CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer.

Background: Colon cancer (CC) is among the top three diseases with the highest morbidity and mortality rates worldwide. Its increasing incidence imposes a major global health burden. Immune checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, can be used for the treatment of CC; however, most patients with CC are resistant to immunotherapy. Therefore, identification of biomarkers that can predict immunotherapy sensitivity is necessary for selecting patients with CC who are eligible for immunotherapy. Methods: Differentially expressed genes associated with the high infiltration of CD8+ T cells were identified in CC and para-cancerous samples via bioinformatic analysis. Kaplan-Meier survival analysis revealed that MS4A1 and TNFRSF17 were associated with the overall survival of patients with CC. Cellular experiments were performed for verification, and the protein expression of target genes was determined via immunohistochemical staining of CC and the adjacent healthy tissues. The proliferation, migration and invasion abilities of CC cells with high expression of target genes were determined via in vitro experiments. Results: Differential gene expression, weighted gene co-expression and survival analyses revealed that patients with CC with high expression of MS4A1 and TNFRSF17 had longer overall survival. The expression of these two genes was lower in CC tissues than in healthy colon tissues and was remarkably associated with the infiltration of various immune cells, including CD8+ T cells, in the tumour microenvironment (TME) of CC. Patients with CC with high expression of MS4A1 and TNFRSF17 were more sensitive to immunotherapy. Quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemical staining validated the differential expression of MS4A1 and TNFRSF17. In addition, Cell Counting Kit-8, wound healing and transwell assays revealed that the proliferation, migration and invasion abilities of CC cells were weakened after overexpression of MS4A1 and TNFRSF17. Conclusions: The core genes MS4A1 and TNFRSF17 can be used as markers to predict the sensitivity of patients with CC to immunotherapy and have potential applications in gene therapy to inhibit CC progression.

Dictamnine inhibits pancreatic cancer cell growth and epithelial-mesenchymal transition by blocking the PI3K/AKT signaling pathway.

Many different treatments are available for pancreatic cancer (PC), including surgical resection, chemotherapy, radiotherapy, and immunotherapy, but these treatments are often ineffective at curing PC. Hence, identifying new and effective agents or strategies to improve therapeutic effects is critical. This study focused on the efficacy of dictamnine (DTM), a furan quinoline alkaloid extracted from Dictamnus dasycarpus Turcz., in treating PC. Our in vitro results showed that DTM can mitigate cell proliferation and induce cell cycle arrest and apoptosis in two different human PC cell lines. Moreover, epithelial-mesenchymal transition (EMT) was prevented during DTM treatment, reflected by reduced cell migration and invasion abilities. In vivo studies demonstrated that DTM treatment led to a remarkable inhibition of tumor growth in a xenograft nude mouse model. Mechanistic investigation showed that DTM might act by restraining constitutive and induced PI3K/AKT activity. In summary, our results demonstrated that DTM slows PC progression by inhibiting the activity of the PI3K/AKT signaling pathway and its downstream effectors and that DTM is effective as a pathway-specific cancer therapy. These findings could provide a greater understanding of the function of anticancer drugs and new options for PC treatment.

Diabetes, disability, and dementia risk: Results from the Hispanic Established Populations for the Epidemiologic Studies of the Elderly (H-EPESE).

BACKGROUND: Emerging research has elucidated pathophysiological relationships among diabetes, disability, cognitive impairment, and incident dementia. However, the relationships between diabetes, disability, and dementia have been largely underexamined in Latino populations, which have a disproportionate prevalence of diabetes and its complications. AIMS: This study examines diabetes as a risk factor for subsequent disability and dementia risk in a Mexican-origin older adult sample. METHODS: The data are drawn from eight waves (1993-2013) of the Hispanic Established Populations for the Epidemiologic Study of the Elderly (HEPESE; N = 3,050, mean age at baseline = 73.6 (±6.8)). Respondents' diabetes status at baseline was ascertained by self-report. Disability was assessed using eight functional domains assessed through the Lawton Instrumental Activities of Daily Living (IADL) Scale. Dementia risk was assessed using a Mini-Mental Status Exam (MMSE) score below 18 and the need for aid with at least two IADLs. We used multivariable Cox proportional hazards models to predict the relation between diabetes and time to disability, cognitive impairment, and incident dementia, adjusting for age at migration, socioeconomic status, acculturation, and health status. RESULTS: At baseline, diabetes prevalence was 28.1%, and 37.7% had IADL disability. Diabetes was associated with a higher risk of developing dementia (Hazard Ratio (HR) = 1.22, p < .001) over the approximetely 20 year study period. In addition, immigrants who migrated at age 50 or older had a higher dementia risk (HR = 1.35, p = .01) when compared to their US-born counterpart. CONCLUSION: Our results highlight the importance of better characterizing the role of diabetes and nativity in the co-occurrence of disability and dementia risk.

