Venlafaxine antagonizes the noradrenaline-promoted colon cancer progression by inhibiting the norepinephrine transporter.

Epidemiological studies have demonstrated that the use of antidepressants is associated with a decreased risk of colorectal cancer (CRC); however, the mechanisms behind this association are yet unknown. Adrenergic system contributes to the stress-related tumor progression, with norepinephrine (NE) mainly secreted from adrenergic nerve fibers. Norepinephrine serotonin reuptake inhibitors are successfully used antidepressants. This study demonstrates that a widely used antidepressant venlafaxine (VEN) antagonizes NE-promoted colon cancer in vivo and in vitro. Bioinformatic analysis suggested that NE transporter (NET, SLC6A2), a target of VEN, was closely associated with the prognosis of clinical patients with CRC. In addition, the knockdown of NET antagonized the effect of NE. The NET-protein phosphatase 2 scaffold subunit alpha/phosphorylated Akt/vascular endothelial growth factor pathway partially mediates the antagonizing effect of VEN on NE's actions in colon cancer cells. These were also confirmed by in vivo experiments. Our findings revealed for the first time that, in addition to its primary function as a transporter, NET also promotes NE-enhanced colon cancer cell proliferation, tumor angiogenesis, and tumor growth. This provides direct experimental and mechanistic evidence for the use of antidepressant VEN in the treatment of CRC and a therapeutic potential for repurposing existing drugs as an anti-cancer approach to improve the prognosis of patients with CRC.

MicroRNA-17 Family Targets RUNX3 to Increase Proliferation and Migration of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one common cancer in the world. Previous studies have shown that miR-17 family members are elevated in most tumors and promote tumor progression. However, there is no comprehensive analysis of the expression and functional mechanism of the microRNA-17 (miR-17) family in HCC. The aim of this study is to comprehensively analyze the function of the miR-17 family in HCC and the molecular mechanism of its role. Bioinfoimatics analysis of the miR-17 family expression profile and its relationship to clinical significance using The Cancer Genome Atlas (TCGA) database, and this result was confirmed using quantitative real-time polymerase chain reaction. miR-17 family members were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability and migration by cell count and wound healing assays. In addition, we using dual-luciferase assay and Western blot demonstrated the targeting relationship between the miRNA-17 family and RUNX3. These members of miR-17 family were highly expressed in HCC tissues, and the overexpression of the miR-17 family promoted the proliferation and migration of SMMC-7721 cells, whereas treatment with anti-miR17 inhibitors caused the opposite effects. Notably, we also found that inhibitors anti-each member of miR-17 can suppress the expression of the entire family member. In addition, they can bind to the 3' untranslated region of RUNX3 to regulate its expression at the translational level. Our results proved that miR-17 family has oncogenic characteristics, overexpression every member of the family contributed to HCC cell proliferation and migration by reducing the translation of RUNX3.

TDG suppresses the migration and invasion of human colon cancer cells via the DNMT3A/TIMP2 axis.

Background: Colorectal cancer (CRC) is one of the most common malignant tumors with high rates of recurrence and mortality. Thymine DNA glycosylase (TDG) is a key molecule in the base excision repair pathway. Recently, increasing attention has been paid to the role of TDG in tumor development. However, the specific functions of TDG in CRC remain unclear. Methods: The biological functions of TDG and DNA methyltransferase 3 alpha (DNMT3A) in CRC were evaluated using migration and invasion assays, respectively. A tumor metastasis assay was performed in nude mice to determine the in vivo role of TDG. The interaction between TDG and DNMT3A was determined via co-immunoprecipitation (Co-IP). Chromatin immunoprecipitation analysis (ChIP) was used to predict the DNA-binding site of DNMT3A. We also performed methylation-specific PCR (MSP) to detect changes in TIMP2 methylation. Results: TDG inhibited the migration and invasion of human colon cancer cells both in vitro and in vivo. TDG promoted the ubiquitination and degradation of DNMT3A by binding to it. Its interference with siDNMT3A also inhibits the migration and invasion of human colon cancer cells. Furthermore, the ChIP, MSP, and rescue experiments results confirmed that TDG accelerated the degradation of DNMT3A and significantly regulated the transcription and expression of TIMP2, thereby affecting the migration and invasion of human colon cancer cells. Conclusion: Our findings reveal that TDG inhibits the migration and invasion of human colon cancer cells through the DNMT3A-TIMP2 axis, which may be a potential therapeutic strategy for the development and treatment of CRC.

