miRNA-377 can target the 3ʹ-UTR of EGFR, PAK2, MAPK1
Eight bioinformatics prediction software programs (miRBase, TargetScan, MiRanda, PicTar, miRWalk, RNA22, PITA and MicroCosm Targets) were applied to predict the best miRNA for silencing ABL2, PAK2, EGFR and MAPK1 genes in ErbB pathway. Four first ranked miRNAs for each gene are shown in Table 1.
hsa-miRNA-377-3p was among the first four miRNAs that could recognize and attach to all four mRNAs. In order to confirm bioinformatics prediction of previous software programs and predict the miRNA-377-target positions interaction and seed matching with ABL2, PAK2, EGFR and MAPK1, prophecy programs including miRWalk, TargetScan, RNA22, DIANA, mirPath, miRmap and miRDIP software programs were used (Table 2). Inhibitory effect of miRNA- 377 on all four genes expression was predicted at least in four prediction software programs with moderate to high score.
In order to validate whether ABL2, PAK2, EGFR and MAPK1 are direct target of miRNA-377, we cloned the 3′-UTR of the ABL2, PAK2, EGFR or MAPK1 and a special scrambled sequence into the Renilla luciferase gene in psiCHECK™-2. This plasmid was co-transfected with miRNA-377 and scrambled in A549 and Calu-6 cells. As shown in Fig. 1a, miRNA-377 significantly reduced the luciferase activity in cells which contain vector harboring, PAK2 3′-UTR, EGFR 3′-UTR or MAPK1 3′-UTR compared with the scrambled oligonucleotide. Transfecting miRNA-377 into cells which contain vector harboring, EGFR 3′-UTR, MAPK1 3′-UTR or PAK2 3′-UTR decreased the luciferase activity to 51% ± 4.623%, 57% ± 3.4631% and 52 %± 4.3272% respectively in comparison with the control (P < 0.001), while in cells which contain vector harboring, ABL2 3′-UTR, the luciferase activity decreased to 90% ± 4.376%. These results suggested that PAK2, EGFR and MAPK1 could be considered as a candidate target gene for miRNA-377 and also their 3′-UTR is a functional target site for this miRNA in A549 and Calu-6 cells.
Expression level of miRNA-377 in lung cancer tissues and lung cancer cell lines was lower than normal tissues
To investigate the level of miRNA-377 in lung cancer samples, A549 and Calu-6 cell lines and normal tissues, total RNA were extracted from thirty-five NSCLC patients and nine normal samples used for real-time PCR. The results are shown that the average miRNA-377 expression in human NSCLC tissues and cell lines was significantly (P < 0.05) lower than that found in non-tumor tissue samples. We compare them due to the fact that we do not have normal cell line (Fig. 1b and c).
There was a negative correlation between the expression of miRNA-377 and some ErbB signaling pathway genes mRNA level in lung cancer tissues
In order to confirm any correlation between the level of miRNA-377 and EGFR, MAPK1, and PAK2 mRNA expression in lung cancer samples and normal tissues, total RNA was extracted from thirty-five NSCLC patients and nine normal samples used for real-time PCR.
The EGFR, MAPK1 and PAK2 mRNAs were significantly increased in the lung tumor tissues compared to normal tissues. (P < 0.05; Fig. 2a).
Statistical analysis further revealed that the expression level of miRNA-377 negatively correlated with EGFR and MAPK1 and PAK2 mRNA expression. These values for EGFR and MAPK1 and PAK2 were r=-0.7699 (P < 0.05), r=-0.7863 (P < 0.05) and r=-0.8990 (P < 0.05) respectively (Fig. 2b).
miRNA-377 introduction to A549 and Calu-6 cells downregulates the expression level of some genes belonging to ErbB signaling pathway.
To determine whether miRNA-377 could target the EGFR, ABL2, MAPK1 and PAK2 mRNAs, we examined the relative expression levels of the target RNAs in transfected A549 and Calu-6 cell lines with miRNA-377 or scrambled oligonucleotide and evaluated with control cells by real-time PCR. In this experiment non-transfected cells and cells transfected with scrambled oligonucleotide were used as controls, respectively. The real-time PCR results demonstrated that miRNA-377 induced a remarkable decrease in levels of EGFR, MAPK1 and PAK2 mRNAs in the two cell lines (Fig. 3). The suppression effect of miRNA-377 on ABL2 levels in A549 and Calu-6 cells was not significant.