Selection of the DEGs increased or decreased in GC
According to the criteria for DEGs selection of log2FC≥2.0 and P value＜1.0E-04, 464 genes increased in GC tissues were set compared with non-cancerous specimens. And, 1136 genes extremely decreased in GC tissues were selected simultaneously.
Among these genes, 46 genes were filtered out via FunRich software, among which 13 genes were over-expressed in tumor tissues, while the other 33 ones were decreased. Respectively, the 13 high expressed genes were HOXA13, SPP1, CST1, COL11A1, COL10A1, CLDN7, FNDC1, CLDN4, CLDN1, CHI3L1, CLDN3, CEMIP and SERPINB5（Fig. 1A）, and the 33 genes decreased were ATP4A, ATP4B, KCNE2, AQP4, GIF, LIPF, SULT2A1, KCNJ16, ADH7, CHIA, GKN1, GKN2, SOSTDC1, ESRRG, SST, FUT9, PDILT, FBP2, CPA2, PDIA2, CCKBR, MT1M, GHRL, CHGA, TFF2, PSCA, SCNN1G, C16orf89, KLK11, RDH12, AKR1B10, SCGB2A1, FGA (Fig. 1B). The expression fold-change of these DEGs, through TCGA database exploration and data mining, demonstrated extremely significant difference at mRNA status between tumor and normal gastric mucosa, partly illustrated in Fig. 1C.
In addition, as Suppl. Fig 1 and Suppl. Table.2 shown, GO and KEGG pathway enrichment analysis were carried out through online tools of the Database for Annotation Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/, Version 6.7) . The Search Tool for the Retrieval of Interacting Genes (STRING) database (Version 10.0, http://string-db.org) was applied for predicting the interaction between proteins derived from the DEGs. PPI network was estiblished through Cytoscape software (http://www.cytoscape.org/, Version 3.4.0).
SPP1 is highly expressed in GC tissues and cells, and is correlated with patients’ survival status
According to the primary exploration of the GEO database, we screened SPP1 out of the 13 extremely highly expressed DGEs in GC tissues (P=2.58E-13 for GSE13911, P=8.32E-5 for GSE19826 and P=2.52E-10 for GSE27342) (Fig. 2A～C). In supplementary, we also analyzed the database of CCLE and observed the obvious inclination of high expression of SPP1 in different human malignancies including GC, and SPP1 presents a commonly incident up-regulation in multiple GC cell lines (Suppl. Fig. 2).
We detected the expression of SPP1 in three GC cell lines (MKN-45, AGS and SNU-16) through qRT-PCR assay and Western-blot analysis, and found SPP1was significantly increased in all the three GC cell lines compared with the control GES-1 cells (Fig. 2D, E) (P＜0.05).
The expression of SPP1 in 105 paired of specimens from GC patients was detected by IHC assay. SPP1 was verified highly expressed in 74.28% tumor tissues (78/105); and only 24.76% (26/105) samples of non-cancerous tissues present a relatively higher expression. Hence, the expression of SPP1 in tumor tissues was frequently and significantly higher than adjacent non-cancerous tissues (P＜0.01). (Fig. 3A, B)
Additionally, Kaplan-Meier plot was generated to indicate the correlation between SPP1 and patents’ over-all survival (OS) or progression free survival rate (PFS). We collected 876 GC cases’ follow-up information, and the generated OS and PFS data indicated that the patients’ are significantly impaired when SPP1 was highly expressed (OS: HR=2.23, P=2.7E-14; PFS: HR=1.98, P=1.1E-10) (Fig. 3C).
High expression of SPP1 is associated with GC clinicopathologic features
The correlation between SPP1 expression status and the clinicopathologic features of the 105 GC cases was analyzed. As Table. 1 shown, there is no significant correlation between OPN expression and the patients’ age, gender or tumor location. On the contrary, high expression of SPP1 inclines toward larger tumor size (P＜0.05), more frequent lymph node metastasis (P＜0.05), deeper local invasion (P＜0.05), and more advanced TNM stages (P＜0.05) in cases with higher SPP1 expression level, which indicates a doubtless correlation with SPP1 over-expression and a part of the GC clinicopathologic features.
