The outcomes suggest that PDGFR and PDGFR- perform an critical function in Path-induced MiR-34a/c and PDGFR-/ are inversely correlated in normal and tumor lung tissue samples

The benefits recommend that PDGFR and PDGFR- enjoy an essential role in Trail-induced MiR-34a/c and PDGFR-/ are inversely correlated in regular and tumor lung tissue samples.order 870281-34-8 (a) qRT-PCR on 18 lung tumor and typical tissues. MiR-34a and miR-34c are downregulated in the tumors when compared to the normal lung tissues. (c) Box plots displaying miR-34a and miR-34c expression in 48 lung typical and most cancers tissues. (d) XY scatter plots demonstrating inverse correlation in between miR-34a/c and PDGFR-/. Two-tailed Student’s t check was used to verify the importance. P<0.05.MiR-34a/c and PDGFR-/ co-expression in vivo. (a) Immunohistochemistry and in situ hybridization on 107 lung cancer tissues samples. MiR-34a (blue) and PDGFR-/ (brown/red, respectively in RGB and each fluorescent red in Nuance converted image) expression was inversely related in lung cancers and the adjacent normal lung tissues. These serial sections were analyzed for miR-34a expression by in situ hybridization, followed by immunohistochemistry for PDGFR-/. (a) Representative example: Co-expression analysis of miR-34a and PDGFR-. Note lack of expression in the merged image (panel b) (fluorescent yellow = co-expression). (b) Representative example: miR-34a= blue (panel a), PDGFR-=red (panel b), co-expression= yellow (panel c). RGB= Regular Green Blue image of the ISH/lHC reaction shown in panels a-c. Scale bar indicates 50 m. The magnification is the same for all the panels apoptosis and that PDGFR inhibitor can sensitize NSCLC cells to TRAIL with important therapeutic consequences.Because PDGFR-/ regulate the PI3K/AKT pathway, notably involved in migration and invasion of different tumors [27,28], we investigated if miR-34a/c could influence NSCLC migration and invasion through PDGFR- and PDGFR- downregulation. To directly test the functional role of miR-34a/c in tumorigenesis, we overexpressed these two miRNAs or silenced PDGFR-/ in Calu-6 or H1703 cells. Intriguingly, we observed a significant decrease of the migratory and invasive capabilities of Calu-6 and H1703 cells after miR-34a or miR-34c overexpression (Figure 6a) as well after PDGFR- and PDGFR- downregulation (Figure 6b), confirmed also by scratch-wound assay (Figure 6c). To further verify that PDGFR- and PDGFRwere involved in tumorigenesis of NSCLC cells, miR-34a and -34c were transfected in Calu-6 cells alone or in combination with a plasmid overexpressing only the coding sequence and not the 3' UTR of PDGFR- and PDGFR-. MiR-34a/c enforced expression reduced migration and invasion of Calu-6 cells but overexpression of PDGFR- or MiR-34a and miR-34c overexpression or PDGFR-/ silencing increases the response of NSCLC cells to TRAILinduced apoptosis. (a) Western blot in Calu-6 cells after miR-34a, -34b and -34c forced expression. MiR-34a or miR-34c and not miR-34b forced expression decreases PDGFR expression levels and reduces the activation of the ERK1/2. (b) Western blot showing the inactivation of the Akt and ERKs pathways after PDGFR- silencing. (c) Caspase 3/7 assay. MiR-34a and -34c enforced expression in Calu-6 and H1703 semi-resistant cells, increases the response to TRAIL-induced apoptosis. (d) Caspase 3/7 assay showing that PDGFR- or PDGFR- silencing increases the response to TRAIL-induced apoptosis. (e) PDGFR- or PDGFR- overexpression in H460 TRAIL-sensitive cells decreases the response to the drug. (f) Combined treatment of PDGFR inhibitor (20 M) and different TRAIL concentrations (0-100-150 ng/ml) for 24h sensitizes NSCLC cells to TRAIL-induced apoptosis. P<0.001, P<0.05.MiR-34a and miR-34c overexpression or PDGFR-/ silencing decreases migratory and invasive capacity of NSCLC cells. (a) MiR-34a and -34c enforced expression reduces migratory and invasive capabilities of H1703 cells. (b) PDGFR- and PDGFR- silencing reduces migratory and invasive capabilities of Calu-6 cells. RFU= Relative Fluorescence Units. (c) Representative photographs of scratched areas of the confluent monolayer of Calu-6 cells transfected with miR-34a/c or control miRNA (miR-Ctr) at 0h, 12h and 24h after wounding with a pipet tip. Scale bar, 100 m. The magnification is the same for all the panels. (d) PDGFR- and PDGFR- overexpression partially rescues migratory and invasive capabilities of Calu-6 cells. P<0.05.PDGFR-, along with the two microRNAs, partially restored the migration and invasion capabilities, suggesting that miR-34a/c regulate NSCLC tumorigenesis, at least in part, through PDGFR-/ (Figure 6 d,e).Lung cancer is the leading cause of cancer death in both men and women worldwide1. The American cancer Society estimates 156,940 deaths from lung cancer in the United States for 2011 alone [29]. Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases and is a leading cause of cancer mortality [30].The high mortality rate associated with lung cancer has prompted numerous exhaustive efforts to identify novel therapeutic targets and treatment modalities for this deadly disease. Platelet-derived growth factor receptors (PDGFRs) and their ligands, platelet-derived growth factors (PDGFs) play critical roles in mesenchymal cell migration and proliferation. Abnormalities of PDGFR/PDGF are thought to contribute to a number of human diseases and especially malignancy [31]. MicroRNAs are small noncoding RNAs that show deregulation in most cancers. There is growing evidence that they play substantial roles in the pathogenesis and prognosis of human malignancies and in the resistance to chemotherapeutic drugs [32,33]. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is ineffective at treating TRAILresistant tumors. This resistance is challenging for TRAILbased anti-cancer therapies. In this study, we found that miR-34a and miR-34c are strongly downmodulated in both NSCLC cells and lung tumors compared to normal tissues. Enforced expression of miR-34a and miR-34c downregulated PDGFR- and PDGFR- mRNA and protein levels. Luciferase and western blot experiments demonstrated that PDGFR- and PDGFR- are direct targets of miR-34a and miR-34c but not of miR-34b. The resistance of many types of cancer to conventional chemotherapies is a major factor undermining successful cancer treatment. AKT activation also contributes to tumorigenesis and tumor metastasis, and as shown most recently, resistance to chemotherapy [26,34]. As a result, both in vitro and in vivo studies combining small molecule inhibitors of the PI3K/Akt pathway with standard chemotherapy have proven successful in attenuating chemotherapeutic resistance. Specifically, inhibiting AKT activity may be a valid approach to treat cancer and increase the efficacy of chemotherapy. Protein kinases are major regulators of most cellular signaling pathways. Among them, receptor tyrosine kinases (RTKs), such as PDGFR, play pivotal roles in promoting cellular growth and proliferation by transducing extracellular stimuli to intracellular signaling circuits [35]. A prominent component of the intracellular signaling machinery is the PI3K/ Akt(PKB)/mammalian target of rapamycin (PI3K/Akt[PKB]/ mTOR) pathway [36,37]. Aberrant activation of this pathway by mutation of any of multiple genes is known to occur in the majority of human cancers through various mechanisms [38,39]. In a previous work [26], we demonstrated that MET, through the activation of the PI3K/AKT pathway, induced tumorigenesis and TRAIL resistance in NSCLC. Therefore, we hypothesized that PDGFR-/, through the activation of the AKT pathway should be involved in TRAIL-induced apoptosis. Indeed, overexpression of miR-34a and miR-34c or downregulation of PDGFR-/ by siRNAs, highly increased the response of semi-resistant NSCLC cells to TRAIL-induced apoptosis. Importantly, combined treatment of a PDGFR inhibitor with TRAIL, increased apoptosis and reduced cell proliferation, as assessed by caspase 3/7 assay and MTT assays. Taken together, the results suggest that combined treatment of TRAIL with PDGFR inhibitors could sensitize a subset of lung tumors, expressing the PDGF receptors, to the drug. Moreover, it is well known that the PI3K/AKT, as well the ERK1/2 pathways regulate cellular migration and invasion of different cancers [40,41]. Here, we reported that miR-34a and miR-34c overexpression or PDGFR-/ silencing inhibited the migration and invasion capacity of Calu-6 and H1703 cells, compared to cells transfected with a scrambled miR or siRNA control. Enforced expression of PDGFR- or PDGFR- partially restored NSCLC migration and invasion supporting that the regulation of the expression of these receptors by miR-34a/c plays an important role in NSCLC tumorigenesis. However, we recognize that other miR-34a/c targets including c-Met [16] and AXL [42] could also be involved. While this manuscript was in preparation Silber et al. reported that miR-34a expression was lower in proneural gliomas compared to other tumor subtypes and identified PDGFR- as a direct target of miR-34a [43].Here, we report that not only miR-34a but miR-34c also downregulates PDGFR- in NSCLC cells. Moreover, we demonstrate that PDGFR- is a miR-34a/c direct target while we did not see any significant effect on the expression of PDGFR- and PDGFR- after miR-34b enforced expression. Remarkably, our study shows that inhibition or downregulation of PDGFR- and PDGFR- by miR-34a/c antagonizes tumorigenicity and increases sensitivity to TRAIL-induced cell death with important therapeutic application for future antitumor therapy of lung cancer.Human H460, A549, H1299, H1703 cell lines were grown in RPMI medium containing 10% heat-inactivated fetal bovine serum (FBS) and with 2mM L-glutamine and 100Uml-1 penicillin-streptomycin. Calu-6 cells were grown in MEM supplemented with 10% fetal bovine serum, 2mM L-glutamine and 100Uml-1 penicillintreptomycin. 