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Open Access Publications from the University of California

Scholarly Works (21 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2016)

Pancreatic ductal adenocarcinoma (PDAC) has a median survival of six months and a five-year survival of <5%, making it one of the most lethal human cancers. This poor prognosis is due to the uniformly advanced disease stage at the time of diagnosis and to its profound resistance to existing therapies. Ductal adenocarcinomas makes up between 75 to 92% of pancreatic neoplasms. Premalignant lesions, known as pancreatic intraepithelial neoplasms (PanINs) are of ductal origin and found in close physical proximity with advanced malignant tumors. PanINs are thought to be precursors of ductal adenocarcinoma, as they progress toward increasingly atypical histological stages. Previous studies have implicated the role of different signaling pathways in the pathogenesis of pancreatic cancer, however the role of transcriptomic alterations have not been well characterized. The aim of this dissertation was to characterize the role and function of a new transcription factor gene, called FOXA2, in the pathogenesis of pancreatic cancer. Our preliminary analysis revealed that FOXA2 gene was highly down-regulated in pancreatic cancer tissues relative to control tissue samples. Interestingly, FOXA2 gene has not been implicated previously in pancreatic oncogenesis. FOXA2 is a transcription factor that was initially identified in hepatocytes, where it binds in the promoter areas of important liver-enriched genes transthyretin, alpha 1-antitrypsin and albumin. Our data revealed that FOXA2 is significantly down-regulated in pancreatic cancer, according to qPCR and immunohistochemical analysis in human pancreatic tissues. In order to study the role of FOXA2 deletion in vivo, we utilized the CRISPR/Cas9 system to delete FOXA2 from the PANC-1 cell line (FOXA2 deletion). Subcutaneous injections in immunodeficient mice demonstrated FOXA2 deletion had a significantly higher weight and volume than the control tumors. Moreover, we identified a negative regulator of FOXA2 expression, microRNA-199a, which targets directly and reduces FOXA2 mRNA and protein expression levels in pancreatic cancer. Overexpression of microRNA-199a was observed in human pancreatic cancer tissue by qPCR and in-situ hybridization methods. In an effort to identify downstream targets of FOXA2, we transiently knocked-down FOXA2 with an siRNA and performed a gene expression array in a human pancreatic cell line. The expression data revealed plasminogen urokinase activator receptor (PLAUR) was significantly up-regulated upon FOXA2 inhibition. PLAUR is a marker of invasiveness and is responsible for the invasive phenotype we observed upon FOXA2 knockdown.

    Cover page: Integration of Transcriptomic and CRISPR-Cas9 Technologies Reveal FOXA2 as a Tumor Suppressor Gene in Pancreatic Cancer
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2016)

    Background

    Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo.

    Methods

    A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors.

    Results

    The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas.

    Conclusions

    Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma.
      Cover page: A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2016)
      Neurotensin (NT) via its receptor 1 (NTR1) modulates the development of colitis, decreases HIF-1α/PHD2 interaction, stabilizes and increases HIF-1α transcriptional activity, and promotes intestinal angiogenesis. HIF-1α induces miR-210 expression, whereas miR-210 is strongly upregulated in response to NT in NCM460 human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1). In this study, we examined whether NT activates a NTR1-HIF-1α-miR-210 cascade using in vitro (NCM460-NTR1 cells) and in vivo (transgenic mice overexpressing [HIF-1α-OE] or lacking HIF-1α [HIF-1α-knockout (KO)] in intestinal epithelial cells and mice lacking NTR1 [NTR1-KO]) models. Pretreatment of NCM460-NTR1 cells with the HIF-1α inhibitor PX-478 or silencing of HIF-1α (small interfering HIF-1α) attenuated miR-210 expression in response to NT. Intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS) administration (2-d model) increased colonic miR-210 expression that was significantly reduced in NTR1-KO, HIF-1α-KO mice, and wild-type mice pretreated intracolonically with locked nucleic acid anti-miR-210. In contrast, HIF-1α-OE mice showed increased miR-210 expression at baseline that was further increased following TNBS administration. HIF-1α-OE mice had also exacerbated TNBS-induced neovascularization compared with TNBS-exposed wild-type mice. TNBS-induced neovascularization was attenuated in HIF-1α-KO mice, or mice pretreated intracolonically with anti-miR-210. Intracolonic anti-miR-210 also reduced colitis in response to TNBS (2 d). Importantly, miR-210 expression was increased in tissue samples from ulcerative colitis patients. We conclude that NT exerts its proinflammatory and proangiogenic effects during acute colitis via a NTR1-prolyl hydroxylase 2/HIF-1α-miR-210 signaling pathway. Our results also demonstrate that miR-210 plays a proinflammatory role in the development of colitis.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2015)

