MiR-301a Promotes Cell Proliferation by Repressing PTEN in Renal Cell Carcinoma

Abstract

Objective: Renal cell carcinoma (RCC) displays an increasing incidence and mortality rate worldwide in recent years. More and more evidence demonstrated microRNAs function as positive or negative regulatory factors in many cancers, while the role of miR-301a in RCC is still unclear.

Material and methods: The expression and clinical significance of miR-301a were assessed via bioinformatic software on open microarray datasets of the Cancer Genome Atlas (TCGA) and then confirmed by quantitative real-time PCR (qRT-PCR) in RCC cell lines. Loss of function assays were performed in RCC cell lines both in vitro and in vivo. Cell Counting Kit-8 (CCK-8), flow cytometry, luciferase reporter assays, Western blotting, and immunohistochemistry were employed to explore the mechanisms of the effect of miR-301a on RCC.

Results: By analyzing RCC clinical specimens and cell lines, we found a uniform increased miR-301a in expression in comparison with normal renal tissue or normal human proximal tubule epithelial cell line (HK-2). In addition, miR-301a upregulation correlated advanced stage and poor prognosis of clear cell RCC (ccRCC). Anti-miR-301a could inhibit growth and cell cycle G1/S transition in RCC cell lines. Moreover, we found that PTEN was identified as a direct target of miR-301a that might partially interrupt miR-301a-induced G1/S transition. Importantly, nude-mouse models revealed that knockdown of miR-301a delayed tumor growth.

Conclusion: These results indicate that miR-301a functions as a tumor-promoting miRNA through regulating PTEN expression, representing a novel therapeutic target for RCC.

Keywords: G1/S transition; PTEN; miR-301a; proliferation; renal cell carcinoma.

Figure 1

Relative miR-301a expression in RCC cell lines and ccRCC tissues and its relationship with the overall survival of ccRCC patients. (A) The expression of miR-301a in the indicated RCC cell lines. (B) Relative expression of miR-301a between ccRCC tissues and their corresponding normal renal tissue samples (n=71) (TCGA). (C–D) miR-301a expression levels in patients with different stages and grades of ccRCC (TCGA). (E) Kaplan–Meier analysis of the correlation between the miR-301a level and overall survival of ccRCC patients (TCGA) with high (>the median, n=258) and low (<the median, n=258) miR-301a expression. The results are presented as the mean±SD.**P≤0.01; ***P≤0.001.

Figure 2

Knockdown of miR-301a suppresses RCC cell proliferation by abrogating the G1/S transition. (A) Knockdown of miR-301a expression in 786–0 and A498 cells by transfecting miR-301a inhibitor was confirmed by qRT-PCR. (B) CCK-8 assays revealed the cell growth curves of the indicated cells. (C) Representative micrographs (left) and relative quantification (right) of crystal violet-stained cell colonies analyzed by colony formation assay. (D) FACS analysis of the cell cycle of the indicated cells. Cells were synchronized by serum deprivation for 24 h, and then stimulated to enter S phrase by serum re-addition. Cells were harvested for FACS analysis at 12 h post release. (E) Representative micrographs (left) and quantification of EdU-incorporated cells in the indicated cells. The results are presented as the mean±SD. *P≤0.05; **P≤0.01.

Figure 3

miR-301a directly targets PTEN and downregulates PTEN expression in RCC. (A) Schematic of miR-301a putative target sites in the 3ʹ-UTR of PTEN. (B) Sequence of miR-301a-mut. (C) Luciferase assay of pGL3-PTEN-3ʹ-UTR reporter cotransfected with increasing amounts (10, 20, and 50 nM) of miR-301a mimic and mutant oligonucleotides in 293-T cells. (D) qRT-PCR analysis of the mRNA levels of PTEN in response to deregulated miR-301a expression in the indicated cells. (E) Western blotting of PTEN expression in the indicated cells. β-actin was used as the internal control. (F) Correlation between miR-301a and PTEN mRNA expression in ccRCC (TCGA, n=501). (G) The effect of restoring the ORF (without the 3ʹ-UTR) of PTEN on the proportions of the indicated cells in distinct cell cycle phases as determined by FACS, and the corresponding change of PTEN protein by Western blotting. The results are presented as the mean±SD. *P≤0.05; **P≤0.01.

Figure 4

Knockdown of miR-301a inhibits tumor growth of RCC cell xenografts in vivo. (A) Representative photograph of 786–0 tumor formation in nude mice. (B) Tumor growth curves of 786–0 cells in nude mice. (C) Each tumor formed by 786–0 cells was weighed. (D) Immunohistochemical analysis of PTEN and Ki-67 in the xenografts of 786–0 cells. The tumors were monitored regularly for 5 weeks (B) and excised at the end of the experiment for weighing (C) and Immunohistochemical staining (D). Each data point represents the mean±SD of six independent xenografts. *P≤0.05; **P≤0.01.