CLEC4A and CLEC12B C-type lectin receptors mediate interactions with Pneumocystis cell wall components.

Introduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF).Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis.Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs.Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing ß-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.

The CT and PET/CT findings in primary pulmonary lymphoepithelioma-like carcinoma with pathological correlation: a study of 215 cases.

AIM: To investigate the computed tomography (CT) and integrated positron-emission tomography (PET)/CT findings of primary pulmonary lymphoepithelioma-like carcinoma (PLELC). MATERIALS AND METHODS: The imaging and histopathological data of 215 patients with PLELC confirmed at histopathology were analysed retrospectively. All patients underwent CT, and 70 underwent PET/CT. None of the cohort had nasopharyngeal lymphoepithelioma-like carcinoma. RESULTS: The PLELC was demonstrated as a solitary nodule/mass in 188 cases (188/215, 87%), multiple nodules/masses in 12 cases (12/215, 6%), lobar or segmental consolidation in 15 cases (15/215, 7%). The tumour showed a well-defined margin in 171 cases (171/215, 80%), lobular sign in 177 cases (177/215, 82%), and spicule sign in 91 cases (91/215, 42%). Most of the cases showed homogeneous density in unenhanced CT (128/215, 60%), and vascular shadows inside the tumour in the arterial stage were found in 105 cases (105/158, 66%). Involvement of the bronchus was found in 154 cases (154/215, 72%). Hilar or mediastinal lymph nodes were enlarged in 160 patients (160/215, 74%). Seventy cases demonstrated avid 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) uptake on PET/CT. The range of maximum standardised uptake values (SUVmax) was 2.1-28.5 (14 ± 5.93). Microscopic pathological classification of 124 resected specimens included 87 cases of the Regaud type and 37 cases of the Schmincke type. Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) was positive in all 215 cases. CONCLUSION: PLELC should be suspected when a large, lobulate, well-defined lung tumour with homogeneous density, vascular encasement, and high 18F-FDG uptake is found. Moreover, EBERs are helpful in patients with suspected PLELC.

The debatable presence of PIWI-interacting RNAs in invasive breast cancer.

Numerous factors influence breast cancer (BC) prognosis, thus complicating the prediction of outcome. By identifying biomarkers that would distinguish the cases with poorer response to therapy already at the time of diagnosis, the rate of survival could be improved. Lately, Piwi-interacting RNAs (piRNAs) have been introduced as potential cancer biomarkers, however, due to the recently raised challenges in piRNA annotations, further evaluation of piRNAs' involvement in cancer is required. We performed small RNA sequencing in 227 fresh-frozen breast tissue samples from the Eastern Finnish Kuopio Breast Cancer Project material to study the presence of piRNAs in BC and their associations with the clinicopathological features and outcome of BC patients. We observed the presence of three small RNAs annotated as piRNA database entries (DQ596932, DQ570994, and DQ571955) in our samples. The actual species of these RNAs however remain uncertain. All three small RNAs were upregulated in grade III tumors and DQ596932 additionally in estrogen receptor negative tumors. Furthermore, patients with estrogen receptor positive BC and higher DQ571955 had shorter relapse-free survival and poorer BC-specific survival, thus indicating DQ571955 as a candidate predictive marker for radiotherapy response in estrogen receptor positive BC. DQ596932 showed possible prognostic value in BC, whereas DQ570994 was identified as a candidate predictive marker for tamoxifen and chemotherapy response. These three small RNAs appear as candidate biomarkers for BC, which could after further investigation provide novel approaches for the treatment of therapy resistant BC. Overall, our results indicate that the prevalence of piRNAs in cancer is most likely not as comprehensive as has been previously thought.

Silencing Ribosomal Protein L22 Promotes Proliferation and Migration, and Inhibits Apoptosis of Gastric Cancer Cells by Regulating the Murine Double Minute 2-Protein 53 (MDM2-p53) Signaling Pathway.

