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Supplementary Fig. S3 legend from Dehydrocostuslactone disrupts signal transducers and activators of transcription 3 through up-regulation of suppressor of cytokine signaling in breast cancer cells

Supplementary Fig. S1 from Dehydrocostuslactone disrupts signal transducers and activators of transcription 3 through up-regulation of suppressor of cytokine signaling in breast cancer cells

This study investigates the anticancer effect of dehydrocostuslactone (DHE), a plant-derived sesquiterpene lactone, on human breast cancer cells. DHE inhibits cell proliferation by inducing cells to undergo cell cycle arrest and apoptosis. DHE suppresses the expression of cyclin D, cyclin A, cyclin-dependent kinase 2, and cdc25A and increases the amount of p53 and p21, resulting in G0/G1-S phase arrest in MCF-7 cells. In contrast, DHE caused S-G2/M arrest by increasing p21 expression and chk1 activation and inhibiting cyclin A, cyclin B, cdc25A, and cdc25C expression in MDA-MB-231 cells. DHE induces up-regulation of Bax and Bad, down-regulation of Bcl-2 and Bcl-XL, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor and endonuclease G. We also found that DHE inhibits survival signaling through the Janus tyrosine kinase-signal transducer and activator of transcription-3 signaling by increasing the expression of suppressors of cytokine signaling (SOCS)-1 and SOCS-3. Reduction of SOCS-1 and SOCS-3 expression by small interfering RNA inhibits DHE-mediated signal transducer and activator of transcription-3 inhibition, p21 up-regulation, and cyclin-dependent kinase 2 blockade, supporting the hypothesis that DHE inhibits cell cycle progression and cell death through SOCS-1 and SOCS-3. Significantly, animal studies have revealed a 50% reduction in tumor volume after a 45-day treatment period. Taken together, this study provides new insights into the molecular mechanism of the DHE action that may contribute to the chemoprevention of breast cancer. [Mol Cancer Ther 2009;8(5):1328–39]

Diabetic nephropathy (DN) is an increasing threat to human health and is regarded as an important public issue. The pathophysiologic mechanisms of DN are complicated. The initiating molecular events triggering the loss function in mesangial cells (MCs) in DN are not well known. In this cross-disciplinary study, transcriptome analysis of high glucose (HG)-treated mouse MCs (MMCs) using next-generation sequencing and systematic bioinformatics analyses indicated that miR-15b-5p and its downstream target B cell lymphoma 2 (BCL-2) contribute to HG-induced apoptosis in MMCs. HG elevated miR-15b-5p expression, which in turn decreased the translation of BCL-2, leading to MMC apoptosis under HG. Apoptosis of MCs was enhanced in the presence of extracellular vesicles isolated from the urine of type 2 diabetic patients with high levels of miR-15b-5p. Furthermore, increased levels of urinary miR-15b-5p were found in db/db mice and type 2 diabetic patients, and such levels correlated with low baseline kidney function and rapid decline in kidney function during a mean of follow-up period of 2.4 ± 0.1 years. Therefore, miR-15b-5p induced mesangial cells apoptosis by targeting BCL-2 under HG. miR-15b-5p has the potential to predict kidney injury in DN. Blocking the miR-15b-5p epigenetic regulatory network could be a potential therapeutic strategy to prevent mesangial apoptosis in DN.

This study has two novel findings: it is not only the first to deduct potential genes involved in scleral growth repression upon atropine instillation from a prevention point of view, but also the first to demonstrate that only slight changes in scleral gene expression were found after atropine treatment as side effects and safety reasons of the eye drops are of concern. The sclera determines the final ocular shape and size, constituting of scleral fibroblasts as the principal cell type and the major regulator of extracellular matrix. The aim of our study was to identify differentially expressed genes and microRNA regulations in atropine-treated scleral fibroblasts that are potentially involved in preventing the onset of excessive ocular growth using next-generation sequencing and bioinformatics approaches. Differentially expressed genes were functionally enriched in anti-remodeling effects, comprising of structural changes of extracellular matrix and metabolic pathways involving cell differentiation. Significant canonical pathways were correlated to inhibition of melatonin degradation, which was compatible with our clinical practice as atropine eye drops are instilled at night. Validation of the dysregulated genes with previous eye growth-related arrays and through microRNA-mRNA interaction predictions revealed the association of hsa-miR-2682-5p-KCNJ5 and hsa-miR-2682-5p-PRLR with scleral anti-remodeling and circadian rhythmicity. Our findings present new insights into understanding the anti-myopic effects of atropine, which may assist in prevention of myopia development.

Metastatic tumors have been shown to establish a supportive pre-metastatic niche (PMN) in distant organs, which in turn determines disseminated tumor cells’ targeting of such organs. PMN is formed through the recruitment of bone-marrow-derived cells (BMDCs); however, the role of BMDCs in PMN formation is not fully understood. On the basis of RNA-seq data and bioinformatic analysis, secretion of extracellular vesicle (EV) miR-92a by BMDCs of lung cancer-bearing mice contributes to the establishment of liver PMN. Both BMDC-derived EVs and miR-92a mimics potentiate the activation of hepatic stellate cells (HSCs), subsequently increasing extracellular matrix (ECM) deposition in mice. Consequently, remodeling of the liver microenvironment enhanced immunosuppressive cell accumulation and cancer cell attachment. EVs miR-92a directly suppressed its target SMAD7, leading to the enhancement of transforming growth factor-β signaling in HSC. Elevated levels of circulating miR-92a are found in the sera of lung cancer patients, and EVs isolated from these patients have a similar ability to increase HSCs activation and ECM protein expression. Our study reveals the sequential steps of liver PMN formation in lung cancer, providing critical mediators that prepare PMN in the liver, and identifies new targets that offer valuable options for diagnosis and therapeutic intervention.

