MiR-21-3p was upregulated in HCC and its clinical significance in HCC based on bioinformatics analysis
Based on our previous chip analysis results, miR-21-3p was significantly upregulated in HCC compared to normal liver tissues (NLTs), which had statistical significance (P=0.028) (Table 1). To further verify the results of chip analysis, the mRNA expression of miR-21-3p was examined in human HCC tissue samples and Wistar rat models, respectively. Results showed that miR-21-3p was significantly enriched at HCC tissues compared to background livers (BLs) (Fig.1A (a)). Its upregulation was also observed in rats with late-stage fibrosis and cirrhosis (Fig.1A (b)). Compared to human relative normal cell L02, miR-21-3p was increased in liver cancer cell lines (Huh-7, Hep-G2, HCCL-M3) (Fig.1A (c)). Considering the well expression consistency in human tissues, rat liver disease models and cell lines, miR-21-3p was selected for subsequent analysis. To explore the clinical significance of the miR-21-3p in human HCCs, the Cancer Genome Atlas (TCGA) data, including 376 HCC patients, whose survival status was available, were analyzed. We found that patients with high miR-21-3p expression were significantly correlated with advanced clinical stages (P = 0.029), especially in T staging (P = 0.026), while no noticeable difference was observed in age and gender. (Figure 1B). Higher miR-21-3p expression level was correlated to shorter ten-year overall survival (OS) time (log-rank P=0.026) (Fig. 1C). GSVA analysis of miR-21-3p displayed that high expression of miR-21-3p was mainly enriched at 20 pathways in HCC (Fig.1D). In particular, the top three gene sets related were nitrogen metabolism, fatty acid metabolism and primary bile acid biosynthesis, severally (Fig.1E). KEGG pathway analysis of miR-21-3p targets revealed that the downstream targets were strongly linked to metabolic pathways, Hippo signaling road and TGF-beta signaling pathway (Table 2). The intersections of miR-21-3p potential targets from three authoritative databases (TargentScan, PicTar, miRDB) were Smad7, HBP1, FBXO11, respectively (Fig.1F). Of these, Smad7 earned the highest score (Table 3), while the potential relationship between miR-21-3p and Smad7 waited to be rectified.
Smad7 as the direct target of miR-21-3p was decreased in HCC
To further verify our analysis, expressions of Smad7 in human tissue samples and rat models were checked. Compared to BLs, Smad7 was obviously decreased in HCCs (9/10) (Fig.2A (a)). Also, the lighter brownish staining was observed in HCCs compared to BLs (Fig.2B (a)). Along with the progressing of liver disease in rat models, a gradually descending trend was observed in Smad7’s protein expression level (Fig.2A (b)), and immunohistochemical staining showed the same trend (Fig.2B (b)). Considering the apparent up-regulation of miR-21-3p and down-regulation of Smad7 in HCC, Dual-Luciferase assay was done in Huh-7 cells to confirm the linear regulation relation between miR-21-3p and Smad7. Prediction websites presented there were two binding sites in the 3’-UTR region of Smad7 with high scores. In the group of co-transfecting miR-21-3p and reporter vector containing wild-type (WT) Smad7, luciferase activity was significantly inhibited, while in the group containing mutation Smad7 reporter vectors (mutation-3 (Mut3) mutating two sites simultaneously), the inhibition efficiency was no statistical significance between miR-NC and miR-21-3p group (Fig.2C). The above results indicated that the two prediction sites might both exert the effect on inhibiting Smad7.
