FEN1 upregulation mediated by SUMO2 via antagonizing proteasomal degradation promotes hepatocellular carcinoma stemness.

PURPOSE: Metastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. METHODS: To rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. RESULTS: Our empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. CONCLUSION: In summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

Long Non-Coding RNA ANRIL Regulates Inflammatory Factor Expression in Ulcerative Colitis Via the miR-191-5p/SATB1 Axis.

Ulcerative colitis, an inflammatory bowel disease, manifests with symptoms such as abdominal pain, diarrhea, and mucopurulent feces. The long non-coding RNA (lncRNA) ANRIL exhibits significantly reduced expression in UC, yet its specific mechanism is unknown. This study revealed that ANRIL is involved in the progression of UC by inhibiting IL-6 and TNF-α via miR-191-5P/SATB1 axis. We found that in patients with UC, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were significantly overexpressed in inflamed colon sites, whereas ANRIL was significantly under-expressed and associated with disease severity. The downregulation of ANRIL resulted in the increased expression of IL-6 and TNF-α in LPS-treated FHCs. ANRIL directly targeted miR-191-5p, thereby inhibiting its expression and augmenting SATB1 expression. Moreover, overexpression of miR-191-5p abolished ANRIL-mediated inhibition of IL-6 and TNF-α production. Dual luciferase reporter assays revealed the specific binding of miR-191-5p to ANRIL and SATB1. Furthermore, the downregulation of ANRIL promoted DSS-induced colitis in mice. Together, we provide evidence that ANRIL plays a critical role in regulating IL-6 and TNF-α expression in UC by modulating the miR-191-5p/SATB1 axis. Our study provides novel insights into progression and molecular therapeutic strategies in UC.

Intestinal Immune Imbalance is an Alarm in the Development of IBD.

Immune regulation plays a crucial role in human health and disease. Inflammatory bowel disease (IBD) is a chronic relapse bowel disease with an increasing incidence worldwide. Clinical treatments for IBD are limited and inefficient. However, the pathogenesis of immune-mediated IBD remains unclear. This review describes the activation of innate and adaptive immune functions by intestinal immune cells to regulate intestinal immune balance and maintain intestinal mucosal integrity. Changes in susceptible genes, autophagy, energy metabolism, and other factors interact in a complex manner with the immune system, eventually leading to intestinal immune imbalance and the onset of IBD. These events indicate that intestinal immune imbalance is an alarm for IBD development, further opening new possibilities for the unprecedented development of immunotherapy for IBD.

Establishment of a lipopolysaccharide-induced inflammation model of human fetal colon cells.

BACKGROUND: Inflammatory bowel disease (IBD) is a global health problem and there are few cell models for IBD at present. To culture a human fetal colon (FHC) cell line in vitro and establish an FHC cell inflammation model that meets the requirements for high expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). METHODS AND RESULTS: FHC cells were cultured with various concentrations of Escherichia coli lipopolysaccharide (LPS) in appropriate media for 0.5, 1, 2, 4, 8, 16 and 24 h to stimulate an inflammatory reaction. The viability of FHC cells was detected by a Cell Counting Kit-8 (CCK-8) assay. The transcriptional levels and protein expression changes of IL-6 and TNF-α in FHC cells were detected by Quantitative Real­Time Polymerase Chain Reaction (qRT-PCR) and Enzyme­Linked Immunosorbent Assay (ELISA), respectively. Appropriate stimulation conditions were selected (i.e., LPS concentration and treatment time), based on changes in cell survival rate, and IL-6 and TNF-α expression levels. An LPS concentration higher than 100 µg/mL or a treatment time longer than 24 h resulted in morphological changes and decreased cell survival. By contrast, expression levels of IL-6 and TNF-α significantly increased within 24 h when LPS concentration lower than 100 µg/mL and peaked at 2 h, whilst maintaining cell morphology and viability in FHC cells. CONCLUSION: The treatment of FHC cells with 100 µg/mL LPS within 24 h was optimal in terms of stimulating IL-6 and TNF-α expression.