A genome-wide analysis reveals the MeCP2-dependent regulation of genes in BGC-823 cells.

Methyl-CpG-binding protein 2 (MeCP2) epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. This study aimed to evaluate the effect of MeCP2 on the global gene expression profile of human gastric adenocarcinoma to determine the potential molecular mechanism of MeCP2. To identify the gene targets of MeCP2 in gastric cancer cells, we combined the expression microarray and chromatin immunoprecipitation approaches of MeCP2, followed by sequencing (ChIP-seq) to define the MeCP2-binding sites across the whole genome. The methylation levels of the promoters in BGC-823 cells were downloaded from the National Center for Biotechnology Information Gene Expression Omnibus database (GSM1093053). A total of 5,684 ChIP-enriched peaks were identified by comparing IP and Input, using a p-value threshold of 10-5 in ChIP-seq. The bioinformatics analysis presented a predictive model of the genome-wide MeCP2-binding pattern, in which the MeCP2 binding site is closely related to the transcription start site region in the genome. The results of motif detection showed that the MeCP2-binding regions contained not only the core CpG motif but also the extended poly (A/T) motifs. Finally, an integrative analysis of the sequence features and DNA methylation states revealed that MeCP2's function as a multifunctional transcriptional regulator may not be directly related to the methylation status of the binding site. The first MeCP2 ChIP-seq and gene expression microarray analysis in BGC-823 cells revealed that MeCP2 plays multiple roles in the regulation of gene expression depending on the microenvironment, such as sequence characteristics and the methylation levels of binding sites.

Nei Endonuclease VIII-Like1 (NEIL1) Inhibits Apoptosis of Human Colorectal Cancer Cells.

The study is aimed at investigating the role of Nei endonuclease VIII-like1 (NEIL1) in the pathogenesis of colorectal cancer (CRC). The human CRC (HCT116 and SW480) cells were subjected to the siRNA silencing and recombinant plasmid overexpression of NEIL1. Transfection of siNEIL1 significantly inhibited the cell growth. It also increased the Bax expression levels, while it decreased the Bcl-2 expression levels in human CRC cells, leading the Bax/Bcl-2 balance toward apoptosis. Moreover, the apoptosis was promoted through the caspase-9 signaling pathway. One the other hand, high expression of NEIL1 promoted the cell viability and reduced the apoptosis, inducing the balance of Bax/Bcl-2 in the human colon cancer cells to be antiapoptotic. In addition, the caspase-9 signaling pathway inhibited apoptosis, contrary to the results obtained by downregulating NEIL1 expression. Furthermore, NEIL1 was negatively regulated by miR-7-5p, indicating that miR-7-5p inhibited the NEIL1 expression after transcription. Overexpression of miR-7-5p reversed the effects of NEIL1 on these CRC cells. In conclusion, NEIL1 promotes the proliferation of CRC cells, which is regulated negatively by miR-7-5p. These findings suggest that NEIL1 is a potential therapeutic target for CRC.

miR-203a suppresses cell proliferation by targeting RING-finger protein 6 in colorectal cancer.

Colorectal cancer (CRC) is one of most common cancers worldwide. Although miR-203a is reported as a tumor suppressor involved in cell progression in some cancers, the role of miR-203a in CRC is still controversial and the underling mechanism of miR-203a in CRC remains unclear. Here, we demonstrated that low expression of miR-203a had poorer survival in CRC patients. miR-203a was down-regulated in most human colon cancer cells. Overexpression of miR-203a could inhibit colon cancer cell proliferation and arrest cell cycle in G1 phase. Bioinformatics and dual luciferase reporter assay confirmed that RING-finger protein 6 (RNF6) was a target gene of miR-203a. Silencing RNF6 inhibited cell proliferation and arrest cell cycle in G1 phase. RNF6 overexpression reversed the effects of miR-203a overexpression in colon cancer cells. Taken together, our data indicate that miR-203a inhibits colon cancer cell proliferation by targeting RNF6, offer novel insights into the regulatory network of miR-203a-modulated cell cycle and proliferation, and suggest that miR-203a a potential therapeutic target in CRC treatment.

Norepinephrine-CREB1-miR-373 axis promotes progression of colon cancer.

