SPP1 functions as an enhancer of cell growth in hepatocellular carcinoma targeted by miR-181c

Abstract

Patients diagnosed with hepatocellular carcinoma (HCC) suffered a high risk of recurrence and poor prognosis. Identification of differentially expressed genes (DEGs) in HCC provides potential biomarkers for evaluating prognosis and specific therapeutic treatments. In this study, DEGs over-expressed in HCC specimens with a fold change over 2.0 were collected through integrative bioinformatics analysis from GEO datasets. Gene ontology and KEGG pathway enrichment were conducted by applying DAVID database. We noticed Secreted phosphoprotein 1 (SPP1) as one of the signature genes up-regulated in HCC tissues with a close relation to the tumor process. Eighty-seven paired HCC specimens from our medical center were explored to verify the aberrant expression of SPP1 by IHC and qRT-PCR assay. Depletion of SPP1 in HCC Hep3B cells was established. The cell proliferation was impaired in SPP1 depleted cells, along with a resistance of cell apoptosis by down-regulating SPP1. Intriguingly, we further validated a direct interaction between miR-181c and SPP1, which indicated a post-transcriptional regulation mechanism of SPP1 in HCC. Thus, our results suggest that SPP1 may function as an enhancer of HCC growth targeted by miR-181c, and probably provide us an innovational target for HCC diagnose and therapeutic treatment.

Keywords: Hepatocellular carcinoma; cell growth; miR-181c; secreted phosphoprotein 1.

Figure 1

DEGs identified through analysis NCBI GEO datasets. A. Venn chart of the significant up-regulated genes in three HCC datasets (GSE6764, GSE14520 and GSE14323) compared with the non-cancerous liver tissues. AKR1B10 and SPP1 were screened out according to the overlapped results. B. Venn chart of the decreased genes among the three datasets. LPA, MT1M, MFAP3L and IL1RAP were collected finally. C. Representative heatmap generated through GEO datasets illustrates the comprehensive expression profiles of the six significant genes including SPP1. SPP1 presents a relatively higher expression in HCC tissues than in the normal liver tissues.

Figure 2

Correlation and interaction between DEGs and the particular analysis of SPP1. A. Correlation matrix of the DEGs. SPP1 presents a highly expression characteristic in HCC tissues sharing co-expression with another HCC promotor, AKR1B10. B. Presentation of the GO and KEGG enrichment analysis indicating the biological process, molecular functions and biological pathways of the DEGs. C. Network that SPP1 involved in including PPI, co-expression and pathway. D. KM plot of the 5 year OS rate generated from 364 HCC cases demonstraed a significant poor outcome of the patients with relatively higher SPP1 expression (HR=2.4, logrank P=1.6e-06).

Figure 3

Expression of SPP1 in either HCC patient’s specimens and the HCC cell lines. A. Statistic of number of cases with higher or lower expression of SPP1 in 87 paired HCC specimens. SPP1 was up-regulated in most of the tumor tissues (61/87), and was expressed at a lower level in most of the adjacent non-cancerous tissues (64/87). SPP1 was frequently and significantly higher expressed in tumor tissues (P<0.01). B. Representative graph of immunohistochemistry analysis on tissue microarray (100 ×). Specimens stained IgG anti-body were applied as control. SPP1 in HCC tumor tissues was significantly higher expressed than that in adjacent non-cancerous tissues. C. Transcription level of SPP1 in cell lines was detected through qRT-PCR assay. The SPP1 mRNA expression is significantly higher in HCC cells than that in L02 cells as control (*P<0.05). D. Detection of protein expression of SPP1 in cell lines by Western-blot analysis. SPP1 protein was significantly up-regulated in HCC cells compared with L02 cells. The numbers above the blot indicate normalized protein amounts relative to the negative control, as determined by densitometry.

Figure 4

Depletion of SPP1 in Hep3B cells impairs the cell growth and promotes cell apoptosis. A. QRT-PCR assay indicated a significant down-regulation of SPP1 mRNA level in Hep3B cells after pGU6/Neo vectors transfection (**P<0.01). B. Western blot analysis validated the decline of SPP1 after transfection. Numbers above the blot indicate the protein amounts normalized. C. CCK8 assay was conducted to illustrate the effect of SPP1 on cell proliferation. Cell proliferation of Hep3B cells was significantly impaired by depleting SPP1 (**P<0.01, *P<0.05). D, E. Representative histograms describing the cell cycle profiles of Hep3B cells. Significantly, the cell cycle was arrested in the G0/G1 phase following depletion of SPP1 in Hep3B cells (*P<0.05). F, G. Apoptosis rate of was detected by flow cytometry. Representative graph of cell apoptosis rate was demonstrated and statistics analysis of cell apoptosis rate indicates that depletion of SPP1 in Hep3B cells significantly promotes cell apoptosis compared with the control ones (**P<0.01).

Figure 5

Analysis of miR-181c expression in HCC and the prediction of its targeting effect on SPP1. A. Calculation of miR-181c expression through dbDEMC2 software. MiR-181c is relatively lower expressed in multiple human maligancies, including HCC. B. qRT-PCR assay indicated significantly lower expression of miR-181c in HCC cell lines compared with L02 cells (*P<0.05). C. The predicted miR-126 binding site in the wild-type SPP1 mRNA 3’-UTR (3’UTR-WT) is visulizated, and the minimum free energy hybridization of miR-181c and SPP1 mRNA 3’-UTR is calculated (Mfe: -18.4 kal/mol).

Figure 6

Modulation of SPP1 in Hep3B cells induced by miR-181c. A. The direct interaction between SPP1 and miR-181c was detected by Dual-luciferase reporter assay. Up-regulation miR-181c through mimics in Hep3B (Hep3B/miR-181c) decreased the luciferase signal of SPP1/pMIR/WT significantly in cells with compared with the negative control (Hep3B/NigmiR), while mutation of the putative miR-181c-binding site abolished this suppressive effect (**P<0.01). B. qRT-PCR assay demonstrated that the mRNA expression of SPP1 was significantly decreased by introducing miR-181c into Hep3B cells (**P<0.01). C. Western blot analysis indicated that the SPP1 protein was significantly decreased by introducing miR-181c into Hep3B cells. D-F. Rescue experiment was conducted due to re-upregulating SPP1 in Hep3B cells transfected with miR-181c. CCK8 assay and the cytometry analysis respectively indicated a partly rescue effect through up-regulating SPP1, which induced recovery of cell proliferation, along with the promotion of cell cycle process and resistence to cell apoptosis (*P<0.05).