Endometrial cancer is one of the most common cancers in women worldwide, affecting more than 300,000 women annually. Dysregulated gene expression, especially those mediated by microRNAs, play important role in the development and progression of cancer. This study aimed to investigate differentially expressed genes in endometrial adenocarcinoma using next generation sequencing (NGS) and bioinformatics. The gene expression profiles and microRNA profiles of endometrial adenocarcinoma (cancer part) and normal endometrial tissue (non-cancer part) were assessed with NGS. We identified 56 significantly dysregulated genes, including 47 upregulated and 9 downregulated genes, in endometrial adenocarcinoma. Most of these genes were associated with defense response, response to stimulus, and immune system process, and further pathway analysis showed that human papillomavirus infection was the most significant pathway in endometrial adenocarcinoma. In addition, these genes were also associated with decreased cell death and survival as well as increased cellular movement. The analyses using Human Protein Atlas, identified 6 genes (PEG10, CLDN1, ASS1, WNT7A, GLDC, and RSAD2) significantly associated with poorer prognosis and 3 genes (SFN, PIGR, and CDKN1A) significantly associated with better prognosis. Combining with the data of microRNA profiles using microRNA target predicting tools, two significantly dysregulated microRNA-mediated gene expression changes in endometrial adenocarcinoma were identified: downregulated hsa-miR-127-5p with upregulated CSTB and upregulated hsa-miR-218-5p with downregulated HPGD. These findings may contribute important new insights into possible novel diagnostic or therapeutic strategies for endometrial adenocarcinoma.

Patients receiving hemodialysis (HD) have a higher risk of cognitive impairment and dementia than the general population. The accumulation of uremic toxins in the brain causes uremic encephalopathy, however, limited data exists to elucidate the effect of protein-bound uremic toxins on cognitive function. Here we investigate the effect of indole-3 acetic acid (IAA) and hippuric acid (HA), two different protein-bound uremic toxins from amino acid derivatives, on cognitive function by Silico and in a clinical study. Prevalent HD patients were enrolled in two independent hospitals. Serum IAA and HA were measured using mass spectrometry. Cognitive performance was measured using Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Cognitive Abilities Screening Instrument (CASI) by trained psychologists. Using silico data to predict the effect of blood-brain barrier penetration was performed. The silico data demonstrated that IAA and HA had positive blood-brain barrier penetration ability. Amongst the 230 HD patients, serum IAA was associated with poor MMSE score (β= -0.90, 95% CI -1.61 to -0.19) and poor CASI score (β= -3.29, 95% CI -5.69 to -0.88) in stepwise multiple linear regression analysis. In logistic regression model, Serum IAA was also associated with cognitive impairment based on MMSE definition (OR, 1.96, 95% CI 1.10, 3.5) and CASI definition (OR, 2.09, 95% CI 1.21, 3.61). There was no correlation between Serum HA levels and cognitive function status. In conclusion, IAA, not HA, was associated with cognitive impairment in HD patients. Further large scale and prospective studies are needed to confirm our findings.

A pre-metastatic niche (PMN) facilitates cancer metastasis through mobilization and recruitment of bone marrow-derived cells (BMDCs) and associated factors. In bone marrow, hematogenous cells, including osteoclasts, macrophages and lymphocytes, and mesenchymal cells, including mesenchymal stem cells, osteoblasts and adipocytes, are involved in PMN formation. Patients with lung cancer and metastasis have a poor prognosis and shortened median survival time. Bone marrow has been considered fertile ground for dormant and proliferating tumor cells, and mobilizing and recruiting BMDCs and immune cells can establish a PMN. However, the role of BMDCs in PMN formation is not yet fully understood. The present study aimed to investigate the association between BMDCs and PMN in bone marrow tissue samples. The results demonstrated that bone marrow served an important role in lung cancer progression and that eight pathways were potentially involved, including 'T-cell receptor signaling pathway', 'osteoclast differentiation', 'MAPK signaling pathway', 'VEGF signaling pathway', 'leukocyte transendothelial migration', 'signaling pathways regulating the pluripotency of stem cells', 'oxytocin signaling pathway' and 'cell adhesion molecules (CAMs)'. In addition, the present study investigated the role of BMDCs in facilitating lung cancer metastasis. In conclusion, the results from the present study suggested that molecular alterations in gene expression may provide a novel signature in lung cancer, which may aid in the development of novel diagnostic and therapeutic strategies for patients with lung cancer and bone metastasis.

Background We aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases. Materials and Methods Using next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity® Pathway Analysis databases, we identified candidate genes. To search for upstream miRNAs with exact target binding sites, we used miRmap, TargetScan, and miRDB to enforce evidence. Then, we clarified gene and protein expression through an in vitro study using western blot analysis and quantitative real-time reverse transcriptase-PCR. Results Interactions between selected target genes obtained using the NGS and miRmap methods were assessed through a Venn diagram analysis. Six potential genes, namely, PDE5A, RECK, ZEB2, NCALD, PLCXD3 and CYBRD1 showed significant differences. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC. In addition, we compared the expression of these genes in various UC cell lines (RT4, BFTC905, J82, T24, UMUC3, 5637, BFTC 909, UMUC14) and found decreased expression of PDE5A in muscle-invasive UC cells compared with the RT4 cell line. Furthermore, by using paired UTUC and normal tissues from 20 patients, lower PDE5A expression was also demonstrated in tumor specimens. Conclusions Our findings suggest these candidate genes may play some roles in UTUC progression. We propose that these markers may be potential targets clarified by in vitro and in vivo experiments. PDE5A also potentially presents tumor suppressor genes, as identified by comparing the expression between normal and tumor specimens.