The adrenergic system contributes to the stress-induced onset and progression of cancer. Adrenergic fibers are the primary source of norepinephrine (NE). The underlying mechanisms involved in NE-induced colon cancer remain to be understood. In this study, we describe the function and regulatory network of NE in the progression of colon cancer. We demonstrate that NE-induced phosphorylation of cAMP response element-binding protein 1 (CREB1) promotes proliferation, migration, and invasion of human colon cancer cells. The downstream effector of NE, CREB1, bound to the promoter of miR-373 and transcriptionally activated its expression. miR-373 expression was shown to be necessary for NE-induced cell proliferation, invasion, and tumor growth. We confirmed that proliferation and invasion of colon cancer cells are regulated in vitro and in vivo by miR-373 through targeting of the tumor suppressors TIMP2 and APC. Our data suggest that NE promotes colon cancer cell proliferation and metastasis by activating the CREB1-miR-373 axis. The study of this novel signaling axis may provide mechanistic insights into the neural regulation of colon cancer and help in the design of future clinical studies on stress biology in colorectal cancer.

Norepinephrine transporter promotes the invasion of human colon cancer cells.

Epidemiological studies suggested the use of antidepressants to be associated with decreased risk of colorectal cancer (CRC). However, the underlying mechanism through which this decreased risk occurs remains elusive. The norepinephrine transporter (NET) is a target of antidepressants that maintains noradrenergic transmission homeostasis; however, little is known about its function in human CRC cells. The present study, using public datasets and immunohistochemistry approaches, revealed that NET was highly expressed in human CRC tissues with metastasis and in human colon cancer cells. Furthermore, knockdown of NET inhibited the invasive capability of human colon cancer cells. Additionally, epithelial (E)-cadherin expression was increased and Notch1 signaling was inhibited in NET-depleted colon cancer cells. These findings suggest that NET is highly expressed in human colon cancer, which is associated with the invasion of human colon cancer cells by influencing cell-cell adhesion through the Notch1-E-cadherin pathway. Thus, the present study revealed a novel function for NET and its downstream effectors in colon cancer cells, which will be valuable for future studies in a clinical setting.

MiR-99b-5p and miR-203a-3p Function as Tumor Suppressors by Targeting IGF-1R in Gastric Cancer.

MicroRNAs (miRNAs) have been explored in many critical cellular processes, including proliferation and apoptosis. The purpose of this study was to detect the biological function and regulation of miR-99b-5p and miR-203a-3p in gastric cancer (GC). Here, we demonstrated that miR-99b-5p/203a-3p were downregulated in both GC tissues and cell lines. MiR-99b-5p/203a-3p overexpression reduced GC cell proliferation and cell cycle progression in vitro. Notably, we combined bioinformatics tools with biological validation assays to demonstrate that insulin-like growth factor 1 receptor (IGF-1R) is a direct co-target and functional mediator of miR-99b-5p/203a-3p in GC cells. Mechanistically, the AKT pathway, which is downstream of IGF-1R, is essential for the functional roles of miR-99b-5p/203a-3p in GC cells. Taken together, our data revealed that IGF-1R is a direct co-target of miR-99b-5p/203a-3p, and miR-99b-5p/203a-3p may function as tumor suppressive miRNAs by negatively regulating IGF-1R expression in GC cells.

The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic ß cells.

Autocrine insulin signaling is critical for pancreatic ß-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic ß cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic ß-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic ß cells.

miR-338-3p confers 5-fluorouracil resistance in p53 mutant colon cancer cells by targeting the mammalian target of rapamycin.

Evidence demonstrate that p53 mutations and microRNAs (miRs) are important components of 5-FU resistance in colorectal cancer (CRC). miR-338-3p has been reported associated with cancer prognosis. However whether or not it influences chemotherapy sensitivity and the underlying mechanisms have not been elucidated. Here, three types of human colon cancer cell lines, HT29 (mutant p53), HCT116 (wild-type p53), and HCT116 p53-/- (deficient p53), were treated with 5-FU. We showed that expression of miR-338-3p was correlated with apoptosis and 5-FU resistance in colon cancer cells. Ectopic expression of miR-338-3p conferred resistance to 5-FU in HCT116 cells. Further experiments indicated that miR-338-3p mediated 5-FU resistance through down-regulation of mTOR expression. Moreover, inhibition of miR-338-3p in HT29 and HCT116 p53-/- cells increased their sensitivity to 5-FU treatment. Furthermore, we detected autophagy changes in our experiment because mTOR was known prominently regulating autophagy and the competition between autophagy and apoptosis in response to 5-FU was a mechanism influencing 5-FU sensitivity. Our results reveal a critical and novel role of miR-338-3p in the correlation of 5-FU resistance with p53 status. Moreover, the miR-338-3p inhibitor has the potential to overcome 5-FU resistance in p53 mutant colon cancer cells.