        Objective

        Neurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo.

        Design

        miR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro.

        Results

        NT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-κB activation, as well as IL-6, IL-8 and IL-1β messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls.

        Conclusions

        NT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2014)
          The expression of neurotensin (NT) and its receptor (NTR1) is up-regulated in experimental colitis and inflammatory bowel disease; NT/NTR1 interactions regulate gut inflammation. During active inflammation, metabolic shifts toward hypoxia lead to the activation of hypoxia-inducible factor (HIF)-1, which enhances vascular endothelial growth factor (VEGF) expression, promoting angiogenesis. We hypothesized that NT/NTR1 signaling regulates intestinal manifestations of hypoxia and angiogenesis by promoting HIF-1 transcriptional activity and VEGFα expression in experimental colitis. We studied NTR1 signaling in colitis-associated angiogenesis using 2,4,6-trinitrobenzenesulfonic acid-treated wild-type and NTR1-knockout mice. The effects of NT on HIF-1α and VEGFα were assessed on human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1) and human intestinal microvascular-endothelial cells. NTR1-knockout mice had reduced microvascular density and mucosal integrity score compared with wild-type mice after 2,4,6-trinitrobenzenesulfonic acid treatment. VEGFα mRNA levels were increased in NCM460-NTR1 cells treated with 10(-7) mol/L NT, at 1 and 6 hours post-treatment. NT exposure in NCM460-NTR1 cells caused stabilization, nuclear translocation, and transcriptional activity of HIF-1α in a diacylglycerol kinase-dependent manner. NT did not stimulate tube formation in isolated human intestinal macrovascular endothelial cells but did so in human intestinal macrovascular endothelial cells cocultured with NCM460-NTR1 cells. Our results demonstrate the importance of an NTR1-HIF-1α-VEGFα axis in intestinal angiogenic responses and in the pathophysiology of colitis and inflammatory bowel disease.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2015)

            Background & aims

            Substance P (SP), a neuropeptide member of the tachykinin family, plays a critical role in colitis. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression. However, whether SP modulates expression of microRNAs in human colonic epithelial cells remains unknown.

            Methods

            We performed microRNA profiling analysis of SP-stimulated human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NCM460-NK-1R). Targets of SP-regulated microRNAs were validated by real time polymerase chain reaction (RT-PCR). Functions of miRNAs were tested in NCM460-NK-1R cells and the TNBS and DSS models of colitis.

            Results

            SP stimulated differential expression of 29 microRNAs, including miR-221-5p, the highest up regulated miR (by 12.6-fold) upon SP stimulation. Bioinformatic and luciferase reporter analyses identified interleukin 6 receptor (IL-6R) mRNA as a direct target of miR-221-5p in NCM460 cells. Accordingly, SP exposure of NCM460-NK-1R cells increased IL-6R mRNA expression, while overexpression of miR-221-5p reduced IL-6R expression. NF-κB and JNK inhibition decreased SP-induced miR-221-5p expression. MiR-221-5p expression was increased in both TNBS- and DSS-induced colitis and colonic biopsies from Ulcerative Colitis, but not Crohn's Disease subjects, compared to controls. In mice, intracolonic administration of a miR-221-5p chemical inhibitor, exacerbated TNBS-and DSS-induced colitis, and increased colonic TNF-α, Cxcl10, and Col2 α 1 mRNA expression. In situ hybridization in TNBS-and DSS-exposed colons revealed increased miR-221-5p expression primarily in colonocytes.

