Pseudogene PTENP1 functions as a competing endogenous RNA to suppress clear-cell renal cell carcinoma progression

Abstract

PTENP1 is a pseudogene of the PTEN tumor suppression gene (TSG). The functions of PTENP1 in clear-cell renal cell carcinoma (ccRCC) have not yet been studied. We found that PTENP1 is downregulated in ccRCC tissues and cells due to methylation. PTENP1 and PTEN are direct targets of miRNA miR21 and their expression is suppressed by miR21 in ccRCC cell lines. miR21 expression promotes ccRCC cell proliferation, migration, invasion in vitro, and tumor growth and metastasis in vivo. Overexpression of PTENP1 in cells expressing miR21 reduces cell proliferation, invasion, tumor growth, and metastasis, recapitulating the phenotypes induced by PTEN expression. Overexpression of PTENP1 in ccRCC cells sensitizes these cells to cisplatin and gemcitabine treatments in vitro and in vivo. In clinical samples, the expression of PTENP1 and PTEN is correlated, and both expressions are inversely correlated with miR21 expression. Patients with ccRCC with no PTENP1 expression have a lower survival rate. These results suggest that PTENP1 functions as a competing endogenous RNA (ceRNA) in ccRCC to suppress cancer progression.

Figure 1

PTENP1 expression is downregulated in ccRCC. A, PTENP1 expression levels were evaluated by real-time PCR in nontumorigenic renal cell line HK-2, renal cancer cell lines, and paired case specimens. B, methylation-specific PCR was performed using primers specific for the unmethylated (U) or methylated (M) PTENP1 promoter region in the genomic DNA deriving from renal cell lines or renal cancer tissues. NC, unmethylated genomic DNA; PC, methylated genomic DNA. C, PTENP1 expression in 5 renal cancer cell lines after 5AZA-C or mock treatment. *, P < 0.05; **, P < 0.01. D, the expression of PTEN and PTENP1 in renal cancer tissues and normal control tissues. The expression of PTEN and PTENP1 is lower in cancer tissues than normal controls. The expression of PTEN and PTENP1 in cancer tissues includes positive correlation (P < 0.01; R² = 0.6374). E, PTEN protein levels were determined by immunoblot analysis in renal cancer cell line ACHN and SN12PM6 after being infected with Lenti-PTENP1 or Lenti-NC.

Figure 2

PTEN expression is regulated by miR21 in ccRCC. A, miR21 expression levels were evaluated by real-time PCR in nontumorigenic renal cell line HK-2, renal cancer cell lines and paired case specimens. B, luciferase signals of wild-type or mutant PTEN reporters in renal cancer cell line ACHN and SN12PM6. miR21 mimics or miR21 inhibitors or miR21 mimics and PTENP1 ormiR21 inhibitors and PTENP1 were cotransfected with the reporter in the cells (a). Sequences of PTEN 3′UTR, miR21, and mutant PTEN 3′UTR (b). The luciferase signals were suppressed by miR21 but partially rescued by PTENP1 expression. **, P < 0.01. C, PTEN expression in ACHN and SN12PM6 cells expressing miR21 mimics or miR21 inhibitors. PTEN expression is suppressed in cells expressing miR21 mimics, but increased in cells expressing miR21 inhibitors. PTENP1 expression rescued the suppression of PTEN expression by miR21.

Figure 3

PTENP1 expression is regulated by miR21 inccRCC. A, luciferase signals of wild-type or mutant PTENP1 reporters in renal cancer cell line ACHN and SN12PM6. miR21 mimics or miR21 inhibitors were cotransfected with the reporter in the cells (a). Sequences of PTENP1 3′UTR, miR21, and mutant PTENP1 3′UTR (b). The luciferase signals were suppressed by miR21. B, PTENP1 expression in ACHN and SN12PM6 cells expressing miR21 mimics or miR21 inhibitors. PTENP1 expression is suppressed in cells expressing miR21 mimics, but increased in cells expressing miR21 inhibitors. C, correlation analysis of the expression of PTENP1 andmiR21 in ccRCC tissues. The expression of PTENP1 andmiR21 is inversely correlated. D, correlation analysis of the expression of PTEN and miR21 in ccRCC tissues. The expression of PTENP1 and miR21 is inversely correlated. **, P < 0.01.

