5p mimics decreased the protein expression of SOX4 compared with the miR-Ctrl group in MDA-MB-231 and MCF7 cells. ***P<0.001, compared with the miR-Ctrl group. In contrast, the miR-340-5p inhibitor increased the expression compared with the inhibitor NC group. ***P<0.001, compared with the inhibitor NC group. (L and M) Expression of SOX4 was upregulated by the overexpression plasmid. ***P<0.001, compared with the vector group. (N and O) Knockdown of lncRNA PCNAP1 decreased invasion which was restored by overexpression of SOX4. ***P<0.001, compared with the siCtrl group. Results are representative of three independent experiments. Data are shown as mean ± SD. BC, breast cancer; lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1; SOX4, SRY-box transcription factor 4.

Figure 7.

Mechanistic model of lncRNA PCNAP1 promoting breast cancer metastasis via miR-340-5p-dependent upregulation of SOX4. lncRNA, long noncoding RNA; PCNAP1, proliferating cell nuclear antigen pseudogene 1; SOX4, SRY-box transcription factor 4; UTR, untranslated region.