Synovitis in osteoarthritis (OA) the consequence of low grade inflammatory process caused by cartilage breakdown products that stimulated the production of pro-inflammatory mediators by fibroblast-like synoviocytes (FLS). FLS participate in joint homeostasis and low grade inflammation in the joint microenvironment triggers FLS transformation. In the current study, we aimed to identify differentially expressed genes and potential miRNA regulations in human OA FLS through deep sequencing and bioinformatics approaches. The 245 differentially expressed genes in OA FLS were identified, and pathway analysis using various bioinformatics databases indicated their enrichment in functions related to altered extracellular matrix organization, cell adhesion and cellular movement. Moreover, among the 14 dysregulated genes with potential miRNA regulations identified, src kinase associated phosphoprotein 2 (SKAP2), adaptor related protein complex 1 sigma 2 subunit (AP1S2), PHD finger protein 21A (PHF21A), lipoma preferred partner (LPP), and transcription factor AP-2 alpha (TFAP2A) showed similar expression patterns in OA FLS and OA synovial tissue datasets in Gene Expression Omnibus database. Ingenuity Pathway Analysis identified the dysregulated LPP participated in cell migration and cell spreading of OA FLS, which was potentially regulated by miR-141-3p. The current findings suggested new perspectives into understanding the novel molecular signatures of FLS involved in the pathogenesis of OA, which may be potential therapeutic targets.

Background. Patients undergoing hemodialysis experience a greater risk of cognitive impairment than the general population, but limited data elucidates the biomarkers on this. We evaluated the association of bone turnover markers on cognitive function among 251 prevalent hemodialysis enrollees in a cross-sectional study. Methods. 251 hemodialysis patients (median age=57.8, 55% men) and 37 control subjects (mean age=61.2, 56% men) without a prior stroke or dementia diagnosis were enrolled. Serum concentrations of 8 bone markers were analyzed as the association of cognitive function (Montreal Cognitive Assessment (MoCA) and Cognitive Abilities Screening Instrument (CASI)) using linear regression analysis. Results. A lower cognitive function was noted in hemodialysis patients compared to control subjects. The receptor activator of nuclear factor kappa-B ligand (RANKL) was the only bone marker found to be associated with cognitive function (MoCA and CASI tests) in hemodialysis patients without a prior stroke or dementia diagnosis. In stepwise multiple linear regression analysis, the association remained significant in MoCA (β=1.14, 95% CI 0.17 to 2.11) and CASI (β=3.06, 95% CI 0.24 to 5.88). Short-term memory (β=0.52, 95% CI 0.01 to 1.02), mental manipulation (β=0.51, 95% CI 0.05 to 0.96), and abstract thinking (β=0.57, 95% CI 0.06 to 1.09) were the significant subdomains in the CASI score related to RANKL. Conclusions. Serum RANKL levels were potentially associated with better cognitive function in hemodialysis patients. Further large-scale and prospective studies are needed to confirm our findings.

Rheumatoid arthritis (RA) is one of the inflammatory joint diseases that display features of articular cartilage destruction. The underlying disturbance results from immune dysregulation that directly and indirectly influence chondrocyte physiology. In the last years, significant evidence inferred from studies in vitro and in the animal model offered a more holistic vision of chondrocytes in RA. Chondrocytes, despite being one of injured cells in RA, also undergo molecular alterations to actively participate in inflammation and matrix destruction in the human rheumatoid joint. This review covers current knowledge about the specific cellular and biochemical mechanisms that account for the chondrocyte signatures of RA and its potential applications for diagnosis and prognosis in RA.

Background We aimed to investigate the long-term outcomes in hepatitis B (HBV)/hepatitis C virus (HCV) dual-infected patients after anti-HCV therapy. Methods A total of 192 HBV/HCV dual-infected patients who had received pegylated interferon treatment were recruited. The investigation outcomes included HBV DNA ≥2000 IU/mL, with or without alanine aminotransferase (ALT) ≥2-fold the upper limit of normal, and hepatitis B surface antigen (HBsAg) seroclearance. Results Four (2.1%) patients developed early HBV reactivation before the end of treatment. Fifty (26.6%) of the remaining patients had an episode of HBV DNA ≥2000 IU/mL in a mean follow-up of 68.8 months. The risk was 4.6 per 100 person years. Only 19 (10.1%) patients developed concomitant ALT flare with oral HBV antiviral therapy; the risk was 1.7 per 100 person years. Despite HBV flare, 67 (34.9%) patients had a favorable outcome of HBsAg seroclearance. The probability was 5.7 per 100 person years. A pretreatment HBV DNA level of 300 IU/mL served as an independent predictor for all the outcomes. The combined pretreatment HBV DNA level and HCV response further enhanced the prediction of HBV flare and HBsAg seroclearance. Conclusions A pretreatment HBV DNA level of 300 IU/mL predicts HBV flare and HBsAg seroclearance after anti-HCV therapy.

Cartilage destruction in rheumatoid arthritis (RA) occurs primarily in the pannus-cartilage interface. The close contact of the synovium-cartilage interface implicates crosstalk between synovial fibroblasts and chondrocytes. The aim of this study is to explore the differentially expressed genes and novel microRNA regulations potentially implicated in the dysregulated cartilage homeostasis in joint destruction of RA. Total RNAs were extracted from human primary cultured normal and RA chondrocytes for RNA and small RNA expression profiling using next-generation sequencing. Using systematic bioinformatics analyses, we identified 463 differentially expressed genes in RA chondrocytes were enriched in biological functions related to altered cell cycle process, inflammatory response and hypoxic stimulation. Moreover, fibroblast growth factor 9 (FGF9), kynureninase (KYNU), and regulator of cell cycle (RGCC) were among the top dysregulated genes identified to be potentially affected in the RA joint microenvironment, having similar expression patterns observed in arrays of clinical RA synovial tissues from the Gene Expression Omnibus database. Additionally, among the 31 differentially expressed microRNAs and 10 candidate genes with potential microRNA-mRNA interactions in RA chondrocytes, the novel miR-140-3p-FGF9 interaction was validated in different microRNA prediction databases, and proposed to participate in the pathogenesis of joint destruction through dysregulated cell growth in RA. The findings provide new perspectives for target genes in the management of cartilage destruction in RA.