Figure 4

PTENP1 serves as a ceRNA in the regulation of tumor growth in RCC. A, cell proliferation in ACHN and SN12PM6 cells expressing miR21 or control. Cell viability was quantified at each time point. miR21 promoted cell growth. Data are plotted as the mean ± SEM of three independent experiments. B, cell proliferation in ACHN and SN12PM6 cells expressing PTEN or control. Cell viability was quantified at each time point. PTEN suppressed cell growth. C, cell proliferation in ACHN and SN12PM6 cells expressing vector control or PTENP1 ormiR21 ormiR21 and PTENP1. Cell viability was quantified at each time point. PTENP1 reversed the promotion of cell growth by miR21.D, we designed five groups in the animal models, and then ACHN cells expressing vector control or miR21 orPTEN,orPTENP1 orcoexpressingmiR21 andPTENPI were transplanted into mice. D, the size of transplanted tumors in different groups (a). Tumor weight of each mouse at the end of 41 days is indicated (b). Tumor volumes were determined at each time point (c). PTEN and PTENP1 suppressed tumor growth in vivo. miR21 promoted tumor growth in vivo. PTENP1 rescued the promotion of tumor growth by miR21 .These data mean PTENP1 serves as a ceRNA in the regulation of tumor growth in RCC. **, P < 0.01.

Figure 5

PTENP1 serves as a ceRNA in the regulation of cell migration and invasion in vitro and metastasis in vivo. A, ACHN and SN12PM6 cells expressing miR21 or control were subjected to migration and invasion assay. Representative photographs were taken at ×200 magnification. C, the number of migrated and invaded cells was quantified in 4 random images from each group. miR21 promoted cell migration and invasion. B, ACHN and SN12PM6 cells expressing PTEN or control were subjected to migration and invasion assay. Representative photographs were taken at ×200 magnification. The number of migrated and invaded cells was quantified in 4 random images from each group (C). PTEN suppressed cell migration and nvasion. D, ACHN and SN12PM6 cells expressing control or miR21 orPTENPI ormiR21 and PTENP1 were subjected to migration and invasion assay. Representative photographs were taken at ×200 magnification. E, the number of migrated and invaded cells was quantified in 4 random images from each group. PTENP1 rescued the promotion of cell migration and invasion by miR21. F, GFP-tagged ACHN cells expressing vector control or miR21 or PTEN or PTENP1 or miR21 and PTENP1 were injected into mouse tail veins. G, GFP signals in lung metastasis were determined by Xenogen bioluminescence system. PTEN and PTENP1 suppressed metastasis, whereas miR21 promoted metastasis. PTENP1 rescued the promotion of metastasis by miR21. **, P < 0.01.

Figure 6

PTENP1 sensitizes renal cancer cells to chemotherapy. A, SN12PM6 cells expressing vector control or PTENP1 were treated with cisplatin (a) and ACHN cells expressing vector control or PTENP1 were treated with cisplatin (b). Cell viability was determined at 24, 48, and 72 hours after treatment. Cell death increased in cells expressing PTENP1 than vector control. Data are plotted as the mean ± SEM of three independent experiments. B, SN12PM6 cells expressing vector control or PTENP1 were treated with gemcitabine (a) and ACHN cells expressing vector control or PTENP1 were treated with gemcitabine (b). Cell viability was determined at 24, 48, and 72 hours after treatment. Cell death increased in cells expressing PTENP1 than vector control. Data are plotted as the mean ± SEM of three independent experiments. C, determination of IC50 in ACHN and SN12PM6 cells with cisplatin or gemcitabine treatment. Cell survival in ACHN and SN12PM6 cells follow the treatment of cisplatin (a and b) or gemcitabine (d and e) of various concentrations. IC50 in cells expressing PTENP1 is significantly lower than that in cells expressing vector control or control cells (c and f). D, ACHN cells expressing PTENP1 or vector control were transplanted into mice that were subjected to cisplatin treatment. Tumor volume was determined at each time point. E, ACHN cells expressing PTENP1 or vector control were transplanted into mice that were subjected to gemcitabine treatment. Tumor volume was determined at each time point. PTENP1 expression sensitizes ACHN cells to cisplatin or gemcitabine treatment in vivo. *, P < 0.05; **, P < 0.01.

Figure 7

PTENP1 and PTEN expression is correlated with survival in RCC. Kaplan-Meier survival curves for patients with renal clear cell cancer according to the expression level of PTENP1 (A) and the expression level of PTEN (B).