TLR4/ROS/miRNA-21 Pathway Underlies Lipopolysaccharide Instructed Primary Tumor Outgrowth In Lung Cancer Patients

Background and purpose:Lung cancer is the leading cause of cancer deaths worldwide. Currently, the outcome of lung cancer patients still remains poor, thus, exploration of crucial effectors involved in cancer progression was urgently needed. Recently,accumulating studies reported that pulmonary infection facilitated lung cancer progression.Lipopolysaccharide (LPS),a major component in the outer membrane of Gram-negative bacteria, directly promotes the tumor progression. Expression of Toll-like receptor 4 (TLR4), which recognizes LPS from Gram-negative bacteria, was increased and reflected disease progress of lung cancer patients. These findings regarded TLR4 activation by LPS as an important stimulus of lung cancer progression.However, whether primary human lung cancer outgrowth respond to LPS and underlying mechanisms remain unclear.Micro RNAs (miRs), which are small non-coding RNA molecules capable of regulating gene expression at posttranscriptional level,have fundamental roles in tumor progression. MiR-21 was described as an oncomiR and implicated in tumor progression of various cancers. Of interest, up-regulation of miR-21 was associated with lung cancer diagnosis and prognosis, indicating a potential role of miR-21 in human lung cancer progression. Recent studies reported the involvement of miR-21 in LPS-induced immune response. These findings indicated a possible involvement of miR-21 in LPS-induced lung cancer progression.In this study, we characterized the effect of LPS on outgrowth of primary human lung cancer cells and evaluated the potential role of miR-21 in this process. We demonstrated that miR-21 was essentially required for tumor growth of primary human lung cancer. TLR4 activation by LPS in primary human lung cancer cells resulted in increased ROS production, which in turn induced up-regulation of miR-21 and led to tumor outgrowth. Our findings could facilitate further understanding of lung cancer pathogenesis and be helpful for developing novel therapeutic strategies for lung cancer.Materials and MethodsEthics statementAll patients provided their written informed consent to participate in this study.Studies with clinical samples were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Animal experiments were performed according to the Guide for Care and Use of Medical Laboratory Animals. Human and animal experiments were performed with an approval from Tongji Institutional Ethics Committee.PatientsTotally 50 human lung cancer patients with pulmonary infection of Gam-negative bacteria were enrolled in this study. The diagnosis of lung cancer was confirmed by pathology. Clinical information of lung cancer patients was summarized in Table 1.Subjects with autoimmune diseases or under immunosuppressive treatment were excluded.MiceFemale BALB/c nude mice of 6-8 weeks old were purchased from Shanghai Laboratory Animal Center, CAS. All mice were housed under specific pathogen-free conditions.Cell culture and reagentsHuman lung cancer cells were freshly isolated from surgical tissues with Clonogenic Tumor Cell Isolation Kit (Cell Biolabs) according to the manual's instructions. Cells were cultured in complete RPMI 1640 medium containing 10%heat-inactivated fetal bovine serum (Gibco) supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate at 37? under 5% C02.Lipopolysaccharide (LPS) was purchased from Sigma. psiRNA vector expressing shRNA targeting human TLR4 gene (psirna42-htlr4) and control were purchased from Invivogen. Expression vector for human miR-21 (MI0000077) and control were purchased from Origene. Human miR-21 shRNA (shADV-215486) and control were purchased from Vector Biolabs. Tempol was purchased from Santa Cruz. All reagents were used according to manual's instructions.Tumor outgrowth in vitroTumor outgrowth in vitro was detected by analyzing proliferative expansion of primary human lung cancer cells with MTT assay. Briefly, cells were seeded at 4×103 cells each well and incubated in the presence or absence of LPS (0.1-10 ?g/ml) in 96-well plates for 72 