Peroxisomal trans-2-enoyl-CoA inhibits proliferation, migration and invasion of hepatocellular carcinoma cells.

OBJECTIVES: Peroxisomal trans-2-enoyl-CoA reductase (PECR) encodes proteins related to fatty acid metabolism and synthesis. It has been confirmed that PECR has decreased expression in colon cancer and breast cancer, while the role of PECR in liver cancer is unknown. We aimed to study the role and mechanism of PECR in the genesis and development of liver cancer. METHODS: In this study, the expression of PECR was queried in the Cancer Genome Atlas Database and Western Blotting and RT-PCR experiments were carried out in paired liver cancer tissues to detect the expression of PECR. Functional tests were evaluated by cell count kit-8 (CCK-8), Flow cytometry, wound healing assay, Transwell, migration. In vivo study, we constructed a nude mouse tumorigenic model to observe the effect of PECR on the proliferation of liver cancer. And the tumor body of the mouse was taken out for histochemistry (IHC). Multiple Cox regression was used to analyze the correlation between PECR and Clinicopathology. RESULTS: We confirmed that the overexpression of PECR inhibited the proliferation, migration and invasion of hepatocellular carcinoma and promoted the apoptosis of hepatocellular carcinoma. The low expression group of PECR promoted the proliferation and metastasis of liver cancer. In vivo, overexpression of PECR inhibits the proliferation of mouse tumors. In addition, the mechanism study shows that PECR may indirectly affect the proliferation of hepatocellular carcinoma cells through ERK pathway. CONCLUSION: In general, PECR may be a new diagnostic marker and a potential therapeutic target for hepatocellular carcinoma.

Visualization system based on hierarchical targeting for diagnosis and treatment of hepatocellular carcinoma.

The accuracy and enrichment rate of targeted drugs largely determine the clinical diagnosis and treatment effect. Therefore, the accuracy and enrichment rate of targeted drugs should be improved. We designed a visual diagnosis and treatment system based on hierarchical targeting. It consists of multifunctional magnetic nanoparticles and a bio magnetic material. Bio-magnet mediated primary targeting can effectively improve the drug enrichment rate in the target tissue. SNF peptide/epithelial cell adhesion molecule antibody mediated targeting liver cancer stem cells (LCSCs) (secondary target) can improve the accuracy of the treatment and its outcomes. Low intensity focused ultrasound irradiation can explode nanoparticles around LCSCs, which can cause physical damage to cells. The combination of released interferon gamma and its receptor (tertiary target) can be used to initiate chemotherapy and immunotherapy. Using the optical properties of Fe3O4 and the phase transformation ability of perfluoropentane, the system can enhance photoacoustic and ultrasonic molecular imaging enabling diagnosis and treatment visualization. Targeting LCSCs can accurately provide physical, chemical, and immune treatment of Hepatocellular carcinoma, making the therapeutic effect more effective and thorough. This system may provide a new method for a more accurate visual diagnosis and treatment of tumors.

Identification and validation of EPHX2 as a prognostic biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer, which is associated with a poor prognosis. It is necessary to identify novel prognostic biomarkers and therapeutic targets to improve the survival of patients with HCC. In the present study, a seven­gene signature associated with HCC progression was identified using weighted gene co­expression network analysis and least absolute shrinkage and selection operator, and its prognostic prediction value was confirmed in The Cancer Genome Atlas­liver HCC and International Cancer Genome Consortium liver cancer­RIKEN, Japan cohorts. Subsequently, a rarely reported gene, epoxide hydrolase 2 (EPHX2), was selected for further validation. Downregulation of EPHX2 in HCC was revealed using multiple expression datasets. Furthermore, reduced expression of EPHX2 was confirmed in HCC tissue samples and cell lines using reverse transcription­quantitative polymerase chain reaction and western blotting. Additionally, Kaplan­Meier survival curves indicated that patients with higher EPHX2 expression exhibited better prognosis, and clinicopathological analysis also revealed elevated EPHX2 levels in patients with early­stage HCC. Notably, EPHX2 was identified as an independent prognostic biomarker for overall survival of patients with HCC. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis and gene set enrichment analysis were performed to elucidate the functions of EPHX2. The results suggested that EPHX2 expression was closely associated with metabolic reprogramming. Finally, the prognostic value of EPHX2 was evaluated using HCC tissue microarrays. In conclusion, downregulation of EPHX2 was significantly associated with the development of HCC; therefore, EPHX2 may be considered a putative therapeutic candidate for the targeted treatment of HCC.