BACKGROUND The aim of this study was to investigate the effect of ribosomal protein L22 (RPL22) on gastric cancer (GC) cell proliferation, migration, and apoptosis, and its correlation with the murine double minute 2-protein 53 (MDM2-p53) signaling pathway. MATERIAL AND METHODS The RPL22 expression in GC tissues and cells was detected by quantitative reverse transcription-polymerase chain reaction and western blotting. RPL22 was overexpressed in the MKN-45 cells by the transfection of a vector, pcDNA3.1 (pcDNA)-RPL22, whereas it was silenced in the MGC-803 cells by the transfection of short interfering (si) RNA (si-RPL22). Flow cytometric analysis, cell viability assays, wound healing assays, and transwell assays were utilized to explore the influences of RPL22 on the apoptosis, proliferation, migration, and invasion. Nutlin-3 (an MDM2-p53 inhibitor) was used to inhibit MDM2-p53 signaling. RESULTS The RPL22 expression was downregulated in GC tissues and cells. It was significantly lower in the advanced GC tissues than in the early GC tissues, and was significantly lower in the lymphatic metastatic tissues than in the non-lymphatic metastatic tissues. The transfection of si-RPL22 accelerated the ability of GC cells to proliferate and metastasize, whereas apoptosis was dampened. The transfection of pcDNA-RPL22 exerted the opposite effect on the GC cells; MDM2 expression was upregulated in RPL22-silenced GC cells, while the expression of p53 was downregulated. In vitro, treatment with nutlin-3 reversed the promoting effects of si-RPL22 on GC progression. CONCLUSIONS In vitro, the silencing of RPL22 aggravates GC by regulating the MDM2-p53 signaling pathway.

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells.

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell-cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.

Deep Sequencing of Small RNAs from Neurosurgical Extracellular Vesicles Substantiates miR-486-3p as a Circulating Biomarker that Distinguishes Glioblastoma from Lower-Grade Astrocytoma Patients.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM; astrocytoma grade IV) biology and are novel sources of biomarkers. EVs released from GBM tumors can cross the blood-brain-barrier into the periphery carrying GBM molecules, including small non-coding RNA (sncRNA). Biomarkers cargoed in circulating EVs have shown great promise for assessing the molecular state of brain tumors in situ. Neurosurgical aspirate fluids captured during tumor resections are a rich source of GBM-EVs isolated directly from tumor microenvironments. Using density gradient ultracentrifugation, EVs were purified from cavitron ultrasonic surgical aspirate (CUSA) washings from GBM (n = 12) and astrocytoma II-III (GII-III, n = 5) surgeries. The sncRNA contents of surgically captured EVs were profiled using the Illumina® NextSeqTM 500 NGS System. Differential expression analysis identified 27 miRNA and 10 piRNA species in GBM relative to GII-III CUSA-EVs. Resolved CUSA-EV sncRNAs could discriminate serum-EV sncRNA profiles from GBM and GII-III patients and healthy controls and 14 miRNAs (including miR-486-3p and miR-106b-3p) and cancer-associated piRNAs (piR_016658, _016659, _020829 and _204090) were also significantly expressed in serum-EVs. Circulating EV markers that correlate with histological, neuroradiographic and clinical parameters will provide objective measures of tumor activity and improve the accuracy of GBM tumor surveillance.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

A kinome-wide shRNA screen uncovers vaccinia-related kinase 3 (VRK3) as an essential gene for diffuse intrinsic pontine glioma survival.

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.

Small RNAs in Rat Sperm Are a Predictive and Sensitive Biomarker of Exposure to the Testicular Toxicant Ethylene Glycol Monomethyl Ether.