h. Assessment of cell proliferation was measured with MTT cell proliferation kit (Cayman Chemical) according to the manual's instructions.Tumor outgrowth in vivoTumor outgrowth of primary human lung cancer cells in nude mice was assessed as previously described. Briefly, groups of nude mice were subcutaneously injected with 4×106 primary human lung cancer cells suspended in 200 ?l of PBS. Control mice receive equal volume of PBS. Immediately before subcutaneous injection, primary human lung cancer cells were treated with LPS (10 ?g/ml) for 24 h or PBS as a control. The size of the tumor was measured at days 3, 6 and 9 after implantation by digital calipers. Each tumor measurement was taken in duplicate by two different investigators.Real-time PCRTotal RNA was extracted with RNeasy Mini Kit (Qiagen). cDNA was synthesized with Maxima First Strand cDNA Synthesis Kits (Thermo Scientific). The primers were obtained from Applied Biosystems. Quantitative PCR analyses were carried out in duplicate to detect mRNA expression with Maxima SYBR Green qPCR Master Mixes (Thermo Scientific). GAPDH was used as an internal control. TaqMan miRNA assays (Applied Biosystems) were used to detect miRNA expression levels with U6 RNA as an internal control.Flow cytometryFor flow analysis, 0.5 million cells were stained with PE-conjugated anti-human TLR4 antibody (12-9917-42, eBioscience) or the isotye control (12-4724-81,eBioscience), and analyzed on a FACSCalibur flow cytometer (BD). ROS levels were detected with CellROX. Deep Red Reagent (Life Technologies) according to manual's instructions. Collected data were analyzed with FlowJo software (Tree Star).Results1.LLPS stimulation promotes tumor outgrowth of primary human lung cancer.To explore the potential role of LPS in outgrowth of primary human lung cancer,freshly isolated human lung cancer cells were stimulated with an increasing dose of LPS and detected for their growth capacity. Results showed that LPS stimulation promoted tumor growth of primary human lung cancer cells in a dose dependent manner. TLR4 was readily detectable in these primary human lung cancer cells and could be up-regulated by LPS stimulation. To detect the potential role of TLR4 signaling in LPS-mediated tumor outgrowth, primary human lung cancer cells were transfected with TLR4 shRNA or control and then stimulated with LPS. We found that transfection of TLR4 shRNA reduced TLR4 expression at both mRNA and protein levels, and limited LPS-induced tumor outgrowth. These data suggest TLR4 as the crucial receptor of LPS-induced primary human lung cancer outgrowth.To confirm the effect of LPS on primary lung cancer outgrowth in vivo, freshly isolated human lung cancer cells were pretreated with or without LPS and injected subcutaneously into nude mice. We found that treatment with LPS promoted growth capacity of primary human lung cancer cells in vivo. Knockdown of TLR4 blocked the enhanced tumor growth of primary human lung cancer in vivo.In essence, these results demonstrate an enhanced outgrowth of primary human lung cancer by LPS trigged TLR4 signaling.2.Mir-21 is required for LPS-mediated primary human lung cancer outgrowthTo evaluate the potential role of miR-21 in LPS-induced primary tumor outgrowth, we detected the expression of miR-21 in freshly isolated human lung cancer cells in response to LPS. We observed that LPS increased miR-21 expression in a dose dependent manner. Knockdown of TLR4 abrogated the increased miR-21 expression by LPS in primary human lung cancer cells. When primary human lung cancer cells were transfected with miR-21 shRNA and stimulated with LPS, we found that transfection with miR-21 shRNA reduced miR-21 expression and blocked LPS-induced tumor outgrowth.To confirm the potential role of miR-21 in LPS-induced primary human lung cancer outgrowth in vivo, nude mice were challenged with freshly isolated human lung cancer cells that were transfected with miR-21 shRNA and pretreated with LPS.Results showed that down-regulation of miR-21 attenuated LPS-enhanced tumor growth of primary human lung cancer in nude mice. Collectively, these results reveal that increased miR-21 is required