            Conclusions

            Our results reveal a novel NK-1R-miR-221-5p-IL-6R network that protects from colitis. The use of miR-221-5p mimics may be a promising approach for colitis treatment.
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2015)

              Background

              Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors.

              Methods

              MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets.

              Results

              A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3'UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3'UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels.

              Conclusions

              Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis.
                Cover page: Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2017)

                Objective

                Chronic HIV-1 infection leads to widespread inflammation and immune dysregulation. The gastrointestinal mucosa, a primary site for HIV-1 replication, is thought to play a significant role in this response. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression, including immune activation and inflammation. Here we investigate miR expression and function in the colonic mucosa during HIV-1 infection.

                Design and methods

                Using miR profiling, we examined miR expression in the colonic mucosa of HIV-infected patients. These miRs were further parsed to identify those that most likely function in HIV-related inflammation. Using bioinformatics tools, we identified potential target genes which were confirmed using in-vitro functional testing.

                Results

                We identified 12 miRs that were differentially expressed in the colonic mucosa of HIV-infected patients with high versus undetectable plasma viral concentrations. Of these, both miR-26a and miR-29a were downregulated in untreated HIV-1 infection, yet not in the colonic mucosa from inflammatory bowel disease. This downregulation occurs within the first hours after infection. These miRs were further shown to directly target IL-6 and STAT3, respectively, with similar changes confirmed in an ex-vivo explant infection model.

                Conclusion

                miR-26a and miR-29a levels are decreased in the colonic mucosa during chronic HIV-1 infection, and this change may be initiated during acute infection. Both miRs de-repress the IL-6/STAT3 signaling pathway, which could contribute to increased inflammation during infection. These miRs may represent novel therapeutic targets for HIV-1-associated inflammation in the colonic mucosa.
                  Cover page: Unique microRNA expression in the colonic mucosa during chronic HIV-1 infection
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2018)
                  Peripheral factors likely play a role in at least a subset of irritable bowel syndrome (IBS) patients. Few studies have investigated mucosal gene expression using an unbiased approach. Here, we performed mucosal gene profiling in a sex-balanced sample to identify relevant signaling pathways and gene networks and compare with publicly available profiling data from additional cohorts. Twenty Rome III+ IBS patients [10 IBS with constipation (IBS-C), 10 IBS with diarrhea (IBS-D), 5 men/women each), and 10 age-/sex-matched healthy controls (HCs)] underwent sigmoidoscopy with biopsy for gene microarray analysis, including differential expression, weighted gene coexpression network analysis (WGCNA), gene set enrichment analysis, and comparison with publicly available data. Expression levels of 67 genes were validated in an expanded cohort, including the above samples and 18 additional participants (6 each of IBS-C, IBS-D, HCs) using NanoString nCounter technology. There were 1,270 differentially expressed genes (FDR < 0.05) in IBS-C vs. HCs but none in IBS or IBS-D vs. HCs. WGNCA analysis identified activation of the cAMP/protein kinase A signaling pathway. Nine of 67 genes were validated by the NanoString nCounter technology (FDR < 0.05) in the expanded sample. Comparison with publicly available microarray data from the Mayo Clinic and University of Nottingham supports the reproducibility of 17 genes from the microarray analysis and three of nine genes validated by nCounter in IBS-C vs. HCs. This study supports the involvement of peripheral mechanisms in IBS-C, particularly pathways mediating neuronal signaling. NEW & NOTEWORTHY Peripheral factors play a role in the pathophysiology of irritable bowel syndrome (IBS), which, to date, has been mostly evident in IBS with diarrhea. Here, we show that sigmoid colon mucosal gene expression profiles differentiate IBS with constipation from healthy controls. These profiling data and analysis of additional cohorts also support the concept that peripheral neuronal pathways contribute to IBS pathophysiology.
                    Creative Commons 'BY-NC' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2015)

                    Introduction

                    Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.

                    Methods

                    Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.

                    Results

                    Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.

                    Conclusion

                    Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.
                      Cover page: Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.
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