The nomogram based on the 6-lncRNA model can promote the prognosis prediction of patients with breast invasive carcinoma.

Long non-coding RNA (lncRNA) is a prognostic biomarker for many types of cancer. Here, we aimed to study the prognostic value of lncRNA in Breast Invasive Carcinoma (BRCA). We downloaded expression profiles from The Cancer Genome Atlas (TCGA) datasets. Subsequently, we screened the differentially expressed genes between normal tissues and tumor tissues. Univariate Cox, LASSO regression, and multivariate Cox regression analysis were used to construct a lncRNA prognostic model. Finally, a nomogram based on the lncRNAs model was developed, and weighted gene co-expression network analysis (WGCNA) was used to predict mRNAs related to the model, and to perform function and pathway enrichment. We constructed a 6-lncRNA prognostic model. Univariate and multivariate Cox regression analysis showed that the 6-lncRNA model could be used as an independent prognostic factor for BRCA patients. We developed a nomogram based on the lncRNAs model and age, and showed good performance in predicting the survival rates of BRCA patients. Also, functional pathway enrichment analysis showed that genes related to the model were enriched in cell cycle-related pathways. Tumor immune infiltration analysis showed that the types of immune cells and their expression levels in the high-risk group were significantly different from those in the low-risk group. In general, the 6-lncRNA prognostic model and nomogram could be used as a practical and reliable prognostic tool for invasive breast cancer.

Metabolic Risk and Depression among Elderly Mexican Americans: The Roles of Nativity Status.

Objective: To evaluate the relationship between metabolic risk (MR) and depression in a sample of older Mexican Americans and examine whether the association differs by age at migration. Methods: Longitudinal study using data from the Hispanic Established Populations for the Epidemiologic Study of the Elderly (HEPESE) (N=807, mean age = 84.3). The analytical sample was compiled from wave 6 (2007) to wave 7 (2010-2011) of HEPESE. Random-effect logistic regression examined the association between MR and depression and tested the model stratified by nativity status and age at migration. Results: MR was associated with higher odds of depression for US-born Mexican Americans after controlling for potential confounders. Similarly, among Mexican Americans who migrated before age 20, MR was associated with higher odds of depression. Conclusion: The findings highlight the importance of age at migration when evaluating the health of foreign-born Mexican Americans from a life-course perspective. Particularly among Mexican Americans who migrated before age 20, those with MR were more vulnerable to depression than their counterparts without MR.

Nativity, Family, Disability: Results from the Hispanic Established Populations for the Epidemiologic Study of the Elderly.

Objectives: ativity and family support may influence attitudes and behaviors that delay or accelerate the disability process in older adults. The objectives of this study were twofold: 1) to evaluate nativity and migration cohort differences in trajectories of disability (assessed by activities of daily living [ADL]) among older Mexican Americans; and 2) to determine the role of objectively measured family support in the association between nativity, migration cohort, and disability changes over time. Methods: This is a longitudinal study with up to 18 years follow-up (1993-2011) using data from the Hispanic Established Populations for the Epidemiologic Study of the Elderly (N=2,785, mean age =72.4 years). Disability was assessed using self-reported limitations in activities of daily living (ADL). Nativity and migration cohort were self-reported. Family support was assessed by marital status and the number of their children participants saw each month. Linear growth curve models evaluated the trajectory of ADL disability over 18 years and assessed variations by nativity status, migration cohort and family support. Results: Foreign-born respondents who migrated before age 20 had more starting ADL limitations (ß= .36, P<.001) and accumulated disability faster (ß=.04, P<.01) compared with their US-born counterparts. In contrast, foreign-born respondents who migrated at later ages showed disability trajectories similar to US-born respondents. Married respondents had a lower level of disability (ß= -.14, P<.01) and a lower rate of accumulation over time (ß= -.02, P=.001) compared with participants who were not married. Discussion: Mexican Americans who migrate at younger ages may experience greater disability over time; however, family support may help mitigate the accumulation of disability among older Mexican Americans.