APPL1 promotes the migration of gastric cancer cells by regulating Akt2 phosphorylation.

As a multifunctional adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and a leucine zipper motif 1) is overexpressed in many cancers, and has been implicated in tumorigenesis and tumor progression. The present study investigated the expression of APPL1 in gastric carcinoma and the function in regulating cell migration. We investigated the expression of APPL1 in gastric carcinoma based upon The Cancer Genome Atlas (TCGA) database. The expression of APPL1 in collected gastric carcinoma tissues and cultured cells was measured by qRT-PCR and western blot analysis. Transwell assay and wound healing assay were used to analyze the effects of APPL1 on tumor cell migration. The statistical results based upon TCGA database showed significantly higher expression of APPL1 in gastric carcinoma compared to adjacent normal tissues, and we confirmed these findings by measuring APPL1 expression in collected gastric carcinoma tissues and cultured cells. The results of transwell assay and wound healing assay showed that when APPL1 was silenced by siRNA, cell migration was inhibited and overexpression of APPL1 promoted migration. Western blot results demonstrated that changes in several mesenchymal markers were consistent with the observed reduction or enhancement of cell migration. Importantly, the expression of APPL1 significantly affected the phosphorylation of Akt2. In addition, MMP2 and MMP9, downstream effectors of Akt2 changed accordingly, which is a critical requirement for Akt2-mediated cell migration. The results demonstrate an important new function of APPL1 in regulating cell migration through a mechanism that depends on Akt2 phosphorylation.

MicroRNA­214 targets Wnt3a to suppress liver cancer cell proliferation.

MicroRNAs (miRNAs/miRs) are crucial molecules that act as tumor suppressor genes or oncogenes in human cancer progression. The dysregulation of miRNA expression has been detected in liver cancer. The present study aimed to explore the molecular mechanisms by which miR­214 affects liver cancer cell proliferation. Reverse transcription­quantitative polymerase chain reaction was used to determine the expression of miR­214 in liver cancer cell lines and hepatocellular carcinoma (HCC) tissues. A luciferase reporter assay was performed to determine whether Wnt3a is a target gene of miR­214. Cell Counting kit­8 and cell cycle analysis were used to explore the effects of miR­214 on liver cancer cell proliferation. Immunohistochemistry was used to detect protein expression levels. Wnt3a knockdown was used to determine the function of Wnt3a in liver cancer cell proliferation. The results demonstrated that the expression levels of human miR­214 were reduced in HCC tissues and liver cancer cell lines compared with in control tissues and cells. Overexpression of miR­214 and Wnt3a silencing each inhibited liver cancer cell growth. Conversely, inhibition of miR­214 promoted liver cancer cell growth. The present study indicated that miR­214 acts as a tumor suppressor and may be considered a promising therapeutic target for the treatment of liver cancer.

Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo.

Curative treatment of patients with hepatocellular carcinoma (HCC) is poor. There is an urgent need to develop more effective strategies for the chemoprevention of HCC. Chlorogenic acid (CGA), a type of polyphenol present in the diet, especially from coffee, has many biological activities. Patients with viral hepatitis who drank coffee everyday experienced a reduction in the incidence of HCC. In the present study, we evaluated the effects of CGA on HCC. CGA inhibited the proliferation of HepG2 cells in vitro and the progression of HepG2 xenograft in vivo. CGA induced the inactivation of ERK1/2 and suppressed the expression of MMP-2 and MMP-9 in HepG2 xenograft tissue. These data demonstrate that CGA can prevent the progression of HCC through multiple pathways. CGA appears to be an effective chemopreventive agent for hepatocellular carcinoma.

MeCP2 Promotes Gastric Cancer Progression Through Regulating FOXF1/Wnt5a/ß-Catenin and MYOD1/Caspase-3 Signaling Pathways.

