MicroRNA-587 Functions as a Tumor Suppressor in Hepatocellular Carcinoma by Targeting Ribosomal Protein SA

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most highly aggressive cancer worldwide with an extremely poor prognosis. Evidence has revealed that microRNA-587 (miR-587) is abnormally expressed in a series of cancers. However, its expressions and functions in HCC have not been clearly acknowledged.

Methods: We detected the expression level of miR-587 both in the Gene Expression Omnibus (GEO) database and 86 paired clinical HCC tissues together with paired adjacent normal tissues by quantitative real-time PCR (qRT-PCR). Afterwards, the transfected HCC cell line SMMC-7721 cells were collected for the cell proliferation assay, cell-cycle arrest, cell migration, and invasion assays to explore the roles of miR-587 in regulating cellular function. In addition, bioinformatics analysis, combined with qRT-PCR and dual-luciferase reporter assays, were performed to confirm whether ribosomal protein SA (RPSA) mRNA was the direct target gene of miR-587. Moreover, the Cancer Genome Atlas (TCGA) and GEO databases as well as 86 paired clinical HCC tissues were used to verify the negative regulation between miR-587 and RPSA.

Results: In the present study, both the GEO database (GSE36915 and GSE74618) analysis and qRT-PCR analysis of 86 paired clinical tissues showed that miR-587 was significantly downregulated in HCC tissues. The overexpression of miR-587 inhibited proliferation, cell cycle, migration, and invasion in SMMC-7721 cells. In addition, miR-587 directly interacted with the 3'-untranslated region (UTR) of RPSA. Moreover, miR-587 overexpression directly suppressed RPSA expression, and the two genes were inversely expressed in HCC based on the analyses in TCGA and GEO (GSE36376) databases and qPCR analysis of 86 paired clinical tissues.

Conclusion: Our results demonstrate that miR-587 is downexpressed in HCC and regulates the cellular function by targeting RPSA.

Figure 1

Downexpression levels of miR-587 in HCC. The gene expression analysis based on (a) GSE36915, (b) GSE74618 datasets from GEO database, and (c) data from 86 paired clinic HCC tissues shows that miR-587 was downexpressed in HCC tissues compared with noncancerous tissues (p < 0.001, p = 0.01, and p = 0.02, respectively). miR: microRNA.

Figure 2

miR-587 suppressed HCC cell proliferation. (a) The transfection efficiency of miR-587 mimic (p = 0.001) and inhibitor (p = 0.037). The MTT assay showed that miR-587 upregulation (b) suppressed the cell viability of SMMC-7721 cells, whereas miR-587 downregulation (c) fostered the cell viability (both p < 0.05). Flow cytometry analysis showed that the number of SMMC-7721 cells at G1 phase increased and those at S phase decreased in the miR-587 mimic group (d) (p = 0.001, p = 0.044, respectively). The number of SMMC-7721 cells at G1 phase decreased and those at S phase increased in the miR-587 inhibitor group (e) (p = 0.028, p = 0.048, respectively). ∗p < 0.05, ∗∗p < 0.01. miR: microRNA.

Figure 3

miR-587 curbed cell migration and invasion in HCC cells. The wound healing migration assay showed that miR-587 upregulation (a) depressed the migration rate of SMMC-7721 cells and miR-587 downregulation (b) elevated the migration rate of SMMC-7721 cells (p < 0.001, p = 0.033, respectively). The Transwell assay showed that miR-587 upregulation (c) suppressed the invasion and miR-587 downregulation (d) enhanced cell invasion of SMMC-7721 cells (p = 0.034, p < 0.001, respectively). ∗p < 0.05, ∗∗p < 0.01. miR: microRNA.

Figure 4

RPSA as a direct target of miR-587. (a) A total of 4884 overexpressed genes in HCC were screened out based on the TCGA database. (b) Thirteen potential target genes of miR-587 were included after intersecting outputs between TCGA database and TargetScan. (c) QRT-PCR assay revealed inverse expressions of RPSA and miR-587 in SMMC-7721 cells in both the mimic group (p = 0.014) and the inhibitor group (p = 0.024). (d) The 3′-UTR region of RPSA had two target sites interacting with miR-587. (e) Dual-luciferase reporter assay showed a specific interaction between miR-587 and the 3′-UTR of RPSA mRNA (p < 0.001). ∗p < 0.05, ∗∗p < 0.01. miR: microRNA.

Figure 5

The negative regulation between miR-587 and RPSA in HCC. The gene expression analysis based on the (a) TCGA database, (b) GEO database (GSE36376), and (c) data from 86 paired HCC tissues showed that RPSA was overexpressed in HCC tissues compared with noncancerous tissues (all p < 0.001). (d) Pearson's analysis uncovered a negative correlation between miR-587 and RPSA expressions in HCC tissues (p = 0.011). miR: microRNA.