Testicular histology and semen parameters are considered the gold standards when determining male reproductive toxicity. Ethylene glycol monomethyl ether (EGME) is a testicular toxicant with well-described effects on histopathology and sperm parameters. To compare the predictivity and sensitivity of molecular biomarkers of testicular toxicity to the traditional endpoints, small RNAs in the sperm were analyzed by next generation RNA-sequencing (RNA-seq). Adult rats were exposed to 0, 50, 60, or 75 mg/kg EGME by oral gavage for 5 consecutive days. Testis histology, epididymal sperm motility, and sperm small RNAs, including microRNAs (miRNAs), mRNA fragments, piwi-interacting RNAs (piRNAs), and tRNA fragments (tRFs), were analyzed 5 weeks after cessation of exposure. Testicular histology showed a significant dose-dependent increase in retained spermatid heads (RSH), while sperm motility declined with increasing dose. RNA-sequencing of sperm small RNAs was used to identify significant dose-dependent changes in percent mRNA fragments (of total reads), percent miRNAs (of total reads), average tRF length, average piRNA length, and piRNA and tRF length-distributions. Discriminant analysis showed relatively low predictivity of exposure based on RSH or motility compared to the average read length of all assigned RNAs. Benchmark dose (BMD) modeling resulted in a BMD of 62 mg/kg using RSH, whereas average read length of all assigned RNAs resulted in a BMD of 47 mg/kg. These results showed that sperm small RNAs are sensitive and predictive biomarkers of EGME-induced male reproductive toxicity.

Mitochondrial PIWI-interacting RNAs are novel biomarkers for clear cell renal cell carcinoma.

PURPOSE: PIWI-interacting RNAs (piRNAs) have been suggested to serve as biomarkers in cancer. In this study, we validated the expression profile of two piRNAs derived from mitochondria, piR-34536 and piR-51810, in tissue and serum of a cohort of clear cell renal cell carcinoma (ccRCC) patients. METHODS: Tissue and serum samples of patients with ccRCC were collected prospectively in our biobank. Total RNA was isolated from 118 ccRCC tissues, 75 normal renal tissues as well as 30 serum samples from patients with ccRCC, and 15 serum samples from patients with non-malignant diseases. The expression of piRNAs was determined using quantitative real-time PCR. RESULTS: Both piR-34536 and piR-51810 were downregulated in ccRCC compared to non-malignant renal tissue. Decreased tissue piRNA levels were significant and independent predictors of shortened progression-free, cancer-specific and overall survival of ccRCC patients. The piRNA levels in serum did not differ in ccRCC patients and control subjects. CONCLUSIONS: The expression of piR-34536 and piR-51810 in ccRCC tissues may be used as prognostic biomarkers in ccRCC.

Functional characterization of RNA fragments using high-throughput interactome screening.

Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules such as miRNAs or siRNAs, also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specific and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interactions of seven RFs originating from tRNA, snoRNA and snRNA. By integrating our results with publicly available data on physical protein-protein interactions, we constructed an RF interactome network. We determined that the RF interactome comprises proteins generally different from those that interact with their parental full length RNAs. Proteins captured by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carboxylic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in recently revealed RNA-protein regulatory networks. SIGNIFICANCE: In the recent decade it has become evident that RNAs with well-known functions (for example tRNA, snoRNA or rRNA) can be cleaved to yield short fragments, whose role in cells remains only partially characterized. At the same time, unconventional interactions between mRNA and proteins without RNA-binding domains have been demonstrated, revealing novel layers of possible RNA-mediated regulation. Considering the above, we hypothesized that RNA fragments (RFs) can be endogenous aptamer-like molecules that unconventionally interact with proteins. In this study we identified protein partners of seven selected RFs. We found that RFs bind different set of proteins than their parental full length RNAs and identified proteins differentially bound by the particular RFs. These observations suggest biological relevance of the discovered interactions. Our data provide a novel perspective on the significance of RFs and point to this pool of molecules as to a rich collection of potential components of the recently discovered RNA-protein regulatory networks.

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

The long non-coding RNA MALAT1 is increased in renal ischemia-reperfusion injury and inhibits hypoxia-induced inflammation.