Methyl-CpG binding protein 2 (MeCP2) has recently been characterized as an oncogene frequently amplified in several types of cancer. However, its precise role in gastric cancer (GC) and the molecular mechanism of MeCP2 regulation are still largely unknown. Here we report that MeCP2 is highly expressed in primary GC tissues and the expression level is correlated with the clinicopathologic features of GC. In our experiments, knockdown of MeCP2 inhibited tumor growth. Molecular mechanism of MeCP2 regulation was investigated using an integrated approach with combination of microarray analysis and chromatin immunoprecipitation sequencing (ChIP-Seq). The results suggest that MeCP2 binds to the methylated CpG islands of FOXF1 and MYOD1 promoters and inhibits their expression at the transcription level. Furthermore, we show that MeCP2 promotes GC cell proliferation via FOXF1-mediated Wnt5a/ß-Catenin signaling pathway and suppresses apoptosis through MYOD1-mediated Caspase-3 signaling pathway. Due to its high expression level in GC and its critical function in driving GC progression, MeCP2 represents a promising therapeutic target for GC treatment.

Chlorogenic acid induces reactive oxygen species generation and inhibits the viability of human colon cancer cells.

Chlorogenic acid (CGA) is one of the polyphenols identified in the human diet. Previous studies have shown that CGA plays a protective role against liver diseases. The colon plays a pivotal role in CGA metabolism. However, little is known about the direct effects and the underlying molecular mechanisms of CGA in colon cancer. Here, we investigate these mechanisms of CGA activity in human colon cancer cells. The effects of CGA on the viability of two human colon cancer cell lines, HCT116 and HT29, were determined using the MTT assay. The intracellular reactive oxygen species (ROS) were detected using fluorescence microscopy and flow cytometry. In addition, changes in cell proliferation were detected by cell cycle analysis. Immunoblotting analysis was used to observe the underlying molecular changes. CGA inhibited the viability of HCT116 and HT29 cells in a dose-dependent manner. CGA induced ROS production, whereas the combined use of ROS scavenger N-acetylcysteine attenuated the CGA-induced viability inhibition. Moreover, CGA induced cell cycle arrest at the S phase and suppressed the activation of extracellular signal-related kinase in both cell types, which likely contributes toward the ROS-induced viability inhibition caused by CGA treatment. CGA-induced ROS production inhibited cell viability in human colon cancer cells. CGA caused S-phase arrest and extracellular signal-related kinase inactivation that may have led to the observed viability inhibition. CGA is therefore a potential treatment against CRC.

MicroRNA-302a enhances 5-fluorouracil-induced cell death in human colon cancer cells.

New therapeutic strategies are needed for colorectal cancer (CRC) treatment. MicroRNAs are involved in cancer­pertinent cellular processes, including chemoresistance. As miR­302a is an embryonic stem cell­specific microRNA, studies on miR­302a have focused on its role in human stem cells. Studies analyzing miR­302 function in cancer are limited. In this study, we used two human colon cancer cell lines, HCT116 and HT29, and evaluated the influence of miR­302a on 5­fluorouracil (5­FU)­induced cell death and viability inhibition. With bioinformatics tools, we hypothesized that insulin­like growth factor­1 receptor (IGF­1R) is a novel target of miR­302a, which we confirmed using a luciferase reporter assay and immunoblotting. Then, we designed siRNA against IGF­1R and found that si­IGF­1R resembled the effect of miR­302a on 5­FU treatment. Both miR­302a and si­IGF­1R inhibited Akt signaling. In conclusion, miR­302a targeted IGF­1R and enhanced 5­FU­induced cell death and viability inhibition in human colon cancer cells. Targeting miR­302a may offer new therapeutic interventions in CRC.

MicroRNA-20a-5p targets RUNX3 to regulate proliferation and migration of human hepatocellular cancer cells.

Growing evidence indicates that some abnormally expressed microRNAs (miRNAs) influence tumorigenesis and progression. Previous studies reported that miR-20a is among the frequently altered miRNAs in human hepatocellular carcinoma (HCC), but its expression pattern and role in HCC remain controversial. In the present study, we demonstrated that miR-20a-5p exhibited aberrant expression in HCC tissues compared with paired non-tumor tissues: 52% of the tumor samples showed a greater increase. Overexpression of miR-20a contributed to HCC cell proliferation and migration in vitro, and treatment with anti-miR20a-5p caused the opposite effects. Further studies revealed RUNX3, an important tumor-suppressor, as a direct target of miR-20a-5p. We observed that the level of RUNX3 was sharply reduced in both mRNA and protein in HCC tissues compared with paired non-tumor tissues. Collectively, our results support the viewpoint that miR-20-5p has an oncogenic property, miR-20a overexpression contributed to HCC cell proliferation and migration through reducing the translation of RUNX3. The data provide a new mechanism of miR-20a regulating RUNX3 in HCC.