Background
Accumulating evidence showed that Toll-like receptors 9 (TLR9), which were mainly expressed on immune cells, were also functional expressed on lung cancer cells [
1‐
3]. And TLR9 signaling could alter biological character of lung cancer cells including promoting the proliferation and enhancing the metastatic potential of tumor cells, indicating that activation of TLRs signaling in lung cancer cells could contribute to the progression of lung cancer [
4‐
8]. Recent literatures further showed that miRNAs, a major class of gene expression regulators, played critical roles in regulating the biological effects of TLR9 signaling pathway on various cells. As such, miR-17-92 cluster might regulate the biological effect of CpG ODNs on chronic lymphocytic leukemia (CLL) cells [
9]. One newly evidence also showed that upregulation of miRNA-574-5p was critical for TLR9 signaling enhanced tumor progression of human lung cancer [
10]. However, the underlying mechanism regulating the expression of TLR9 signaling-associated miRNAs in lung cancer cells remains largely unknown.
MicroRNA-7 (miR-7), a unique member of miRNAs, played an important role in the progression of various tumors including lung cancer [
11‐
13]. Mechanistic evidence showed that miR-7 could regulate the transduction of Akt pathway, which was critical for growth and metastasis of tumor cells [
14,
15]. Our most recent study also showed that downregulation of intrinsic miR-7 was important for TLR9 signaling enhanced growth and metastatic potential of human lung cancer cells [
16]. However, the mechanism that downregulation of miR-7 in TLR9 signaling treated lung cancer cells remains to be investigated. Recent evidence showed that Human antigen R (HuR), a post-transcriptional regulator of gene expression, played a key role in stabilizing multiple mRNAs in cellular biology [
17‐
19]. Interestingly, one research work further showed that HuR could regulate the expression of miR-7 in nonneural cells in brain [
20]. However, whether HuR was also involved in the expression of miR-7 in TLR9 signaling treated lung cancer cells still remains to be elucidated. Here, we carefully evaluated the potential role of HuR in the expression of miR-7 on TLR9 signaling treated human lung cancer cells.
Discussion
Accumulating evidence showed that HuR was expressed in various tumor cells and played an important role in the biology of various tumor cells through post-transcriptionally regulating the stabilization of multiple AU-rich element-bearing mRNAs [
17,
22,
23]. Such as, Kurosu et al. reported that HuR could keep an angiogenic switch on by stabilising mRNA of VEGF and COX-2 in tumor endothelium [
18]. Moreover, Blaxall et al. found that the expression of HuR was important for the maintenance and progression of tumor cells in neoplastic lung tissue [
24]. Recently, Kim et al. further reported that HuR was highly expressed on clinical lung cancer tissues and stabilizes the expression of cyclooxygenase-2 (Cox2) [
25]. Our present work extended these previous works by demonstrating that TLR9 signaling could enhance the expression of HuR. Importantly, we further found that up-regulation of HuR was contributed to TLR9 signaling enhanced growth and metastatic potential of human lung cancer cells. These finding might support the fact that HuR could be an important intrinsic regulator in distinct tumor cells, which ultimately contributed to tumor biology.
Recently, miR-7 was reported played an important role in regulating the biology of various tumor cells through repressing the expression of different target molecules. In previous study, we reported down-regulation of intrinsic miR-7 was critical for TLR9 signaling enhanced progression of human lung cancer cells through altering the expression of PIK3R3 [
16]. As a tumor suppressor, the expression of miR-7 was commonly repressed in tumor cells. Such as, Kong et al. reported that activated macrophage-derived small molecule could reduce the expression of miR-7 in gastric tumor cells [
26]. Reddy et al. reported that homeodomain transcription factor (HoxD10) could regulate the expression of miR-7 through binding to the promoter site of miR-7 in breast cancer cells [
27]. Our current work further reported that HuR could regulate the expression of miR-7 in human lung cancer cells. Consistently, Choudhury et al. found that HuR could bind to the conserved terminal loop of pri-miR-7 and regulate the expression of miR-7 in nonneural cells in brain tissue [
21]. In addition, it should be noted that our previous data also showed the activity of miR-7 promoter also decreased in TLR9 signaling treated human lung cancer cells [
16]. Combining these data suggested that the underlying mechanism regulating expression of distinct miRNAs such as miR-7 in different cells was distinct and complex, which related to different transcriptional and post-transcriptional mechanisms. Therefore, the related transcriptional mechanism still remains to be further elucidated.