BACKGROUND: To investigate the expression of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in renal ischemia-reperfusion injury and explore its role in acute kidney injury. METHODS: 18 mice were randomly divided into a sham operation group (Sham) and an ischemia-reperfusion group (IR) in which animals were sacrificed at 6 h or 12 h after surgery. The kidneys were harvested to measure the expression of MALAT1 mRNA. HK2 cells were treated with cobalt chloride (CoCl2) to mimic hypoxia or transfected with siRNA to knockdown MALAT1 before CoCl2 treatment. After that, MALAT1 was analyzed by RT-PCR (reverse transcription-polymerase chain reaction). HIF-1ɑ (hypoxia-inducible factor-1 alpha) and NF-κB (nuclear factor-kappa B) was measured by Western blot. The concentrations of IL-6 (interleukin-6) and TNF-ɑ (tumor necrosis factor-alpha) in the media were detected by ELISA (enzyme-linked immunosorbent assay). RESULTS: The expression of MALAT1 in the IR (6 h/12 h) group was significantly higher than that in the sham group. In HK2 cells, MALAT1 was significantly increased at 1 h, 3 h, and 6 h after CoCl2 treatment but had reduced to the basal level at 12 h and 24 h. Knockdown of MALAT1 by siRNA significantly up-regulated the expression of HIF-1ɑ and NF-κB proteins in CoCl2-treated HK2 cells. In addition, the concentrations of IL-6 and TNF-ɑ were increased by MALAT1 siRNA transfection in CoCl2-treated HK2 cells. CONCLUSION: The expression of MALAT1 is increased in renal ischemia-reperfusion injury. It is likely that MALAT1 inhibits the hypoxia-induced inflammatory response through the NF-κB pathway.

Non-coding RNAs in Various Stages of Liver Disease Leading to Hepatocellular Carcinoma: Differential Expression of miRNAs, piRNAs, lncRNAs, circRNAs, and sno/mt-RNAs.

Hepatocellular carcinoma (HCC) was the fifth leading cause of cancer death in men and eighth leading cause of death in women in the United States in 2017. In our study, we sought to identify sncRNAs in various stages of development of HCC. We obtained publicly available small RNA-seq data derived from patients with cirrhosis (n = 14), low-grade dysplastic nodules (LGDN, n = 9), high grade dysplastic nodules (HGDN, n = 6), early hepatocellular carcinoma (eHCC, n = 6), and advanced hepatocellular carcinoma (HCC, n = 20), along with healthy liver tissue samples (n = 9). All samples were analyzed for various types of non-coding RNAs using PartekFlow software. We remapped small RNA-seq to miRBase to obtain differential expressions of miRNAs and found 87 in cirrhosis, 106 in LGDN, 59 in HGDN, 80 in eHCC, and 133 in HCC. Pathway analysis of miRNAs obtained from diseased samples compared to normal samples showed signaling pathways in the microRNA dependent EMT, CD44, and others. Additionally, we analyzed the data sets for piRNAs, lncRNAs, circRNAs, and sno/mt-RNAs. We validated the in silico data using human HCC samples with NanoString miRNA global expression. Our results suggest that publically available data is a valuable resource for sncRNA identification in HCC progression (FDR set to <0.05 for all samples) and that a data mining approach is useful for biomarker development.

LncRNA ENST00113 promotes proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway in atherosclerosis.