Overexpression of Smad7 partly abrogates the progressive tumor effect of miR-21-3p on cell malignant phenotypes in HCC
Mimics and inhibitors of miR-21-3p were transfected into Huh-7 and Hep-G2 cells to gain and lose its function separately. Plasmids of Smad7 (P-Smad7) was constructed and transfected into Huh-7 and Hep-G2 to overexpress Smad7, and Plasmid-NC (P-NC) served as the negative control. Smad7 expression was promoted after transfecting miR-21-3p inhibitors and decreased following transfecting miR-21-3p mimics in Huh-7 (Fig.3A (a)) and Hep-G2 cells (Fig.3A (b)). To investigate the effect of the miR-21-3p/ Smad7 on tumor cell malignant phenotypes in HCC, biomarkers related to cell apoptosis, migration and invasion were examined. Pro-apoptotic protein Bax and epithelial signature protein E-cadherin (E-cad) were upregulated while anti-apoptotic protein Bcl-2, mesenchymal characteristic protein N-cadherin (N-cad) and Vimentin were down-regulated after transfecting miR-21-3p inhibitors or Smad7 plasmids in Huh-7 and Hep-G2 respectively (Fig.3A-B). The effect of miR-21-3p on cell malignant biomarkers was partly attenuated by co-transfecting Smad7 in both two cell lines (Fig.3B). Flow cytometry results displayed that the early apoptotic rate stained green was increased by transfecting Smad7 (P<0.01)and deceased by up-regulating miR-21-3p (P<0.05) (Fig.4A). The migration (Fig.4B (a-b)) and invasion abilities (Fig.4B (c-d)) enhanced by transfecting miR-21-3p mimics were partly reversed by co-transfecting Smad7.
MiR-21-3p could enhance the expression of Yap1 partly via down-regulating Smad7
The KEGG pathway enrichment analysis displayed that miR-21-3p was significantly related to the Hippo signaling pathway (Table 2). Besides, a previous study confirmed that Yap1 could enhance the binding of Smad7 to TβRⅠ (18). To further investigate the potential link among, miR-21-3p, Smad7 and Yap1 in HCC, the expression of Yap1 was detected in HCC tissues and rat models. Compared to BLs, Yap1 was enriched at human HCC tissues (8/10) (Fig.5A), and advanced stages of liver fibrosis and cirrhosis compared to F0 (F: fibrosis) (Fig.2A (b)). The darker brownish staining in immunohistochemistry images was observed in HCC tissues (Fig.5B (a)) and the stage of F3 and F4 in rats compared to F0 (Fig.5B (b)). In both Huh-7 and Hep-G2 cells, Yap1’s expression was promoted through enhancing miR-21-3p and inhibited when cells were transfecting miR-21-3p inhibitors or up-regulating Smad7 (Fig.5C (a,b)). In protein level, the promotion of expression of Yap1 was partly attenuated by co-transfecting Smad7 (Fig.5C (b)). However, in the mRNA level, the influence of miR-21-3p and Smad7 on Yap1 had no statistical significance (Fig 5C (c,d)). Large tumor suppressor 2 (LATS2) as the upstream regulator of Yap1 was decreased by up-regulating Smad7 (Fig.5C(b), S1(A-B)). Besides, mRNA (Fig.S2A-B) and protein (Fig.5C(b)) expression trend of connective tissue growth factor (CTGF) which was proven to be the direct nuclear target of nuclear Yap1 was consistent with Yap1 here (22) (Fig.5C (b)). Therefore, we deduced that miR-21-3p could promote the expression of Yap1 partly through downregulating Smad7.
The clinical significance of Smad7/Yap1 based on bioinformatics analysis
Data about 376 HCC patients was downloaded from the Cancer Genome Atlas (TCGA) database. Wilcoxon rank-sum test results displayed that Smad7 was decreased (P=4.578e-04) and Yap1 (P=3.6e-05) was increased in HCC compared to adjacent normal liver tissues (Fig.6A). Combining RNA-seq data with patients’ clinical traits, low Smad7/ high Yap1 was related to low grading and staging (Fig.6B). Co-survival analysis results indicated that Lower miR-21-3p/Higher Smad7 (P=0.049) and lower miR-21-3p/lower Yap1 (P=0.038) were related to a better five-year OS rate (Fig.6C). GSVA volcano map showed that Smad7 was mainly involved in 18 pathways (Fig.6D). Of note, TGF-β signaling pathway, Notch signaling pathway, adherens junction were most relevant in HCC (Fig.6E).