Some literatures showed that the expression of HuR was regulated through transcriptional and post-transcriptional mechanisms [
28,
29]. For example, Mansfield et al. reported that Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation [
30]. Dai et al. further found that HuR could autoregulate its expression by promoting alternative polyadenylation site usage [
31]. However, the possible signaling 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 [
16]. In present study, we further demonstrated that TLR9 signaling could enhance the expression of HuR through Akt pathway, which ultimately 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 [
21]. 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 (Additional file
1: Figure S1), through inhibiting the transduction of PI3K/Akt pathway [
16]. 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 [
20], 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 signaling 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
Reagents and cell line
The following oligonucleotides (ODNs) were used and purchased from Integrated DNA Technologies (Coralville, IO): CpG ODNs 2216 5′-GGGGGACGATCGTCGGGGG-3′; control, ODNs1612: 5′-GCTAGAGCTTAGGCT-3′. Human 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 (GIBICO, Grand land, NY, USA) medium. HuR RNAi and corresponding control RNAi were purchased from Novus Biologicals (No.H00001994-R01). Akt inhibitor IV, PI3K inhibitor (LY294002) and specific MEK (Mitogen-activated protein kinase) inhibitor (U0126) 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 [
16]. HuR levels were measured by SYBR Green-based Realtime PCR using Light Cycler (Roche, USA). Reverse transcriptase reactions and real-time PCR were performed according to the manufacturer’s protocols. The sequences were as follows: HuR primers: 5′- CCTGTTCAGCAGCATTG-3′ and 5′- GGCGAGCATACGACAC-3′. Cycle threshold (CT) values were compared against a standard curve to estimate starting amounts of mRNA, and the relative expression of HuR mRNA was estimated by normalizing these values against 18S rRNA CT values were generated using a preoptimized 18S rRNA primer set (Applied Biosystems, FosterCity, CA). The relative expression of miR-7 was performed according to our previous description [
16].
Plasmid construction and preparation
The gene for the HuR (NM_001419.2) were expanded by RT-PCR from human mRNA derived from 95D cells using forward primer (5′- CGGAATTCAATACAATGTCTAATGGTTATG-3′) and a reverse primer (5′-GGGGTACCATTGGCGCAAAATGAG-3′) and then subcloned into EcoR I and Kpn I sites of pcDNA3.1 vector (Invitrogen Corp., San Diego, California, USA) to generate pcDNA3.1-HuR plasmid (termed as pHuR). Clone identity was verified using restriction digest analysis and plasmid DNA sequencing. Endotoxin-free plasmids were obtained using Endofree plasmid mega kit (QIAGEN GmbH, Hilden, Germany). Then, plasmids were transiently transferred into the 95D cells using Lipofectamine-2000 (Invitrogen) in different following experiments according to the manufacturer’s instruction.
Cell proliferation assays
95D cells transiently transfected with 10 nmol HuR RNAi or Scramble control using Lipofectamine-2000 (Invitrogen) were seeded at 3 × 103 cells each well and incubated in the presence of 10 μg/ml CpG ODNs at 37°C in 5% CO2 in 96 well plates for 72 hrs. Assessment of cell proliferation was measured in terms of optical absorbance (OD) per well by a semi-automated tetrazolium-based colorimetric assay using MTT.
BrdU labeling
95D cells transiently transfected with 10 nmol HuR RNAi or Scramble control were treated with CpG ODNs as described in the previous report [
7]. After 48 hrs, final concentration of 5 mmol/ml BrdU (5-bromo-2-deoxyuridine; Sigma) was added. 4 hrs later, 95D cells were collected and the proliferation was analyzed by FACS.
Invasion assay
The invasive assay was done as described previously [
16]. 95D cells transiently transfected with 10 nmol HuR RNAi or control RNAi were placed in the upper wells in the presence of 10 μg/ml CpG ODNs or control ODNs and the lower wells were filled with fibroblast-conditioned medium. After incubation for 24 hrs, cells on the lower surface of the membrane were stained by the H&E method and counted under a light microscope (×200).
Western blotting
Western blotting was performed on cytosolic cellular extracts as described previously [
16]. The membrane was washed in 5% skim milk in phosphate buffered saline + 0.03% Tween 20 (PBS-T) for 1 hrs in order to block nonspecific protein binding sites on the membrane. Immunoblotting was performed using a monoclonal antibody to HuR (Santa Cruz, SC-5261) at a dilution of 1/1000 in a nonfat milk-Tris buffer. The membrane was then washed and subsequently probed with a corresponding secondary anti-mouse antibody conjugated to horseradish peroxidase (Amersham Life Sciences) at a dilution of 1:5000 and developed with chemiluminescence (Pierce, IL). The membrane was then exposed to X-ray film (Kodak, NY) which was subsequently developed.
Statistical analyses
Statistical analyses of the data were performed with the aid of analysis programs in SPSS12.0 software. Statistical evaluation was performed using one-way analysis of variance (ANOVA; p < 0.05) using the program PRISM 5.0 (GraphPad Software Inc., San Diego, CA, USA).
Acknowledgements
This work was supported by International Cooperation Foundation of Guizhou Province (2010–7031), National Natural Science foundation of China (31370918, 81260398), Program for New Century Excellent Talents in University, Ministry of Education of China (NCET-12-0661), Specific Foundation for the Scientific Educational Talent of President of Guizhou Province (09C457), Project of Guizhou Provincial Department of Science and Technology (2009C491) and Zunyi Medical College Start-up Fund (2008 F-329).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LYJ and WCH executed the western blotting, Realtime-PCR experiments and drafted the manuscript. ZY and LZY both participated in the RNAi experimental procedures. HY and CC performed MTT and Invasion assay. ZSF and LJM performed inhibition assay and BrdU labeling assay. WZK contributed to the design of the study, the analysis of the data, and drafted the manuscript. XL supervised the project, making substantial contributions to the concept and design of the study, analyzing and interpreting the data, and writing the manuscript. All authors read and approved the final manuscript.