Hypoxia, the most commonly observed characteristic in cancers, is implicated in the establishment of an immunosuppressive niche. Recent studies have indicated that extracellular vesicle (EV)-mediated cancer-stroma interactions are considered to play a critical role in the regulation of various cellular biological functions, with phenotypic consequences in recipient cells. However, the mechanisms underlying the relationship between EVs and hypoxia during cancer progression remain largely unknown. In this study, we found that EVs derived from hypoxic lung cancers increased M2-type polarization by miR-103a transfer. Decreased PTEN levels caused by hypoxic cancer-cell-derived EV miR-103a increased activation of AKT and STAT3 as well as expression of several immunosuppressive and pro-angiogeneic factors. In contrast, inhibition of miR-103a by an miRNA inhibitor effectively decreased hypoxic cancer-mediated M2-type polarization, improving the cytokine prolife of tumor infiltration macrophages. Macrophages received cancer-cell-derived EV miR-103a feedback to further enhance cancer progression and tumor angiogenesis. Finally, circulating EV miR-103a levels were higher in patients with lung cancer and closely associated with the M2 polarization. In conclusion, our results delineate a novel mechanism by which lung cancer cells induce immunosuppressive and pro-tumoral macrophages through EVs and inspire further research into the clinical application of EV inhibition or PTEN restoration for immunotherapy., Graphical Abstract, Extracellular vesicle (EV) miR-103 can be transferred from hypoxic cancer cells to macrophages, resulting in the enhancement of M2 polarization by the downregulation of miR-103a’s direct target PTEN. EV miR-103a increases the stimulatory effects of macrophages on cancer progression and angiogenesis.

// Ming-Lun Yeh 1, 2, 3 , Ching-I Huang 2 , Chung-Feng Huang 2, 3 , Meng-Hsuan Hsieh 2, 3, 5 , Ming-Yen Hsieh 2 , Zu-Yau Lin 2, 3 , Shinn-Cherng Chen 2, 3 , Jee-Fu Huang 2, 3 , Po-Lin Kuo 1 , Hsing-Tao Kuo 6, 7, * , Chia-Yen Dai 1, 2, 3, 4, 5, * , Ming-Lung Yu 2, 3, 4, 8 and Wan-Long Chuang 2, 3, 4 1 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Lipid Science and Aging Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Department of Occupational and Environmental Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 6 Division of Hepatogastroenterology, Department of Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan 7 Department of Senior Citizen Service Management Chia Nan University of Pharmacy and Science, Tainan, Taiwan 8 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan * These authors contributed equally to this work Correspondence to: Chia-Yen Dai, email: d820195@gmail.com Hsing-Tao Kuo, email: kht@mail.chimei.org.tw Keywords: hepatocellular carcinoma, incidence, hepatitis B virus, hepatitis C virus, dual infection Received: August 02, 2017 Accepted: September 21, 2017 Published: January 13, 2018 ABSTRACT We investigated the long-term risk of hepatocellular carcinoma (HCC) in dual-infected hepatitis B and C patients after eradication of hepatitis C virus (HCV). A total of 164 (62% male, median age, 50.5 years) hepatitis B and C dual-infected patients who achieved HCV sustained virological response were recruited. Half the patients were HCV genotype 1 with a median viral load of 5.5 log10 IU/mL, and 22.6%had an HBV DNA level ≥ 2000 IU/mL before therapy. HCC developed in 14 patients (8.5%), with an annual incidence of 1.38% per person-year. The 3-year, 5-year, 10-year, and 15-year cumulative probabilities were 2.5%, 5.1%, 12.6%, and 22.7%, respectively. Six months after treatment, a Cox regression hazard analysis revealed platelet level (HR: 0.98, 95% CI: 0.957–0.999, P = 0.038) and AFP level (HR: 1.20, 95% CI: 1.031–1.400, P = 0.019) to be independent factors in HCC. A higher 10-year cumulative risk of HCC was detected in patients with 6-month post-treatment AFP levels > 5.0 ng/mL and platelet levels < 130 x1000/μL (54.9%), compared to patients with neither (8.6%). Although the risk of HCC is low, surveillance of HCC is encouraged in dual-infected patients after eradication of HCV. Post-treatment AFP and platelet levels predict HCC development.

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer, mainly serves as an additive to render polyvinyl chloride (PVC) soft and flexible. PVC plastics have become ubiquitous in our modern society. Yet, the leaching of DEHP from PVC-based consumables ultimately results in the deposition in certain tissues via inadvertent applications. Health risks for human populations exposed to DEHP has been assumed by studies on rodents and other species, including the DEHP-induced developmental dysregulation, reproductive impairments, tumorigenesis, and diseases in a transgenerational manner. In this review, we comprehensively summarize the accumulated literature regarding the multifaceted roles of DEHP in the activation of the nuclear receptors, the alteration of the redox homeostasis, epigenetic modifications and the acquisition of chemoresistance.

During the aging process, impaired osteoblastic function is one key factor of imbalanced bone formation and age-related bone loss. The aim of this study is to explore the differentially expressed genes in normal and aged osteoblasts and to identify genes potentially involved in age-related alteration in bone physiology. Based on next generation sequencing and bioinformatics analysis, 12 differentially expressed microRNAs and 22 differentially expressed genes were identified. Up-regulation of miR-204-5p was validated in an array of osteoporotic hip fracture in the Gene Expression Omnibus database (GSE74209). The putative targets for miR-204-5p were Kruppel-like factor 7 (KLF7) and SRY-box 11 (SOX11). Ingenuity Pathway Analysis identified SOX11, involved in osteoarthritis pathway and differentiation of osteoblasts, together with miR-204-5p, a potential upstream regulator, suggesting the critical role of miR-204-5p-SOX11 regulation in the aging process of human bones. In addition, as semaphorin 3A (SEMA3A) and ephrin type-A receptor 5 (EPHA5) were involved in nervous system related biological functions, we postulated a potential linkage between SEMA3A, EPHA5 and development of neurogenic heterotopic ossification. Our findings implicate new candidate genes in the diagnosis of geriatric musculoskeletal disorders, and provide novel insights that may contribute to the elaboration of new biomarkers for neurogenic heterotopic ossification.