Regulation of Pancreatic Fibrosis by Acinar Cell-Derived Exosomal miR-130a-3p via Targeting of Stellate Cell PPAR-γ.

INTRODUCTION: As endogenous miRNA carriers, exosomes play a role in the pathophysiological processes of various diseases. However, their functions and regulation mechanisms in pancreatic fibrosis remain unclear. METHODS: In this study, an RNA microarray was used to detect differentially expressed exosomal miR-130a-3p in AR42J cells before and after taurolithocholate (TLC) treatment. mRNA-seq was used to screen differentially expressed genes before and after pancreatic stellate cell (PSC) activation. We used the STRING database to construct a protein-protein interaction (PPI) network for differentially expressed genes, used CytoNCA to analyze the centrality of the PPI network, and identified 10 essential proteins in the biological network. Then, the TargetScan and miRanda databases were used to predict the target genes of miR-130a-3p. The intersections of the target genes and the mRNAs encoding the 10 essential proteins were identified to construct miR-130a-3p/peroxisome proliferator-activated receptor gamma (PPAR-γ) pairs. Fluorescence labeling of exosomes and dynamic tracing showed that exosomes can fuse with the cell membranes of PSCs and transport miR-130a-3p into PSCs. A luciferase reporter gene assay was used to confirm that miR-130a-3p can bind to PPAR-γ to inhibit PPAR-γ expression. In vitro and in vivo functional experiments were performed for gain-of-function studies and loss-of-function studies, respectively. RESULTS: The studies showed that acinar cell-derived exosomal miR-130a-3p promotes PSC activation and collagen formation through targeting of stellate cellular PPAR-γ. Knockdown of miR-130a-3p significantly improved pancreatic fibrosis. Notably, miR-130a-3p knockdown reduced serum levels of hyaluronic acid (HA) and ß-amylase and increased the C-peptide level to protect endocrine and exocrine pancreatic functions and the function of endothelial cells. CONCLUSION: This study revealed that the exosomal miR-130a-3p/PPAR-γ axis participates in PSC activation and the mechanism of chronic pancreatitis (CP) with fibrosis, thus providing a potential new target for the treatment of chronic pancreatic fibrosis.

Maternal human capital accumulation and children's well-being.

Research examining the intergenerational transmission of human capital is subject to two limitations. First, for the parental generation, most studies focus on formal education but fail to consider vocational training experience, which has more variation than formal educational attainment over the life course. Second, most studies have found consistent conclusions using income and occupation for the children's outcomes but have generated mixed findings regarding health and cognitive ability. This study aims to answer whether mothers' additional vocational training beyond formal education is beneficial to children's health and cognitive ability. Applying fixed-effects regression to the National Longitudinal Survey of Youth 1979 and the National Longitudinal Survey of Youth Child and Young Adults datasets, this study finds that mothers' human capital accumulation is positively associated with higher cognitive scores for both boys and girls, but does not significantly predict children's illnesses or behavior problems. These findings bear implications for policy aimed at mitigating the intergenerational cycles of disadvantages.

HIV and three dimensions of Wisdom: Association with cognitive function and physical and mental well-being: For: Psychiatry Research.

Wisdom is a unique human personality trait with cognitive, affective or compassionate, and reflective dimensions. We evaluated relationships of three specific dimensions of wisdom with cognitive function and physical and mental well-being in people with HIV (PWH) and HIV-negative (HIV-) participants. Subjects included 138 adults (61 PWH, 77 HIV-) from the San Diego community. Validated measures were used to assess wisdom and well-being. Cognitive function was assessed via the Montreal Cognitive Assessment. We conducted multivariate linear regressions to evaluate the associations of wisdom dimensions with cognitive function and physical and mental well-being. Compared to the HIV- group, PWH had lower mean scores on cognitive function, and physical and mental well-being, and cognitive and reflective dimensions of wisdom, but similar scores on affective or compassionate wisdom. Among PWH, higher total wisdom scores were associated with older age, lower likelihood of substance dependence, greater mental well-being, better cognitive function, higher resilience, social support, and optimism scores, as well as lower levels of perceived stress and nadir CD4 count. Our findings of an association of different dimensions of wisdom with physical and/or mental well-being in PWH would point to a possibility that enhancing these dimensions of wisdom might improve health outcomes in PWH.

