Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells

Previously, we found that the expression of microRNA-146a (miR-146a) was downregulated in hepatocellular carcinoma (HCC) formalin-fixed paraffin-embedded (FFPE) tissues compared to the adjacent noncancerous hepatic tissues. In the current study, we have explored the in vitro effect of miR-146a on the malignant phenotypes of HCC cells. MiR-146a mimic could suppress cell growth and increase cellular apoptosis in HCC cell lines HepG2, HepB3, and SNU449, as assessed by spectrophotometry, fluorimetry, and fluorescence microscopy, respectively. Furthermore, western blot showed that miR-146a mimic downregulated EGFR, ERK1/2, and stat5 signalings. These effects were less potent compared to that of a siRNA targeting EGFR, a known target gene of miR-146a. Moreover, miR-146a mimic could enhance the cell growth inhibition and apoptosis induction impact of various EGFR targeting agents. The most potent combination was miR-146a mimic with cetuximab, presenting a synergistic effect. In conclusion, miR-146a plays a vital role in the cell growth and apoptosis of HCC cells and inducing miR-146a level might be a critical targeted molecular therapy strategy for HCC.


Introduction
Hepatocellular carcinoma (HCC) represents a major form of primary liver malignancy in adults worldwide. The tumorigenesis and development of HCC is typical of a multistage process. Major risk factors for HCC include infection with HBV or HCV, alcoholic liver disease, and most probably nonalcoholic fatty liver disease [1][2][3][4][5][6][7]. The progression is considered to deregulate genes that are critical to biological cellular procedures such as cell cycle control, apoptosis, cell migration, and metastasis [8][9][10][11][12][13][14][15]. However, the sensitivity and specificity of these markers remain imperfect [15]. Therefore, new biomarkers are needed to help to understand the causes of hepatocarcinogenesis and to predict response possibilities towards different therapeutic methods.
MicroRNAs (miRs), small noncoding single-stranded RNAs of 19-24 nucleotides in length, negatively regulate the 2 BioMed Research International expression of numerous target genes at the posttranscriptional and/or translational levels and play a vital role in the initiation and deterioration of HCC. Extensive profiling studies over the past several years have indicated that many miRNAs are abnormally expressed in HCC tissues [30][31][32][33][34]. Previously, we found that miR-146a expression was lower in HCC than that in their paracancerous liver tissues. The lower miR-146a expression was also related to advanced clinical TNM stage and distant metastasis [35]. In order to investigate the role of miR-146a on the malignant phenotypes of HCC cells, we performed in vitro experiments in the current study to explore the effect of miR-146a on the cell proliferation, viability, caspase-3/7 activity, apoptosis, and regulation of cellular signals in HCC cells lines Hep3B, HepG2, and SNU449. Furthermore, we studied the potential of miR-146a mimic as a therapeutical strategy by combining it and different EGFR target agents, including tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and monoclonal antibody cetuximab.

Inhibition and Reexpression of mir-146a in HCC Cells.
The human HCC-derived cell lines HepG2 (HB-8065), HepB3 (HB-8064), and SNU449 (CRL-2234) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). They were cultured as previously described [42][43][44]. All in vitro experiments were performed in triplicate. HCC cells were planted in 24-well plates (2.5 × 10 4 cells per well) or 96-well plates (2.5 × 10 3 cells per well) and incubated at 37 ∘ C for 24 h. The cells were transfected with miR-146a inhibitor, miRNA inhibitor negative control, miR-146a mimic, and miRNA mimic negative control, respectively (Ambion, Life Technologies Grand Island, NY, USA) at a final concentration of 200 nmol/L for 96 h using combiMAGnetofection (OZ BIOSCIENCES,  Marseille Cedex 9, France) in accordance with manufacturer's procedure. MiRNA mimics are small, chemically modified double-stranded RNA molecules designed to specifically bind to and mimic endogenous miRNA molecules. They can enable miRNA functional analysis by upregulation of miRNA activity. MiRVana miRNA Mimic Negative Control Number 1 was used as miRNA mimic negative control in the current study and it is a random sequence miRNA mimic molecule that has been extensively tested in human cell lines and tissues and validated not to produce identifiable effects on known miRNA function. MiRNA inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNA molecules. These ready-to-use inhibitors can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs. Anti-miR Negative Control Number 1 was used as inhibitor negative control in the present study and it is a random sequence anti-miR molecule that has been extensively tested in human cell lines and tissues and validated to produce no identifiable effects on known miRNA function. EGFR TKIs gefitinib, erlotinib, and EGFR-specific monoclonal antibody cetuximab (2 mg/mL, 13.719 M) were purchased from Selleckchem, Munich, Germany. All agents were prepared as described previously [36][37][38][39][40][41]. The EGFR specific siRNA was described previously [36][37][38][39] (sequence: GCAAAGTGTGTAACGGAATAGGTAT). The EGFR specific siRNA was transfected into HCC cells with the same method as above.