BACKGROUND: Atherosclerosis is one of the most common cardiovascular disorders. The dysfunction of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are 2 key factors in the formation of atherosclerosis. This study aims to find strategies to prevent VSMCs and ECs dysfunction for the treatment of atherosclerosis. METHODS: We investigated the expression patterns of long noncoding RNAs (lncRNAs) in 2 pairs of serum samples from both atherosclerosis patients and healthy volunteers through microarray analysis. Then we selected the most up-regulated lncRNA ENAST00113 (lnc00113) to further verify its roles in atherosclerosis. VSMCs, and human umbilical vein endothelial cells (HUVECs) transfected with small interfering RNA (siRNAs) (si-00113-1, si-00113-1) and a negative control (si-NC) were cultured. MTT assay, Caspase 3 enzyme-linked immunosorbent assay (ELISA) assay, and wound healing assay were performed to evaluate whether lnc00113 had an effect on proliferation, apoptosis, and migration ability. Further, the correlation between lnc00113 and PI3K/Akt/mTOR signaling pathway was explored. RESULTS: Microarray results indicated that 243 lncRNAs were up-regulated and 187 lncRNAs were down-regulated. Therefore, we chose the most up-regulated lncRNA ENST (lnc00113) to further explore its roles in atherosclerosis. Real-time polymerase chain reaction (RT-qPCR) results showed that the expression of lnc00113 was highly increased in atherosclerosis patients. In vitro experiment demonstrated that lnc00113 down-regulation significantly suppressed VSMCs and HUVECs proliferation, survival, and migration. Furthermore, we found that the protein expressions of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt), phosphorylated-mTOR (p-mTOR), and bcl-2 in HUVECs cells transfected with si-00113-1 or si-00113-2 were dramatically decreased compared with si-NC-transfected cells and control cells. However, the total- PI3K (t-PI3K), total-Akt (t-Akt), and total-mTOR (t-mTOR) protein expressions were not changed, indicating that lnc00113 could activate PI3K/Akt/mTOR signaling pathway in atherosclerosis. CONCLUSIONS: This finding identified an important role of lnc00113 in VSMCs and HUVECs that promotes cell proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway, which could probably serve as a promising therapeutic target for atherosclerosis.

Cyclodextrin-siRNA conjugates as versatile gene silencing agents.

Functional siRNAs (luciferase and PLK1) have been conjugated to ß-cyclodextrin and the ability of the conjugates to retain gene knockdown activity has been assessed by delivery to cancer cell lines using various formulations. Initially two formulations used complexation with polycations, namely Lipofectamine 2000 and an amphiphilic polycationic cyclodextrin. Gene knockdown results for human glioblastoma cells (U87) and prostate cancer cells (PC3, DU145) showed that conjugation to the cyclodextrin did not reduce gene silencing by the RNA. A third mode of delivery involved formation of targeted nanoparticles in which the conjugate was first complexed with adamantyl-PEG-ligands (targeting ligand RVG peptide or dianisamide) by adamantyl inclusion in the cyclodextrin cavities of the conjugates, followed by charge neutralisation with the cationic polymer chitosan. Enhanced knockdown was achieved by these ligand-targeted formulations. In summary, while this study illustrated the gene silencing efficacy of a simple cyclodextrin-siRNA conjugate it is envisaged that future studies will explore the use of conjugates with a modified cyclodextrin which would be self-delivering. Detailed data such as stability, lysosomal escape etc. will then be reported for each conjugate, since this will be appropriate for conjugates which are intended to exploit, rather than merely demonstrate, the concept. The present paper was intended to demonstrate the viability and generality of this novel concept.

Analysis of Chromatin Opening in Heterochromatic Non-Small Cell Lung Cancer Tumor-Initiating Cells in Relation to DNA-Damaging Antitumor Treatment.