AQP9 suppresses hepatocellular carcinoma cell invasion through inhibition of hypoxia-inducible factor 1α expression under hypoxia.

BACKGROUND AND AIM: Intratumor hypoxia is a hallmark of hepatocellular carcinoma (HCC) and is associated with an aggressive tumor phenotype. Although it has been shown that AQP9 plays an important role in HCC, the relevance between hypoxia and AQP9 is still unknown. METHODS: We established in vitro normoxic or hypoxic models to investigate the role of AQP9 in the regulation of hypoxia-inducible factor 1α (HIF-1α) and hypoxia-enhanced invasion of hepatoma cells. Molecular expression was detected using western blot or quantitative polymerase chain reaction. Cell invasion ability was determined using Transwell invasion assay. In vivo xenograft experiment was used to detect the role of AQP9 on tumor growth. RESULTS: Our present study revealed a decrease in the expression levels of AQP9 in hypoxic microenvironments. Overexpression of AQP9 led to a decreased expression of HIF-1α; conversely, suppression of AQP9 in HCC cells had an opposite effect. Furthermore, up-regulated AQP9 blocked the hypoxic-enhanced invasion of HCC cells. The overexpression of AQP9 inhibited the growth of tumors and HIF-1α expression in vivo. CONCLUSIONS: These data suggest that AQP9 acts as a tumor suppressor in HCC invasion via the regulation of HIF-1α expression in the tumor hypoxic microenvironment.

Aquaporin 9 inhibits growth and metastasis of hepatocellular carcinoma cells via Wnt/ß-catenin pathway.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide, and it is the second leading cause of cancer-related mortality. Aquaporin 9 (AQP9) is an essential aquaporin in the liver and located in the basolateral membrane of hepatocytes, but its roles on HCC has not been completely elucidated. This study investigated the regulatory functions of AQP9 in the pathogenesis of HCC. The expression levels of AQP9 were significantly down-regulated in HCC tissues and cells, which was also correlated with tumor size and number, TNM stage, five-year survival rate, lymphatic and distal metastasis within the patients. Furthermore, overexpressed AQP9 suppressed the proliferation, migration and invasion of HCC cells. The levels of PCNA, E-cad, N-cad, α-SMA, DVL2, GSK-3ß, cyclinD1 and ß-catenin in HCC cells were reduced by overexpressed AQP9, while cell apoptosis was remarkably enhanced. Additionally, following the treatment with Wnt/ß-catenin signaling inhibitor (XAV939), the proliferative activity of HCC cells was significantly inhibited; PCNA and EMT-related markers were down-regulated; migration and invasion of cells were notably suppressed; cell apoptotic rate was decreased. Vice versa, after the cells were treated with Wnt/ß-catenin inducer (SKL2001), the effects caused by overexpressed AQP9 were abrogated. In vivo studies indicated that tumor volume and weight were remarkably decreased in AQP9 overexpression group, where the levels of Wnt/ß-catenin signaling- and EMT-associated molecules were also reduced. Taken together, our results suggested that overexpressed AQP9 could inhibit growth and metastasis of HCC cells via Wnt/ß-catenin pathway. AQP9 may be a promising therapeutic target for the treatment of patients with HCC.

Long non­coding RNA H19 regulates cell growth and metastasis via the miR­22­3p/Snail1 axis in gastric cancer.

