Dai et al. further found that HuR could autoregulate its expression MG132 by promoting alternative polyadenylation site usage. However, the possible sig naling pathway involved in regulation on HuR expression remains largely unknown. It was well documented that PI3K/Akt pathway was a critical for tumor biology. Our previous study also showed that PI3KAkt pathway was critical for TLR9 signaling enhanced metastatic potential of lung cancer cells. In present study, we further demonstrated that TLR9 signaling could enhance the expression of HuR through Akt pathway, which ultim ately reduce the expression of miR 7, suggesting that PI3K/ Akt pathway was important for the expression of HuR in cancer cells.

Similarly, one most newly work also reported that Akt signaling could enhance the expression of HuR, which binded to Grb10 and inhibits apoptosis of renal proximal tubule cells by amplifying Akt signaling through a positive feedback loop. In additions, we also found that overexpression of miR 7 could significantly reduce the expression of HuR in CpG ODNs treated human lung cancer cells, through inhibiting the transduction of PI3K/Akt pathway. Therefore, as shown in Figure 5B, we presumed that during the treatment of TLR9 agonist CpG ODNs on human lung cancer cells, TLR9 signaling induced the expression of HuR via PIK3/Akt pathway. Up regulated HuR could bind to the loop sites of pri miR 7 and reduce the expression of miR 7, thereby synergizing the transduction of PI3K/ Akt pathway as a positive feedback loop, which ultimately resulted in enhanced growth and metastatic potential of human lung cancer cells.

Conclusions To our knowledge, it is the first time TLR9 signaling was identified could enhance the expression of HuR in human lung cancer cells. Importantly, in contrast to previous findings, we characterized that up regulation of HuR was contributed to TLR9 signaling enhanced growth and metastatic potential of human lung cancer through altering the expression of miR 7. Our findings indicated that HuR could act as regulator in regulating TLR9 signal ing associated biological effect in human lung cancer cells through a positive feedback loop, which might be helpful for the understanding of the potential role of HuR in tumor biology. Materials and methods Hu man lung cancer cell line 95D cells, NCI H727 cells, BE1 cells and SPCA/I cells were cultured at 37 C under 5% CO2 in completed RPMI 1640 medium.

HuR RNAi and corresponding control RNAi were purchased from Novus Biologicals. Akt inhibitor IV, AV-951 PI3K inhibitor and specific MEK inhibitor was purchased from Merck. All other reagents were purchased from Sigma Aldrich unless stated otherwise. Real time PCR assay Total cellular RNA and cDNA were prepared as previously described. HuR levels were measured by SYBR Green based Realtime PCR using Light Cycler.