Effects of Egr1 on pancreatic acinar intracellular trypsinogen activation and the associated ceRNA network.

Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3­sulfate (TLC­S)­treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNA­transfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNA­transfected cells, indicating that in TLC­S­induced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNA­microRNA (miRNA/miR)­long non­coding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)­miR­214­3p and rno­miR­764­5p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.

Intestinal flora imbalance affects bile acid metabolism and is associated with gallstone formation.

BACKGROUND: The gut microbiota participates in the metabolism of substances and energy, promotes the development and maturation of the immune system, forms the mucosal barrier, and protects the host from pathogen attacks. Although the pathogenesis of cholesterol gallstones is still not clear, studies have suggested that gut microbiota dysbiosis plays an important role in their formation. METHODS: Microbial DNA from faeces of normal control patients and those of patients with calculi was subjected to 16S rRNA gene sequencing to detect gene expression changes in intestinal microbes. ELISA kits were used to measure free bile acids, secondary bile acids and coprostanol according to the manufacturer's instructions. The relationship between flora and their metabolites was then analysed. RESULTS: In the gallstone group, the diversity of intestinal bacteria and the abundances of certain phylogroups were significantly decreased (p < 0.05), especially Firmicutes (p < 0.05), the largest phylum represented by the gut microbiota. This study found an increase in free bile acids (p < 0.001) and secondary bile acids (p < 0.01) in the enterohepatic circulation. Bile salt hydrolase activity was not related to the abundances of BSH-active bacteria. 7a-dehydroxylating gut bacteria were significantly increased (p < 0.01), whereas cholesterol-lowering bacteria were significantly reduced (p < 0.05). The Ruminococcus gnavus group could be used as a biomarker to distinguish the gallstone group from the control group. CONCLUSION: We conclude that intestinal flora imbalance affects bile acid and cholesterol metabolism and is associated with gallstone formation.

The Regulatory Effect of the Kinase Inhibitor PD98059 on Autophagic Flux During Trypsinogen Activation in Pancreatic Acinar Cells.

OBJECTIVES: To study the role of kinase inhibitor PD98059 on autophagy flow in the process of trypsinogen activation in pancreatic acinar cell and its related mechanism. METHODS: In the present study, bioinformatics analysis was used to predict kinases and their most relevant inhibitor (PD98059) which participates in autophagy of acute pancreatitis (AP). The rat pancreatic acini AR42J cells were divided into 4 groups: control group, sodium taurocholate hydrate (TLC) group, PD98059 group, and TLC + PD group. Twenty-seven Sprague-Dawley rats were divided into 3 groups (n = 9), including control group, severe AP (SAP) group, and SAP + PD group. We detected trypsinogen activation, autophagic activation, lysosome pH, and cathepsin-L activity in vivo and in vitro. RESULTS: Results revealed trypsinogen activation was significantly inhibited in mitogen-activated protein kinase 1, JAK2, LYN, and their common inhibitor was PD98059. The trypsinogen activation, Beclin1, and light chain 3 II expressions were reduced, whereas the expressions of lysosomal-associated membrane protein 2, cathepsin L1, and cathepsin-L activity is upregulated after the PD98059 pretreatment, both in vivo and in vitro. CONCLUSIONS: Lysosomal dysfunction blocked autophagy flux, accompanied by increasing pancreatic acinar cell autophagy in the process of trypsinogen activation. PD98059 inhibited AP occurrence and pancreatic injury via improving the blocked autophagic pathway and reducing trypsinogen activation.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor.

The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

miR­92a­3p regulates trypsinogen activation via Egr1 in AR42J cells.

Acute pancreatitis (AP) exhibits high morbidity and mortality rates. The onset of AP is characterized by early trypsinogen activation.The present study aimed to investigate the expression of microRNA (miR)­92a­3p and early growth response protein 1 (Egr1), and the effect of miR­92a­3p on trypsinogen activation in the pancreatic exocrine cell line AR42J. mRNA and miRNA microarrays were used to identify differentially expressed mRNAs and miRNAs in AR42J cells. A miRNA­mRNA network was constructed using bioinformatics software, and Egr1 and its regulated miRNA subnetworks were identified by reviewing previous literature. The results suggested that miR­92a­3p could bind to Egr1 3'untranslated region sequence. Subsequently, miR­92a­3p mimic and inhibitor were used to transfect AR42J cells. Following transfection, reverse transcription­quantitative PCR and western blotting were performed to detect Egr1 expression. Furthermore, AR42J cells were cotransfected with miR­92a­3p inhibitor and small interfering (si)­Egr1. The trypsinogen activation rate of AR42J cells was measured by flow cytometry. Microarrays and bioinformatics results indicated that Egr1 may be a target gene of miR­92a­3p. In addition, the present study suggested that miR­92a­3p downregulated Egr1 in vitro and that miR­92a­3p and Egr1 expression was associated with trypsinogen activation. Furthermore, miR­92a­3p inhibitor reversed the effect of si­Egr1 on trypsinogen activation. In conclusion, miR­92a­3p may negatively regulate the activation of trypsinogen in AR42J cells via Egr1.