PURPOSE: We previously reported that sphere-forming non-small cell lung cancer (NSCLC) tumor-initiating cells (TICs) have an altered activation of DNA damage response- and repair proteins and are refractory to DNA-damaging treatments. We analyzed whether chromatin organization plays a role in the observed refractoriness. METHODS AND MATERIALS: Bulk cells and TICs from the NSCLC H23 and H1299 cell lines were examined using cell viability, clonogenic survival, Western blot, short interfering RNA analysis, and micronucleus assay. RESULTS: NSCLC TICs displayed elevated heterochromatin markers trimethylated lysine 9 of histone H3 and heterochromatin protein 1γ relative to bulk cells and reduced cell viability upon histone deacetylase inhibition (HDACi). Vorinostat and trichostatin A increased the euchromatin markers acetylated lysine 9/14 of histone H3 and lysine 8 of histone H4, and HDACi pretreatment increased the phosphorylation of the DNA damage response proteins ataxia telangiectasia mutated and DNA-dependent protein kinase, catalytic subunit, upon irradiation in TICs. HDACi sensitized TICs to cisplatin and to some extent to ionizing irradiation. The protectiveness of a dense chromatin structure was indicated by an enhanced frequency of micronuclei in TICs following irradiation, after knockdown of heterochromatin protein 1γ. CONCLUSIONS: Although confirmatory studies in additional NSCLC model systems and with respect to analyses of other DNA damage response proteins are needed, our data point toward a heterochromatic structure of NSCLC TICs, such that HDACi can sensitize TICs to DNA damage.

Semaphorin4D Drives CD8+ T-Cell Lesional Trafficking in Oral Lichen Planus via CXCL9/CXCL10 Upregulations in Oral Keratinocytes.

Chemokine-mediated CD8+ T-cell recruitment is an essential but not well-established event for the persistence of oral lichen planus (OLP). Semaphorin 4D (Sema4D)/CD100 is implicated in immune dysfunction, chemokine modulation, and cell migration, which are critical aspects for OLP progression, but its implication in OLP pathogenesis has not been determined. In this study, we sought to explicate the effect of Sema4D on human oral keratinocytes and its capacity to drive CD8+ T-cell lesional trafficking via chemokine modulation. We found that upregulations of sSema4D in OLP tissues and blood were positively correlated with disease severity and activity. In vitro observation revealed that Sema4D induced C-X-C motif chemokine ligand 9/C-X-C motif chemokine ligand 10 production by binding to plexin-B1 via protein kinase B-NF-κB cascade in human oral keratinocytes, which elicited OLP CD8+ T-cell migration. We also confirmed using clinical samples that elevated C-X-C motif chemokine ligand 9/C-X-C motif chemokine ligand 10 levels were positively correlated with sSema4D levels in OLP lesions and serum. Notably, we determined matrix metalloproteinase-9 as a new proteolytic enzyme for the cleavage of sSema4D from the T-cell surface, which may contribute to the high levels of sSema4D in OLP lesions and serum. Our findings conclusively revealed an amplification feedback loop involving T cells, chemokines, and Sema4D-dependent signal that promotes OLP progression.

Inhibition of FKBP10 Attenuates Hypertrophic Scarring through Suppressing Fibroblast Activity and Extracellular Matrix Deposition.

Hypertrophic scar is a pathogenic form of scar formation with no recognized treatment to date. Its molecular mechanism is related to the abnormal proliferation and transition of fibroblasts and overproduction of extracellular matrix. FKBP10 is a molecular chaperone able to regulate α-smooth muscle actin expression and pro-collagen maturation in fibroblasts. However, to our knowledge, no research has investigated the biological function of FKBP10 in scar formation to date. In this study, we aim to assess the expression and function of FKBP10 in hypertrophic scarring. Through microarray analysis, real-time reverse transcriptase-PCR and immunohistochemistry, we discovered that FKBP10 is up-regulated in human and mouse hypertrophic scars. Then we evaluated hypertrophic scar formation in mouse models treated with FKBP10 small interfering RNA and found that knockdown of FKBP10 could attenuate hypertrophic scar formation in vivo. To further explore the underlying mechanism, FKBP10 was knocked down in human hypertrophic scar fibroblasts. The in vitro results showed that FKBP10 siRNA could inhibit fibroblast activity, reduce the expression of α-smooth muscle actin and extracellular matrix components, and attenuate transforming growth factor-ß1 expression and the activation of the Smad signaling pathway. In conclusion, FKBP10 plays a crucial role in hypertrophic scar formation and might be a therapeutic target for hypertrophic scars.