9 lung tumors (including adenocarcinoma and squamous cell carcinoma) and their normal counterparts were kindly provided by Dr. S.P. NanaSinkam, Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH. 48 lung normal and tumor tissue samples were provided from the Department of Pathology, Ohio State University. All human tissues were obtained according to a protocol approved by the Ohio State Institutional Review Board.Calu-6 cells were cotransfected with 1g of p3'UTR-PDGFR, p3'UTR-PDGFR- or with p3'UTRmut-PDGFR- and p3'UTRmut-PDGFR-, 1 g of a Renilla luciferase expression construct pRL-TK (Promega) by using Lipofectamine 2000 (Invitrogen). Cells were harvested 24h post-transfection and assayed with Dual Luciferase Assay (Promega) according to the manufacturer's instructions. Three independent experiments were performed in triplicate(s.d.). Cell viability was examined with 3- (4,5dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide (MTT)Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer's protocol. Metabolically active cells were detected by adding 20 l of MTT to each well. After 1 h of incubation, the plates were analyzed in a Multilabel Counter (Bio-Rad Laboratories).Cells were cultured to 50% confluence and transiently transfected for 72h using Lipofectamine 2000 with 100 nM antiPDGFR- and/or with 100nM anti-PDGFR- SMARTpool siRNAs or control siRNAs (Dharmacon), a pool of four target specific 205 nt siRNAs designed to knock down gene expression.Total proteins from NSCLC were extracted with radioimmunoprecipitation assay (RIPA) buffer (0.15mM NaCl, 0.05mM Tris-HCl, pH 7.5, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate and 1% Nonidet P40). Sample extract (50 g) was resolved on 7.52% SDSolyacrylamide gels (PAGE) using a mini-gel apparatus (Bio-Rad Laboratories) and transferred to Hybond-C extra nitrocellulose. Membranes were blocked for 1h with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20, incubated overnight with primary antibody, washed and incubated with secondary antibody, and visualized by chemiluminescence. The following primary antibodies were used: anti-PDGFR-, anti-PDGFR-, antiERK1/2, anti-p-ERKs, anti-pAKT, anti-total AKT, anti-GAPDH antibodies (Cell Signaling). A secondary anti-rabbit or antimouse immunoglobulin G (IgG) antibody peroxidase conjugate (Chemicon) was used.In situ hybridization (ISH) was carried out on deparaffinized human lung tissues using previously published protocol[44], which includes a digestion in pepsin (1.3 mg/ml) for 30 minutes.The probe cocktail and tissue miRNA were co-denatured at 60 for 5 minutes, followed by hybridization at 37 overnight and a stringency wash in 0.2X SSC and 2% bovine serum albumin at 4 for 10 minutes. The probe-target complex was seen due to the action of alkaline phosphatase on the chromogen nitroblue tetrazolium and bromochloroindolyl phosphate (NBT/BCIP). Negative controls included the use of a probe, which should yield a negative result in such tissues (scrambled miRNA). No counterstain was used, to facilitate co-labeling for PDGFR- and PDGFR- protein. After in situ hybridization for the miRNAs, as previously described (Nuovo et al., 2009), the slides were analyzed for immunohistochemistry using the optimal conditions for PDGFR- (1:100, cell conditioning for 30 minutes) and PDGFR- (1:200, cell conditioning for 30 minutes). For the immunohistochemistry, we used the Ultrasensitive Universal Fast Red or DAB systems from Ventana Medical Systems. The percentage of tumor cells expressing PDGFR-, PDGFR- and miR-34a, was then analyzed with emphasis on co-localization of the respective targets. Co-expression analysis was done with the Nuance system (Cambridge Research Institute) per the manufacturer's recommendations.Real-time PCR was performed using a standard TaqMan PCR Kit protocol on an Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems). 17947497The 10 l PCR reaction included 0.67 l RT product, 1 l TaqMan Universal PCR Master Mix (Applied Biosystems), 0.2 mM TaqMan probe, 1.5 mM forward primer and 0.7 mM reverse primer. The reactions were incubated in a 96-well plate at 95 for 10 min, followed by 40 cycles of 95 for 15 s and 60 for 1 min. All reactions were run in triplicate. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The comparative CT method for relative quantization of gene expression (Applied Biosystems) was used to determine miRNA expression levels. The y axis represents the 2(-CT), or the relative expression of the different miRs. MiRs expression was calculated relative to U44 and U48 rRNA. Experiments were carried out in triplicate for each data point, and data analysis was performed by using software (Bio- Rad).For detection of caspase 3/7 activity, cells were cultured in 96-well plates, in triplicate, treated with TRAIL (100-150ng/ml) and analyzed using Caspase-Glo 3/7 Assay kit (Promega) according to the manufacturer’s instructions.