Background and Aim Hepatitis B virus (HBV) may reactivate when treating chronic hepatitis C (CHC) with direct acting antivirals (DAA). We aim to investigate the risk of HBV reactivation during DAA therapy. Methods Chronic hepatitis C patients receiving pan-oral DAA therapy from December 2013 to August 2016 were evaluated. Fifty-seven patients that had a past HBV infection (negative hepatitis B surface antigen [HBsAg] and positive hepatitis B core antibody) and seven patients that had a current HBV infection (positive HBsAg) were enrolled. Serum HBV and hepatitis C virus (HCV) markers were regularly measured. The endpoints were the HCV sustained virological response (SVR) and the HBV virological/clinical reactivation. Results The overall SVR12 rate was 96.9%, and two patients, one with positive HBsAg, had a relapse of HCV. No episodes of HBV virological reactivation were observed among the patients with a past HBV infection. For the seven patients with a current HBV infection, HBV virological reactivation was found in four (57.1%) of the seven patients. Clinical reactivation of HBV was observed in one patient with pretreatment detectable HBV DNA and recovered after entecavir administration. For the other three patients with HBV virological reactivation, the reappearance of low level HBV DNA without clinical reactivation was observed. HBsAg levels demonstrated only small fluctuations in all the patients. Conclusions There was a minimal impact of hepatitis B core antibody seropositivity on HCV efficacy and safety. For CHC patients with current HBV infection, the risk of HBV reactivation was present, and monitoring the HBV DNA level during therapy is warranted.

Lung adenocarcinoma is one of the leading causes of cancer-related death worldwide. We showed transcriptomic profiles in three pairs of tumors and adjacent non-tumor lung tissues using next-generation sequencing (NGS) to screen protein-coding RNAs and microRNAs. Combined with meta-analysis from the Oncomine and Gene Expression Omnibus (GEO) databases, we identified a representative genetic expression signature in lung adenocarcinoma. There were 9 upregulated genes, and 8 downregulated genes in lung adenocarcinoma. The analysis of the effects from each gene expression on survival outcome indicated that 6 genes (AGR2, SPDEF, CDKN2A, CLDN3, SFN, and PHLDA2) play oncogenic roles, and 7 genes (PDK4, FMO2, CPED1, GNG11, IL33, BTNL9, and FABP4) act as tumor suppressors in lung adenocarcinoma. In addition, we also identified putative genetic interactions, in which there were 5 upregulated microRNAs with specific targets - hsa-miR-183-5p-BTNL9, hsa-miR-33b-5p-CPED1, hsa-miR-429-CPED1, hsa-miR-182-5p-FMO2, and hsa-miR-130b-5p-IL33. These 5 microRNAs have been shown to be associated with tumorigenesis in lung cancer. Our findings suggest that these genetic interactions play important roles in the progression of lung adenocarcinoma. We propose that this molecular change of genetic expression may represent a novel signature in lung adenocarcinoma, which may be developed for diagnostic and therapeutic strategies in the future.

// Chau-Chyun Sheu 1, 2, 3, * , Ming-Ju Tsai 1, 2, 3, 5, * , Feng-Wei Chen 1 , Kuo-Feng Chang 4 , Wei-An Chang 1, 2 , Inn-Wen Chong 2, 5 , Po-Lin Kuo 1, 6, 7 and Ya-Ling Hsu 1, 7 1 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 Department of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Welgene Biotech Inc, Taipei, Taiwan 5 Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan * These authors have contributed equally to this work Correspondence to: Po-Lin Kuo, email: kuopolin@seed.net.tw Ya-Ling Hsu, email: hsuyl326@gmail.com Keywords: next-generation sequencing, bioinformatics, microRNA, airway remodeling, asthma Received: April 23, 2017 Accepted: June 19, 2017 Published: July 31, 2017 ABSTRACT Airway epithelial cells play important roles in airway remodeling. Understanding gene regulations in airway epithelial homeostasis may provide new insights into pathogenesis and treatment of asthma. This study aimed to combine gene expression (GE) microarray, next generation sequencing (NGS), and bioinformatics to explore genetic regulations associated with airway epithelial homeostasis. We analyzed expression profiles of mRNAs (GE microarray) and microRNAs (NGS) in normal and asthmatic bronchial epithelial cells, and identified 9 genes with potential microRNA-mRNA interactions. Of these 9 dysregulated genes, downregulation of MEF2C and MDGA1 were validated in a representative microarray (GSE43696) from the gene expression omnibus (GEO) database. Our findings suggested that upregulated mir-203a may repress MEF2C , a transcription factor, leading to decreased cellular proliferation. In addition, upregulated mir-3065-3p may repress MDGA1 , a cell membrane anchor protein, resulting in suppression of cell-cell adhesion. We also found that KCNJ2 , a potassium channel, was downregulated in severe asthma and may promote epithelial cell apoptosis. We proposed that aberrant regulations of mir-203a- MEF2C and mir-3065-3p- MDGA1 , as well as downregulation of KCNJ2 , play important roles in airway epithelial homeostasis in asthma. These findings provide new perspectives on diagnostic or therapeutic strategies targeting bronchial epithelium for asthma. The approach in this study also provides a new aspect of studying asthma.

Pleural effusion is associated with multiple benign and malignant conditions. Currently no biomarkers differentiate malignant pleural effusion (MPE) and benign pleural effusion (BPE) sensitively and specifically. The present study identified a novel combination of biomarkers in pleural effusion for differentiating MPE from BPE by enrolling 75 patients, 34 with BPE and 41 with MPE. The levels of lactate dehydrogenase, glucose, protein, and total cell, neutrophil, monocyte and lymphocyte counts in the pleural effusion were measured. The concentrations of interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-α, interferon γ, transforming growth factor-β1, colony stimulating factor 2, monocyte chemoattractant protein-1 and vascular endothelial growth factor (VEGF) were detected using cytometric bead arrays. Protein and VEGF levels differed significantly between patients with BPE and those with MPE. The optimal cutoff value of VEGF and protein was 214 pg/ml and 3.35 g/dl respectively, according to the receiver operating characteristic curve. A combination of VEGF >214 pg/ml and protein >3.35 g/dl in pleural effusion presented a sensitivity of 92.6% and an accuracy of 78.6% for MPE, but was not associated with a decreased survival rate. These results suggested that this novel combination strategy may provide useful biomarkers for predicting MPE and facilitating early diagnosis.