Clinicopathological and prognostic significance of octamer-binding transcription factor 4 in patients with gastric cancer: a systematic review and meta-analysis.

AIM: The role of octamer-binding transcription factor 4 (Oct4) in gastric cancer (GC) progression is still under debate and reported results are inconsistent. Therefore, we conducted a meta-analysis to evaluate the clinicopathological and prognostic significance of Oct4 expression in patients with GC. MATERIALS & METHODS: Relevant articles were retrieved from a diverse number of databases, and meta-analysis was completed using STATA software 12.0. RESULTS: Total of 21 studies were included in this analysis (3209 samples). Expression of Oct4 was associated with incidence, tumor size, lymph node metastasis, histological differentiation, pTNM stage, tumor depth of infiltration, vascular invasion and distal metastasis. Additionally, Oct4 expression was correlated with poor overall survival rate. CONCLUSION: The Oct4 overexpression suggested aggressive biological behaviors and imply that Oct4 may be a useful prognostic biomarker in gastric cancers.

The interaction between ANXA2 and lncRNA Fendrr promotes cell apoptosis in caerulein-induced acute pancreatitis.

BACKGROUND: Annexin A2 (ANXA2) plays a crucial role in acute pancreatitis (AP). However, its potential mechanism remains unclear. METHODS: In the present study, we used caerulein-treated AR42J rat pancreatic acinar cells as cell model of AP to investigate the potential functions of ANXA2 and its predicted long noncoding RNA (lncRNA) FOXF1 adjacent noncoding developmental regulatory RNA (lncRNA Fendrr). Cell apoptosis was evaluated by flow cytometry using annexinV-fluorescein isothiocyanate/propidium iodide staining. The expressions of ANAX2 and lncRNA Fendrr were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, Western blot analysis was performed to determine the protein levels of ANXA2, Bcl-2, and Bax. The association between lncRNA Fendrr and ANXA2 was disclosed by RNA pull-down, RNA immunoprecipitation, and electrophoretic mobility shift assays. RESULTS: ANXA2 was elevated in caerulein-induced AP model and promoted apoptosis of AR42J cells. LncRNA Fendrr was also upregulated in AP cell model and directly bound ANXA2 protein. Further studies indicated that the interaction between ANXA2 and lncRNA Fendrr contributed to the apoptosis of AR42J cells in AP cell model. CONCLUSION: Our study demonstrated that ANXA2 promoted AP progression via interacting with lncRNA Fendrr in vitro, which will provide a novel insight into the therapeutic target for AP.

Modular bioinformatics analysis demonstrates that a Toll­like receptor signaling pathway is involved in the regulation of macrophage polarization.

In recent years, an increasing number of studies on the roles of macrophages in tumors, immune responses and metabolism have been published, in which macrophage polarization has been an extensively discussed topic. In the present study, differentially expressed genes in various types of macrophages were analyzed using the Gene Expression Omnibus database. Cluster analysis of differentially expressed genes was conducted, and a protein­protein interaction (PPI) network was constructed. Finally, modular analysis and functional enrichment analysis revealed that a Toll­like receptor (TLR) signaling pathway is involved in the regulation of macrophage polarization. Furthermore, the high­degree proteins in the PPI network that are involved in the molecular regulation of macrophage polarization are closely associated with proteins of the TLR signaling pathway. These results suggested that the TLR signaling pathways may be a principal direction of future research on the regulation of macrophage polarization.

miR-352 participates in the regulation of trypsinogen activation in pancreatic acinar cells by influencing the function of autophagic lysosomes.