for LPS to enhance tumor growth of primary human lung cancer.3.Mir-21 licenses outgrowth of primary human lung cancerGiven the important role of miR-21 in LPS-induced tumor outgrowth, we detected the function of miR-21 itself in primary lung cancer growth. Transfection of freshly isolated human lung cancer cells with miR-21 expression vector resulted in elevated expression of miR-21 and enhanced tumor outgrowth. Transfection with miR-21 shRNA ameliorated outgrowth of primary human lung cancer. To confirm these results in vivo, nude mice were injected with primary human lung cancer cells that were transfected with miR-21 expression vector or miR-21 shRNA respectively.We found that increased miR-21 expression enhanced tumor outgrowth, while decreased miR-21 expression attenuated tumor outgrowth in vivo. These findings regard miR-21 as an essential oncomiRNA for tumor growth of primary human lung cancer.Phosphatase and tensin homolog (PTEN) andprogrammed cell death 4 (PDCD4)are well-known targets of miR-21. We determined whether PTEN and PDCD4 were critical targets of miR-21 in primary human lung cancer cells. Results showed that transfection with miR-21 expression vector decreased expressions of PTEN and PDCD4. Vice versa, transfection with miR-21 shRNA increased expressions of PTEN and PDCD4. Of important, overexpression of PTEN and PDCD4 blocked the pro-cancer activity of miR-21 in lung cancer outgrowth. These data identify PTEN and PDCD4 as important targets of miR-21 in primary human lung cancer.4.Elevated ros production confers enforced mir-21 expression by LPSReactive oxygen species (ROS) were involved in TLR4-triggered immune response and gastric cancer progression. We detected the ROS levels in freshly isolated human lung cancer cells in response to LPS. Results showed that LPS induced ROS production in primary human lung cancer cells. Knockdown of TLR4 abrogated the LPS-increased ROS production. To evaluate the function of ROS in LPS-induced primary human lung cancer outgrowth, freshly isolated human lung cancer cells were treated with LPS in the presence of Tempol as a ROS scavenger.Results showed that Tempol abrogated LPS-enhanced primary lung cancer outgrowth.We detected whether ROS was required for LPS-induced miR-21 expression, and found that Tempol inhibited the elevated miR-21 expression in response to LPS.To determine the effect of ROS on LPS-induced primary human lung cancer outgrowth in vivo, nude mice were injected with freshly isolated human lung cancer cells that were pretreated with LPS in the presence or absence of Tempol. Results showed that Tempol abrogated the enhanced tumor outgrowth induced by LPS in vivo.Collectively,elevated ROS production is critical for LPS to induce miR-21 expression and primary lung cancer outgrowth.5.TLR4 expression is correlated with mir-21 expression and ROS production in lung cancer patientsTo elucidate the in vivo relevance of our findings in clinical patients, we detected expressions of TLR4 and miR-21 in tumor tissues and adjacent tissues. Results showed that TLR4 and miR-21 expressions were significantly higher in tumor tissues.Further, freshly isolated, untreated human lung cancer cells were assayed for expressions of TLR4, miR-21 and ROS levels. Analysis showed that TLR4 expression levels were correlated with their miR-21 expressions and ROS levels. ROS levels in primary human lung cancer cells were correlated with their miR-21 expressions.These results suggest an involvement of TLR4/ROS/miR-21 pathway in tumor progression of lung cancer patients.ConclusionActivation of TLR4 with LPS resulted in elevated ROS production in primary human lung cancer cells, which increased miR-21 expression and tumor outgrowth.The close correlation of TLR4 expression with miR-21 and ROS levels in freshly isolated, untreated human lung cancer cells suggested a TLR4/ROS/miR-21 pathway through which Gram-negative bacteria facilitates lung cancer progression in clinical patients. These results reveal a new mechanism through which Gram-negative bacteria facilitate lung cancer pathogenesis and provide clues for developing new therapeutics for lung cancer patients.