// Jen-Yu Hung 1, 2 , Meng-Chi Yen 3 , Shu-Fang Jian 4 , Cheng-Ying Wu 4 , Wei-An Chang 2, 4 , Kuan-Ting Liu 1, 3, 4 , Ya-Ling Hsu 5 , Inn-Wen Chong 2, 6 and Po-Lin Kuo 4, 7 1 School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Inn-Wen Chong, email: chong@kmu.edu.tw Po-Lin Kuo, email: kuopolin@seed.net.tw Keywords: SPARC, WNK1, lung cancer, EMT, migration Received: March 24, 2017 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT The extracellular matrix is a component of physiological microenvironment and a regulator of cellular processes such as migration and proliferation. Secreted Protein Acidic and Rich in Cysteine (SPARC/osteonectin) is an extracellular matrix-associated glycoprotein involved in the regulation of cell proliferation and cell migration in several types of cancers. However, the role of SPARC in lung cancer is paradoxical and details of the regulatory mechanism are not well-known. In this study, we investigated novel SPARC-mediated signaling pathways. Treatment of SPARC increased cell proliferation, migration, and mesenchymal phenotype in two non-small cell lung cancer cell lines, CL1-5 and H1299. We found that these phenotypes were not regulated by focal adhesion kinase and Src kinase, but were mediated by with no lysine (K) kinase 1 (WNK1). Suppression of WNK1 expression decreased the expression of SPARC-induced N-cadherin and smooth muscle actin. Moreover, Snail, an important transcription factor for regulating epithelial–mesenchymal transition, is also involved in SPARC/WNK1 pathway. In a murine tumor model, SPARC treatment significantly induced phosphorylation of Akt and WNK1 in lung tumor nodules when compared to control mice. In conclusion, these data suggest that WNK1 is a novel molecule in SPARC-mediated mesenchymal signaling pathway in non-small cell lung cancer.

The lipid metabolic enzymes are considered candidate therapeutic targets for breast cancer. Long-chain acyl-coenzyme A (CoA) synthase (ACSL) is one of lipid metabolic enzymes and converts free-fatty acid to fatty acid-CoA. Five ACSL isoforms including ACSL1, ACSL3, ACSL4, ACSL5 and ACSL6 are identified in human. High ACSL4 expression has been observed in aggressive breast cancer phenotype. However, the role of other isoforms is still little-known. We therefore, analyzed the expression of ACSL isoforms in each subtype of breast cancer within METABRIC dataset and cancer cell line encyclopedia dataset. The expression levels of ACSL1, ACSL4 and ACSL5 in estrogen receptor (ER)-negative group were higher than that in ER-positive group. Similar expression pattern was detected among breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative). Treatment of ACSL inhibitor triacsin C which inhibited enzyme activity of ACSL 1, 3, 4 and 5 suppressed cell growth of MCF-7 and MDA-MB-231. Our results further showed that high ACSL5 expression was associated with good prognosis in patients with both ER-positive and ER-negative breast cancer through KM plotter analysis. These results suggest that ACSL1, ACSL4 and ACSL5 expression is regulated by ER signaling pathways and ACSL5 is a potential novel biomarker for predicting prognosis of breast cancer patients.

// Leong-Perng Chan 1, 2 , Cheng Liu 3, 4 , Feng-Yu Chiang 2 , Ling-Feng Wang 2 , Ka-Wo Lee 2 , Wan-Ting Chen 5 , Po-Lin Kuo 1, 6 and Chia-Hua Liang 5 1 Graduate Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Division of Plastic Surgery & HBOT Center, Chi Mei Medical Center, Tainan, Taiwan 4 Department of Electrical Engineering, Southern Taiwan University of Science & Technology, Tainan, Taiwan 5 Department of Cosmetic Science and Institute of Cosmetic Science, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 6 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Po-Lin Kuo, email: kuopolin@seed.net.tw Chia-Hua Liang, email: tinna_ling@mail.cnu.edu.tw Keywords: HNSCC, IL-8, inflammation, MAPK Received: November 30, 2016 Accepted: March 08, 2017 Published: April 07, 2017 ABSTRACT This investigation identifies interleukin 8 (IL-8) as the main inflammatory mediator in head and neck squamous cell carcinoma (HNSCC). The expressions of chemokines of IL-8, IL-1β and IL-6 and the cytokines of tumor necrosis factor-α (TNF-α) were higher in HNSCC patient tissues than in non-cancerous matched tissues (NCMT) whereas the expression of IL-10 was lower. IL-8 is most highly expressed in the tissues of patients with HNSCC. Treatment of HNSCC cells with IL-8 increased the secretion of IL-1β, IL-6 and TNF-α and reduced IL-10 expression; the increase in the expression of IL-1β was particularly considerable. IL-8 silencing by siRNA reduced IL-1β expression in HNSCC cells, suggesting that IL-8 as a main inflammatory mediator improved IL-1β expression in HNSCC. The expressions of p-p38 mitogen-activated protein kinases (MAPK) and p-extracellular signal regulated kinase (p-ERK) were higher and that of p-c-Jun-NH2-terminal kinase (p-JNK) was lower in HNSCC patient tissues than in NCMT. IL-8 treatment induced p-p38 MAPK and p-ERK expression, but reduced p-JNK expressions in HNSCC cells. IL-8 siRNA suppressed p38 MAPK and ERK but increased JNK expression in HNSCC cells. Exposure of SCC25 cells to IL-8, increased the expressions of p-IκB-α and nuclear factor (NF)-κB, suggesting that IL-8 regulates inflammatory response by modulating the MAPK and NF-κB pathway in HNSCC cells. IL-8 promotes the migration of SCC25 cells and increases matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. These results reveal that IL-8 is the major stimulus of inflammatory mediation in HNSCC progression and migration by activating the p38 MAPK/ERK-NF-κB pathway and reducing JNK.