SPP1 depletion suppresses cell proliferation, arrests the cell cycle and promotes cell apoptosis in MKN-45 cells
MKN-45 cells was found expressing the highest level of SPP1 among the three GC cell lines, and was selected and transfected with pGU6/Neo vectors for SPP1 effect. The effect of transfection was validated through RT-qPCR and Western blot analysis (Fig. 4A, B).
CCK8 assay was applied for evaluating the effect of SPP1 on GC cell proliferation. A significant suppression of cell proliferation in SPP1 depleted MKN-45 cells compared with the control MKN-45 cells, as P value＜0.05 for d1, and P value＜0.01 for d2 to d4 (Fig. 4C). Likewise, the flow cytometry analysis for cell cycle demonstrated that the cell cycle of MKN-45 cells was arrested at G0/G1 phase via SPP1 depletion (Fig. 4D, E). The percentage of MKN-45 cells in G0/G1 phase was raised from 46.29% to 64.78% (P＜0.01); Cell portion distributing at S phase was decreased from 32.69% to 25.98%; and for G2/M phase, was decreased from 21.02% to 12.25%. Whilst, we also noticed an increased apoptosis rate of MKN-45 treated with SPP1 depletion, from 7.92% to 14.12% (P＜0.01) (Fig. 4F, G).
All these results above indicate that SPP1 depletion significant defects the tumor cell growth in GC.
SPP1 is a post-transcriptionally targeted by miR-4262 in GC cells
MiRNAs are short non-coding RNAs consist of around 22 nucleotides, inducing post-transcriptional regulation of transcribed genes. By using online bioinformatics and prediction tools described in methods parts above, we found miR-4262, a miRNA aberrantly expressed in multiple human malignancies, is potentially function as the upstreaming modulator of SPP1 (Fig. 5A). Online software of dbDEMC (Version 2.0) indicates the expression of miR-4262 in human malignancies, which presents miR-4262 as a remarkably down-regulated miRNA in GC (Fig. 5B). On this basis, we detected the expression of miR-4262 in the three GC cell lines, and found that the expression of miR-4262 was significantly decreased in GC cells compared with GES-1 cells (P＜0.01) (Fig. 5C).
The direct interaction between SPP1 mRNA and miR-4262 was detected via dual-luciferase reporter assay. As Fig 5D shown, luciferase signal for SPP1/pMIR/WT was significantly impaired when miR-4262 was introduced into MKN-45 cells (MKN-45/miR-4262). In contrast, this suppressive effect induced by miR-4262 could be remarkably abolished in MKN-45 cells with mutation of the putative miR-4262 binding site (SPP1/pMIR/MUT) (Fig. 5D). And the SPP1 expression at either mRNA status or protein status was significantly decreased in MKN-45/miR-4262 cells (Fig. 5E, F). These findings above suggest that SPP1 is one of the direct targets suppressed by miR-4262 in GC.
Re-upregulation of SPP1 in MKN-45 cells rescues the phenotype of cell growth and apoptosis induced by introducing miR-4262
In GC, miR-4262 had been verified significantly down-regulated in tumor tissues and cells, and plays a suppressive role in tumor progress, remarkably inhibiting the cell proliferation, and promotes the cell apoptosis (14).
Since we have validated the direct interaction between miR-4262 and SPP1 mRNA, we assumed that by re-introducing SPP1 into MKN-45 cells could rescue the phenotypes induced by miR-4262. In our study, we discovered that the suppressing function of miR-4262 of cell proliferation was significantly reversed by re-introducing SPP1 in MKN-45 cells (P value＜0.05 for d1～d2, and P value＜0.01 for d3～d4). Simultaneously, the cell cycle arrest and increase of cell apoptosis in MKN-45 cells were also partly rescued (Fig. 6).
Thus, we suggest that SPP1 is an enhancer of cell growth in GC, targeted by miR-4262.