Statistical
Analysis. SPSS19.0 (Munich, Germany) was performed for statistical analysis. Results were representative of minimal three independent experiments unless stated otherwise. Values were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) test was used to analyze significance between different groups. The least significant difference (LSD) method of multiple comparisons between two groups was applied when the probability for ANOVA was statistically significant. Statistical significance was determined at a < 0.05 level. In the analysis of additivity and synergism, the theoretical zero-interaction (exactly additive) dose-response curve for each miR-146a mimic + other agent combination was calculated by applying Bliss independence criterion [45] and was also assessed by the Biosoft CalcuSyn program (Ferguson, MO, USA). The combination index (CI) was used to express synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1) [37,38,40,41,43,46].

MiR-146a Inhibited Cell Growth in HCC Cells.
Transfection efficiency was first confirmed using real time RT-qPCR ( Figure 1). The effect of miR-146a on cell growth was detected using three independent assays, including MTS tetrazolium  assay, fluorimetric resorufin viability assay, and Hoechst 33342/propidium iodide (PI) double fluorescent chromatin staining, respectively. MTS tetrazolium assay revealed that cell proliferation increased slightly in HepG2 cells 96 h after transfection compared to blank and negative controls with miR-146a inhibitor. MiR-146a inhibitor exerted no influence on HepB3 or SNU449 cells. After transfection with the miR-146a mimic, a large reduction in proliferation was examined at 72 and 96 h in all the three cell lines tested, although less than the effect of siRNA targeting EGFR (Figure 2). The cell growth inhibitory effect showed a time dependent manner. To verify these results, the effect on cell viability was assessed by using a fluorimetric resorufin viability assay ( Figure 3) and Hoechst 33342/PI double fluorescent chromatin staining (data not shown), which largely mirrored the results from MTS assay. The effect of miR-146a on cell growth suppression showed also a dose dependent manner (Figure 4).

MiR-146a Mimic Induced Apoptosis in HCC Cells.
To validate whether miR-146a is able to influence apoptosis, the CellTiter-Blue assay was multiplexed with a fluorescent caspase-3/7 assay. The results showed that with the miR-146a inhibitor, caspase-3/7 activity was slightly less than the blank and negative controls, but indicated no significant

Mock control Negative inhibitor control miR-146a inhibitor
Negative mimic control miR-146a mimic EGFR siRNA

Contribution of miR-146a in Relevant Cellular Signaling.
To investigate the contribution of miR-146a in the regulation of cellular signaling, we examined the signaling of EGFR, AKT, ERK, and stat pathways by using western blot, which are related to cell survival, apoptosis, and invasion. These pathways were influenced little with miR-146a inhibitor transfection. However, the phospho-EGFR, phospho-ERK1/2, and phospho-stat5 were downregulated by miR-146a mimic 96 h after transfection, although weaker than EGFR siRNA (Figure 9).

Synergistic Effect of miR-146a Mimic and Cetuximab.
EGFR is a confirmed target gene of miR-146a as previously reported [41,47,48]. We desired to explore the combinatorial effect of miR-146a mimic and agents targeting EGFR (small molecular inhibitors or monoclonal antibody), using the colorimetric MTS formazan proliferation assay and caspase activity assay. The inhibition of cell proliferation was much stronger with miR-146a mimic in combination with gefitinib, erlotinib, or cetuximab, compared to single drug or single miR-146a mimic in HepG2 cells ( Figure 10). However, for gefitinib and erlotinib, the proliferation curve of the combinatorial treatment was not significantly higher than the Bliss independence curve, which indicated an additive effect. Surprisingly, the combination of miR-146a mimic and cetuximab showed an extremely higher inhibitory effect compared to the Bliss independence curve, which suggested a possible synergy in cell growth suppression ( Figure 10). Furthermore, the combination of miR-146a mimic and cetuximab also gave a significantly higher caspase-3/7 signal than single treatment ( Figure 11). In addition, western blot proved that with the combination of miR-146a mimic and cetuximab, the caspase-3 protein level was reduced and cleaved caspase-3 expression was increased ( Figure 12). Meanwhile, the EGFR and downstream pathway signals were also further downregulated with miR-146a mimic combined with cetuximab, especially p-EGFR and p-ERK1/2 ( Figure 13). To verify the additive or synergistic nature of combining EGFR targeted agents with the miR-146a mimic, a CI was calculated [37,38,40,41,45]. This unambiguously showed that the effect of gefitinib or erlotinib and miR-146a was additive (data not shown). With regard to the combination of cetuximab and miR-146a mimic, the effect was indeed entirely synergistic ( Figure 14).