Background and objectives: Bladder urothelial carcinoma is the most common type of genitourinary cancer. Patients with bladder cancer may have limited treatment efficacy related to drug toxicity, resistance or adverse effects, and novel therapeutic strategies to enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance. Epigallocatechin gallate (EGCG) is a polyphenolic compound found in green tea leaves, and a potential anti-cancer agent in various cancer types through modulating and regulating multiple signaling pathways. The current study aimed to explore the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma. Materials and Methods: The BFTC-905 cells, human urinary bladder transitional cell carcinoma (TCC) cell line, were treated with EGCG or water for 24 hours, and the expression profiles of mRNAs and microRNAs were analyzed using next generation sequencing (NGS). The enriched biological functions were determined using different bioinformatics databases. Results: A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, which were mainly involved in nicotinamide adenine dinucleotide (NAD) biogenesis, inflammatory response and oxidation-reduction metabolism. Moreover, several microRNA-mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p- ARRB1 (arrestin beta 1), miR-3116- MGAT5B (alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase B), miR-31-5p-TNS1 (tensin 1), miR-642a-5p-TNS1, miR-1226-3p- DLG2 (discs large homolog 2), miR-484-DLG2, and miR-22-3p- PPM1K (protein phosphatase 1K). Conclusions: The current findings provide insights into novel therapeutic targets and underlying mechanisms of action of EGCG treatment in bladder cancer.

Introduction: Lipoteichoic acid (LTA) is a major cell wall component of Gram-positive bacteria. As an agonist of toll-like receptor-2, LTA might stimulate immune cells and contribute to the pathogenesis of sepsis. This study aimed to explore the possible miRNA-mediated gene expression alterations in neutrophils stimulated with LPA. Methods: The neutrophils isolated from peripheral blood of a healthy donor were treated with either Staphylococcus aureus LTA or water for 16 hours, and the miRNA and mRNA expression profiles were assessed and analyzed with next-generation sequencing and bioinformatics approaches. Results: A total of 38 differentially expressed miRNAs and 342 differentially expressed genes were identified between LTA-treated and vehicle-treated neutrophils. Using gene ontology analysis of the differentially expressed genes, the significantly enriched biological processes, such as defense response and positive regulation of cell migration, and significantly enriched pathways, such as cytokine-cytokine receptor interaction, were identified. Gene ontology analysis of differentially expressed miRNAs found the significantly enriched biological processes included signal transduction and cell communication. Using miRNA target-predicting tools, we found 4 microRNAs, including hsa-miR-34a-5p, hsa-miR-34c-5p, hsa-miR-708-5p, and hsa-miR-1271-5p, and five target genes, including CACNB3, HBEGF, MET, TNS3, and TTYH3, might play important roles in LTA-induced changes in neutrophils. Conclusions: We identified some changes of miRNAs and mRNAs in neutrophils treated with LTA. Further studies are needed to understand detailed mechanisms.

Background and Objectives: Atropine is a nonselective muscarinic antagonist which has been used to prevent worsening of myopia in children. Different concentrations of atropine were used for myopia, ranging from 0.01% to 1.0%. However, there are still potential toxicity of different doses of atropine to the cornea. Here, we present a study of investigating novel genes potentially involved in the effects of very low dose atropine treatment (0.003%) on corneal epithelial cells using next-generation sequencing (NGS) and bioinformatics approaches. Materials and Methods: Human corneal epithelial cells were treated with 0.003% atropine, cultured until confluence, and RNA extracted for differential expression profiling of mRNA and microRNA (miRNA) between control and atropine-treated corneal epithelial cells. The functional enrichment analysis for differentially expressed genes was performed using two bioinformatics databases, including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity&reg, Pathway Analysis (IPA). In addition, potential miRNA-mRNA interactions involved in atropine-treated corneal epithelial cells were predicted and validated using different miRNA target prediction databases. Results: Our results showed 0.003% atropine might suppress the apoptosis of corneal epithelial cells, potentially through Ras and protein kinase A signaling pathways. We also validated the possible miRNA regulations by using TargetScan and miRDB databases. Hsa-miR-651-3p-EPHA7, hsa-miR-3148-TMEM108 and hsa-miR-874-5p-TBX6 were validated as possible miRNA regulations involved in corneal epithelial cells treated with 0.003% atropine. Conclusions: These findings may contribute novel insights into therapeutic strategies for treating cornea with 0.003% atropine.

Using next-generation sequencing to decipher the molecular mechanisms underlying aberrant rheumatoid arthritis synovial fibroblasts (RASF) activation, we performed transcriptome-wide RNA-seq and small RNA-seq on synovial fibroblasts from rheumatoid arthritis (RA) subject and normal donor. Differential expression of mRNA and miRNA was integrated with interaction analysis, functional annotation, regulatory network mapping and experimentally verified miRNA&ndash, target interaction data, further validated with microarray expression profiles. In this study, 3049 upregulated mRNA and 3552 downregulated mRNA, together with 50 upregulated miRNA and 35 downregulated miRNA in RASF were identified. Interaction analysis highlighted contribution of miRNA to altered transcriptome. Functional annotation revealed metabolic deregulation and oncogenic signatures of RASF. Regulatory network mapping identified downregulated FOXO1 as master transcription factor resulting in altered transcriptome of RASF. Differential expression in three miRNA and corresponding targets (hsa-miR-31-5p:WASF3, hsa-miR-132-3p:RB1, hsa-miR-29c-3p:COL1A1) were also validated. The interactions of these three miRNA&ndash, target genes were experimentally validated with past literature. Our transcriptomic and miRNA interactomic investigation identified gene signatures associated with RASF and revealed the involvement of transcription factors and miRNA in an altered transcriptome. These findings help facilitate our understanding of RA with the hope of serving as a springboard for further discoveries relating to the disease.

