PTENP1/miR-20a/PTEN axis contributes to breast cancer progression by regulating PTEN via PI3K/AKT pathway

Abstract

Background: Long non-coding RNA PTENP1, the pseudogene of PTEN tumor suppressor, has been reported to exert its tumor suppressive function via modulation of PTEN expression in many malignancies, including breast cancer (BC). However, whether the PTENP1/miR-20a/PTEN axis exists and how it functions in BC progression remains elusive.

Methods: The levels of PTENP1, PTEN and miR-20a were measured by qRT-PCR. Furthermore, the breast cancer cells proliferation was further measured by CCK8 assay, colony formation assays, EDU and Ki67 staining. The migratory and invasive ability was determined by transwell assay. Flow cytometry, JC-1 and TUNEL assays were conducted to show the occurrence of apoptosis. Xenograft model was used to show the tumorigenesis of breast cancer cells.

Results: We analyzed PTENP1 and PTEN levels in clinical BC samples and cell lines, and found that PTENP1 and PTEN were confirmed and closely correlated with the malignancy of BC cell lines and poor clinical prognosis. Moreover, alteration of PTENP1 affects BC cell proliferation, invasion, tumorigenesis and chemoresistance to adriamycin (ADR). Bioinformatic analysis and dual-luciferase reporter gene assay predicted that PTENP1 was a direct target of miR-20a, which was clarified an alternative effect on BC aggressiveness phenotype. In addition, PTENP1 functioned as an endogenous sponge of miR-20a to regulate PTEN expression, which mediated BC cells proliferation, invasion and drug resistance via activation the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. PI3K inhibitor LY294002 or siAkt also prevented BC cells progression.

Conclusion: Collectively, these data indicated that PTENP1/miR-20a/PTEN axis involved in the malignant behaviors of BC cells, illuminating the possible mechanism mediated by PTEN via PI3K/Akt pathway. Targeting PTENP1/miR-20a/PTEN may provide a potential diagnosis and treatment strategy for BC.

Keywords: Breast cancer; PI3K/AKT pathway; PTEN; PTENP1; miR-20a.

Fig. 1

PTENP1 and PTEN are concomitantly downregulated in BC tissues and cell lines. a Higher PTENP1 was observed in the nontumor tissues than BC tissues. PTEN was downregulated in BC tissues. b The levels of PTENP1 and PTEN were identified in MCF-10A and BC cell lines. c Relative PTENP1 and PTEN levels showed lower tendency in advanced stages. d The overall survival curves (OS) of PTENP1 and PTEN were presented. e PTENP1 expression was extremely higher with transfection of PTENP1 compared to the control groups. f Decreased PTENP1 level was detected in the transfected MCF-7 and T47D cells. Data are the means ± SD of triplicate determinants (*P < 0.05)

Fig. 2

Overexpression of PTENP1 suppresses BC progression. a CCK8 assays were conducted to detect the growth of MDA-MB-231 and MCF-7/ADR cells. b The inhibitory proliferative effect of PTENP1 on MDA-MB-231 and MCF-7/ADR cells was determined by colony formation assay. c The proliferation of BC cells was measured by Edu staining. Green fluorescence: Edu, blue fluorescence: DAPI (Scale bar = 20 μm). d Ki67 staining also showed the repressed proliferation of PTENP1 transfected BC cells. Red fluorescence: Ki67, blue fluorescence: DAPI (Scale bar = 20 μm). e The migration and invasion of transfected MDA-MB-231 cells were suppressed by transwell experiment (Scale bar = 20 μm). f The chemoresistance to ADR of MCF-7/ADR cells was assessed by CCK8 assays. g The IC₅₀ value was computed in the transfected MCF-7/ADR cells. h With ADR treatment, the colony formation of PTENP1 transfected MCF-7/ADR cells was more inhibited. i With ADR treatment, the apoptotic rate was determined by flow cytometry. j The mitochondrial membrane potential was measured by JC-1 staining. Green fluorescence: the monomer, red fluorescence: the J-aggregates, orange fluorescence: merged photo (Scale bar = 20 μm). k The occurrence of apoptosis was further confirmed by TUNEL assay (Scale bar = 200 μm). l The expression of caspase-related apoptotic molecules was determined by western blot. m The tumorigenesis was detected by xenograft model. The nude mice treated with PTENP1 revealed smaller tumor volume compared to the control group. The tumor was further inhibited in response to ADR treatment. n The levels of PTEN and Ki67 were determined by IHC staining. Data are the means ± SD of triplicate determinants (*P < 0.05), (Scale bar = 200 μm)