Discussion
Previously, we found that the relevant miR-146a expression in the HCC tissues and cultured cells was significantly lower than that in the adjacent noncancerous hepatic tissues. Moreover, miR-146a expression in early clinical stages (I and II) was remarkably higher than that in advanced stages (III and IV). Lower level of miR-146a was also found in HCC patients with metastasis compared with those without metastasis [35]. These results strongly suggest that miR-146a acts as a tumor suppressor miRNA in HCC. To further explore the function of miR-146a on the malignant phenotypes of HCC cell, we performed a series of in vitro experiments. We first investigated the contribution of miR-146a to cell growth and apoptosis in HCC cells and also compared the effect of miR-146a mimic to the siRNA specially targeting EGFR mRNA, since EGFR was documented as a target of miR-146a in various cancers [41,[47][48][49][50]. In the current study, the EGFR protein was downregulated in HCC HepG2 cells after transfection of miR-146a mimic, which implies that EGFR is a target of miR-146a in HCC. The miR-146a mimic decelerated the cell growth in all the cell lines tested (HepG2, HepB3, and SNU449), using three independent assays. Additionally, miR-146a mimic enhanced the caspase-3/7 activity and induced apoptosis in HCC cell lines. This could partially be explained by the downregulation of EGFR and the downstream pathways as shown by western blot, especially ERK1/2  and stat5, though HCC HepG2 expresses low level of p-stat5 at the basic line. However, the effect of miR-146a mimic on inhibiting cell growth and inducing apoptosis was much weaker than that of an EGFR-specific-siRNA. Since different mechanism could be involved in the process of EGFR gene silencing, probably much higher dosage of miR-146a mimic is needed to reach the same effect of EGFR siRNA. The miR-146a inhibitor had little effect on the function of HCC cells, which could be related to the limited downregulation of miR-146a level. With miR-146a inhibitor, the ΔΔCq was about 2 times higher than the negative controls, in contrast with miR-146a mimic; the ΔΔCq was about 9 times lower than the controls. The suppressive role of miR-146a on the malignant phenotype of HCC cells makes it tempting to develop novel therapeutical strategies of inducing miR-146a levels. When we combined the miR-146a mimic together with different EGFR TKIs or cetuximab in HCC cells, miR-146a mimic was found to enhance the cell proliferation inhibition and apoptosis induction by TKIs and cetuximab, the strongest effect obtained when combined with cetuximab. Synergistic effect was obtained with the combination of miR-146a mimic and cetuximab. Western blot also confirmed that the EGFR, ERK1/2, and stat5 pathways were strongly inhibited with this combination. Since EGFR siRNA has stronger effect than miR-146a mimic, we wondered if the combination of EGFR siRNA and cetuximab could produce even more potent synergistic effect. Indeed, EGFR siRNA plus cetuximab have very powerful effect on cell growth inhibition and apoptosis induction (unpublished data on file). The level of miR-146a in cancer cells can also be upregulated by other agents. For example, Li et al. found that reexpression of miR-146a by nontoxic "natural agents, " including 3,3 -diindolylmethane, and isoflavone, has antitumor effects in pancreatic cancer [47].

Conclusions
Since miR-146a was found to be related to the HCC metastases, further in vitro studies are planned to identify the mechanism of miR-146a in metastasis. Other possible target genes of miR-146a, such as TRAF6, IRAK1, and NUMB, will also be tested. On the other hand, cell growth inhibition and apoptosis induction by miR-146a mimic represent great relevance due to its possible therapeutic potential. The application of miR-146a mimic might thus be a promising approach to HCC therapies in the future, for both in vivo studies and clinic trials.