Toll-like receptors (TLRs), which are a class of pattern-recognition receptors, can sense specific molecules of pathogens and then activate immune cells, such as neutrophils. The regulation of TLR signaling in immune cells has been investigated by various studies. However, the interaction of TLR signaling-activated microRNAs (miRNAs) and genes has not been well investigated in a specific type of immune cells. In the present study, neutrophils were isolated from peripheral blood of a healthy donor, and then treated for 16 h with Staphylococcus aureus lipoteichoic acid (LTA), which is an agonist of TLR2. The miRNA and mRNA expression profiles were analyzed via next-generation sequencing and bioinformatics approaches. A total of 290 differentially expressed genes between LTA-treated and vehicle-treated neutrophils were identified. Gene ontology analysis revealed that various biological processes and pathways, including inflammatory responses, defense response, positive regulation of cell migration, motility, and locomotion, and cell surface receptor signaling pathway, were significantly enriched. In addition, 38 differentially expressed miRNAs were identified and predicted to be involved in regulating signal transduction and cell communication. The interaction of 4 miRNAs (hsa-miR-34a-5p, hsa-miR-34c-5p, hsa-miR-708-5p, and hsa-miR-1271-5p) and 5 genes (MET, CACNB3, TNS3, TTYH3, and HBEGF) was proposed to participate in the LTA-induced signaling network. The present findings may provide novel information for understanding the detailed expression profiles and potential networks between miRNAs and their target genes in LTA-stimulated healthy neutrophils.

Patients with chronic kidney disease have a greater risk of cognitive impairment. Cerebral uremic solute accumulation causes uremic encephalopathy; however, the association of protein-bound uremic toxins on cognitive function remains unclear. The present study aimed to investigate the association of two protein-bound uremic toxins, namely indoxyl sulfate (IS) and p-cresyl sulfate (PCS), on cognitive function in patients receiving hemodialysis (HD) for at least 90 days. Circulating free form IS and PCS were quantified by liquid chromatography/mass spectrometry. Mini-Mental State Examination (MMSE) and Cognitive Abilities Screening Instrument (CASI) were used to evaluate cognitive function. In total, 260 HD patients were recruited with a mean age of 58.1 ± 11.3 years, of which, 53.8% were men, 40% had diabetes, and 75.4% had hypertension. The analysis revealed that both free IS and free PCS were negatively associated with the CASI score and MMSE. After controlling for confounders, circulating free IS levels persisted to be negatively associated with MMSE scores [β = −0.62, 95% confidence interval (CI): −1.16 to −0.08] and CASI scores (β = −1.97, 95% CI: −3.78 to −0.16), mainly in the CASI domains of long-term memory, mental manipulation, language ability, and spatial construction. However, there was no correlation between free PCS and total MMSE or total CASI scores after controlling for confounders. In conclusion, circulating free form IS, but not PCS is associated with lower cognitive function test scores in HD patients. Thus, a further study is needed to evaluate whether a decrease in free IS levels can slow down cognitive decline in HD patients.

Idiopathic pulmonary fibrosis (IPF) is a disabling and lethal chronic progressive pulmonary disease. Epigallocatechin gallate (EGCG) is a polyphenol, which is the major biological component of green tea. The anti-oxidative, anti-inflammatory, and anti-fibrotic effects of EGCG have been shown in some studies, whereas its effects in altering gene expression in pulmonary fibroblasts have not been systematically investigated. This study aimed to explore the effect of EGCG on gene expression profiles in fibroblasts of IPF. The pulmonary fibroblasts from an IPF patient were treated with either EGCG or water, and the expression profiles of mRNAs and microRNAs were determined by next-generation sequencing (NGS) and analyzed with the bioinformatics approach. A total of 61 differentially expressed genes and 56 differentially expressed microRNAs were found in EGCG-treated IPF fibroblasts. Gene ontology analyses revealed that the differentially expressed genes were mainly involved in the biosynthetic and metabolic processes of cholesterol. In addition, five potential altered microRNA&ndash, mRNA interactions were found, including hsa-miR-939-5p&ndash, PLXNA4, hsa-miR-3918&ndash, CTIF, hsa-miR-4768-5p&ndash, PDE5A, hsa-miR-1273g-3p&ndash, VPS53, and hsa-miR-1972&ndash, PCSK9. In summary, differentially expressed genes and microRNAs in response to EGCG treatment in IPF fibroblasts were identified in the current study. Our findings provide a scientific basis to evaluate the potential benefits of EGCG in IPF treatment, and warrant future studies to understand the role of molecular pathways underlying cholesterol homeostasis in the pathogenesis of IPF.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease. Therapeutic options for IPF remain limited. Nintedanib, a tyrosine kinase inhibitor approved for IPF treatment, is known to inhibit fibroblasts proliferation, migration and transformation to myofibroblasts. However, how nintedanib changes gene regulations in IPF has never been systematically investigated. We conducted a next-generation sequencing and bioinformatics study to evaluate the changes of mRNA and miRNA profiles in IPF fibroblasts treated with 2 &micro, M and 4 &micro, M nintedanib, compared to those without treatment. We identified 157 upregulated and 151 downregulated genes and used STRING and DAVID databases for analysis of protein&ndash, protein interactions, biological pathways, and molecular functions. We found strong protein&ndash, protein interactions within these dysregulated genes, mostly involved in the pathways of cell cycle and mitotic cell cycle. We also discovered 13 potential miRNA&ndash, mRNA interactions associated with nintedanib treatment. After validation using miRDB, TargetScan, and RT-qPCR, we identified 4 downregulated genes (DDX11, E2F1, NPTX1, and PLXNA4) which might be repressed by the upregulated hsa-miR-486-3p. According to the proposed functions of DDX11, E2F1, and PLXNA4 reported in previous studies, these gene expression changes together might contribute to decreased proliferation of fibroblasts and decreased angiogenesis in the microenvironment of IPF. Our findings need further studies to confirm.