This study was performed to screen miRNAs and mRNAs that are differentially expressed during trypsinogen activation in acute pancreatitis and to verify their role in the process of trypsinogen activation. The function enrichment analysis showed that the functions of miR-352 and its regulatory targets lysosome-associated membrane protein 2 (LAMP2) and cathepsin L1 (CTSL1) were lysosome related. The results of the verification experiment showed that in the TLC-S-treated AR42J (pancreatic cell line) cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, and the autophagy pathway was blocked. In the miR-352 mimic-transfected cells, miR-352 expression increased, expression levels of LAMP2 and CTSL1 were significantly reduced, trypsinogen activation was increased, intracellular lysosomal pH increased, cathepsins L activity decreased and the amount of autophagolysosomes increased. In the miR-352 inhibitor-transfected cells, miR-352 expression was reduced, expression levels of LAMP2 and CTSL1 were significantly increased, trypsinogen activation was decreased, intracellular lysosomal pH decreased, cathepsins L activity increased and the amount of autophagolysosomes decreased. In the process of taurolithocholic acid 3-sulfate (TLC-S) induced trypsinogen activation, overexpression of miR-352 could down-regulate LAMP2 and CTSL1, resulting in the dysfunction of autophagic lysosome. Thus, the autophagy pathway was blocked, and trypsinogen activation was enhanced.

Gene Silencing of Human Sertoli Cells Utilizing Small Interfering RNAs.

Sertoli cells, as the unique somatic cells within the seminiferous tubules, play essential roles in regulating normal spermatogenesis. In addition, recent studies have demonstrated that Sertoli cells could have significant applications in regenerative medicine due to their great plasticity. However, the roles of genes in controlling the fate determinations of human Sertoli cells remain largely unknown. Silencing genes of human Sertoli cells utilizing small interfering RNAs (siRNAs) is an important method to explore their functions and mechanisms in human Sertoli cells. We isolated and identified human Sertoli cells. RNA interference (RNAi) was employed to probe the roles and signaling pathways of BMP6 and BMP4 in mediating the proliferation and apoptosis of human Sertoli cells. Specifically, siRNAs against BMP6 and BMP4 were used to knock down the expression levels of BMP6 and BMP4 and examine the function and mechanism in controlling the fate decisions of human Sertoli cells. In this chapter, we provided the detailed methods of RNAi in silencing BMP6 gene of human Sertoli cells. Quantitative real-time PCR demonstrated that the designed BMP6 siRNAs apparently silenced BMP6 mRNA in human Sertoli cells at 24 h after transfection. Western blots showed that the siRNAs silenced the expression of BMP6 protein effectively at 48 h after transfection. In summary, siRNAs can effectively and specifically knock down targeting genes at both transcriptional and translational levels utilizing RNAi in human Sertoli cells.

Role of the c-Jun N-terminal kinase signaling pathway in the activation of trypsinogen in rat pancreatic acinar cells.

Bile acid causes trypsinogen activation in pancreatic acinar cells through a complex process. Additional research is required to further elucidate which signaling pathways affect trypsinogen activation when activated. the changes in the whole­genome expression profile of AR42J cells under the effect of taurolithocholic acid 3­sulfate (TLC­S) were investigated. Furthermore, gene groups that may play a regulatory role were analyzed using the modular approach of biological networks. The aim of the present study was to improve our understanding of the changes in TLC­S­stimulated AR42J cells through a genetic functional modular analysis. whole­genome expression profile chip arrays were applied to detect genes that were differentially expressed in pancreatic acinar AR42J cells treated with TLC­S for 20 min. Based on the human protein reference database, a protein­protein interaction network was obtained, which was then processed by CFinder software to derive 14 modules. Among these 14 modules, the gene ontology biological processes enrichment analysis identified two as modules of interest. Kyoto encyclopedia of genes and genomes map analysis revealed that MAP2K4, MAPK8 and FLNA are part of the c-Jun N-terminal kinase (JNK) pathway. The JNK signaling pathway is involved in regulating trypsinogen activation in rat pancreatic AR42J cells. Next, a regulatory network of seven kinase inhibitors was constructed. SP600125 is an ATP­competitive, efficient, selective and reversible inhibitor of JNK. the results were verified by four sets of experiments and demonstrated that trypsinogen activation is mediated by the JNK signaling pathway in the pathogenesis of acute pancreatitis (AP). The present study provided a useful reference for better understanding the pathogenesis of AP and identifying new targets to regulate trypsinogen activation, in addition to providing valuable information for the treatment of AP.