Fig. 3

Low PTENP1 level enhances the malignant behavior of BC cells. a The viability of transfected BC cells were detected by CCK8 assays at 0, 24, 48,72, 96 h. b Knockdown of PTENP1 enhanced the colony formation in BC cells. c The proliferation of siPTENP1 transfected cells was increased by Edu staining (Scale bar = 20 μm). d Ki67 staining also showed intensive proliferation (Scale bar = 20 μm). e The aggressiveness was enhanced with knocking down PTENP1 in MCF-7 cells (Scale bar = 20 μm). f The siPTENP1-MCF-7 cells revealed more resistance to ADR. g Higher IC₅₀ value was also proved the enhanced chemoresistance to ADR. h Weakened colony formation ability was shown in response to ADR. i More resistance to ADR was shown in siPTENP1-MCF-7 cells. Low apoptosis rate was detected by flow cytometry. j JC-1 staining assay showed altered mitochondrial membrane potential with siPTENP1 transfection. Green fluorescence: the monomer, red fluorescence: the J-aggregates, orange fluorescence: merged photo (Scale bar = 20 μm). k TUNEL assay confirmed the incidence of apoptosis (Scale bar = 200 μm). l Apoptosis-related molecules expression was determined by western blot. m The xenografted tumors were presented with or without ADR treatment. n PTEN and Ki67 levels were determined by IHC staining. Data are the means ± SD of triplicate determinants (*P < 0.05) (Scale bar = 200 μm)

Fig. 4

PTENP1 is a direct target of miR-26a and a positive regulator of PTEN. a Relative miR-20a expression was identified by qRT-PCR between BC tumor tissues and corresponding nontumor tissues. b Relative miR-20a expression was detected in MCF-10A and BC cell lines. c The negative correlation between PTENP1 and miR-20a was analyzed. d The predicted sequence aligment was shown, and dual-luciferase reporter assay confirmed the direct binding between PTENP1 and miR-20a. e Overexpressed miR-20a enhanced PTENP1 level in transfected MCF-7 and T47D cells. f MiR-20a inhibitor promoted PTENP1 expression in transfected MDA-MB-231, MCF-7/ADR and T47D/ADR cells. g The co-precipitated RNA was identified by RNA immunoprecipitation experiment. PTENP1 and miR-20a were shown as fold enrichment in Ago2. h The negative correlation was confirmed by Spearman’s correlation analysis. i The predicted binding sites were presented. The directed binding was confirmed by dual-luciferase reporter assay. j Downregulation of PTEN was detected in miR-20a mimic transfected MCF-7 and T47D cells. k Inhibition of miR-20a increased PTEN expression in MDA-MB-231, MCF-7/ADR and T47D/ADR cells. Data are the means ± SD of triplicate determinants (*P < 0.05)

Fig. 5

MiR-20a reverses the tumor suppressive function of PTENP1 by regulating PTEN expression in BC progression. a Relative PTEN expression was measured by qRT-PCR in the cells transfected with miR-20a mimic or PTENP1. b PTEN protein level was detected by western blot. c The variety of growth rates were confirmed by CCK8 assay. d Colony formation assay was conducted to show the proliferative formation with different treated BC cell lines. e The altered migration and invasion were detected by transwell assay (Scale bar = 20 μm). f The chemoresistance to ADR was identified by CCK8 assay. g The IC₅₀ values of different groups were calculated. h The colony formation was shown in MCF-7/ADR cells in response to ADR. i The altered apoptotic rate was detected by flow cytometry. Data are means ± SD of three independent assays (*P < 0.05)

Fig. 6

Inhibition of miR-20a reverses the promotional effect of siPTENP1 by mediating PTEN expression in BC progression. a PTEN mRNA expression was identified with the treatment of miR-20a inhibitor or siPTENP1. b PTEN protein level was detected by western blot. c The proliferation was measured by CCK8 assays. d Colony formation assay was used to measure the colony formation of transfected cells. e The aggressiveness was determined by transwell assay (Scale bar = 20 μm). f CCK8 assays were carried out to assess the chemoresistance to ADR with different treated BC cells. g IC₅₀ values were calculated in differential treated MCF-7 cells. h In response to ADR, the colony formation was measured in transfected MCF-7 cells. i The AnnexinV and PI staining was used to determine the occurrence of apoptosis. Data are means ± SD of three independent assays (*P < 0.05)

Fig. 7

PTENP1/miR-20a/PTEN axis activates PI3K/Akt pathway in PI3K/AKT- mediated BC cell progression. a The main molecules of PI3K/Akt pathway were determined by western blot after transfection with miR-20a mimic or PTENP1. b The main molecules of PI3K/Akt pathway were measured with treatment of miR-20a inhibitor or siPTENP1. c The PI3K/Akt pathway was repressed by LY294002 and Akt siRNA. d Using colony formation assay and transwell assay, the attenuated proliferation and aggressiveness were shown in the cells transfected with LY294002, Akt siRNA or combination of siAkt and PTENP1. Data are means ± SD of three independent assays (*P < 0.05) (Scale bar = 20 μm)