Gastric cancer (GC) is the fifth most prevalent type of malignancy and the third leading cause of cancer­related mortality worldwide, with the prognosis of patients with late­stage GC remaining at poor levels. Long non­coding RNA (lncRNA) H19 (H19) is involved in the growth and metastasis of tumors, and it is upregulated under hypoxic conditions and in certain types of cancer; however, the underlying mechanisms of action of this lncRNA as regards the initiation and development of GC remain unknown. Thus, in the present study, we aimed to determine the role of lncRNA H19 in GC and to elucidate the underlying mechanisms. H19 was found to be upregulated in GC tissues and cells compared with the para­cancerous tissues, and an elevated expression of H19 was associated with lymph node metastasis and TNM stage. Furthermore, the downregulation of H19 suppressed the proliferation, invasion, migration and epithelial­mesenchymal transition of GC cells in vitro and suppressed tumor growth in vivo. H19 was also found to be able to bind with miR­22­3p, and H19­induced cell growth and metastasis were shown to be reversed by the upregulation of miR­22­3p; the miR­22­3p level was found to inversely correlate with H19 expression in GC tissues. Furthermore, the overexpression of miR­22­3p notably suppressed the proliferation, migration and invasion of GC cells, and these effects were enhanced by the downregulation of Snail1. In addition, cell growth and metastasis induced by miR­22­3p downregulation were partially reversed by the knockdown of Snail1. Furthermore, a negative correlation was observed between the mRNA expression levels of miR­22­3p and Snail1 in GC tissues. On the whole, the findings of the present study revealed that H19 was upregulated in GC tissues, which promoted tumor growth and metastasis via the miR­22­3p/Snail1 signaling pathway. In summary, these findings provide novel insight into the potential regulatory roles of H19 in GC, and suggest that the H19/miR­22­3p/Snail1 axis may prove to be a promising therapeutic target for the treatment of patients with GC.

Clinical application of combined laparoscopic surgery in the treatment of primary hepatocellular carcinoma with portal hypertension: a report of 16 cases.

Primary hepatocellular carcinoma (HCC) is a common malignant tumor in China and is often accompanied by portal hypertension (PHT). Whether simultaneous operation can be used in patients with liver cirrhosis and PHT accompanied by HCC is controversial. The aim of the present study was to investigate the safety and feasibility of simultaneous laparoscopic hepatectomy, splenectomy and pericardial devascularization in the treatment of HCC with PHT. A total of 16 patients were treated with laparoscopic surgery for PHT and HCC from April 2011. The operation time, volume of intraoperative blood loss, red blood cell transfusion, plasma transfusion, time for diet intake, drainage time, duration of hospital stay and occurrence of complications were observed. The follow-up time was 3 years, and long-term outcomes included overall survival rates and recurrence-free survival rates. The operation time and volume of blood loss were 336±18 min and 337±351 mL, respectively. Of the patients, 2 received intraoperative homologous blood transfusion and 9 received plasma transfusion. The time for diet intake, drainage time and duration of hospital stay were 3.5±0.5, 7.3±1.0 and 13.6±3.6 days, respectively. The postoperative complications included 1 patient with anastomosis-site bleeding, 2 patients with abdominal effusion and 3 patients with portal vein thrombosis, which were treated conservatively. The overall survival rates at 1 and 3 years were 100% (16/16) and 87.5% (14/16), respectively. The recurrence-free survival rates at 1 and 3 years were 87.5% (14/16) and 62.5% (10/16), respectively. In conclusion, simultaneous laparoscopic hepatectomy, splenectomy and pericardial devascularization is an effective treatment for PHT and primary HCC, and provides a potential new therapy for patients.

Inhibition of GSK3 and MEK induced cancer stem cell generation via the Wnt and MEK signaling pathways.

Cancer stem cells (CSCs) are considered to be tumor­initiating cells, responsible for tumor invasive growth and dissemination to distant organ sites. Typically, radiation treatment and chemotherapy should target CSCs. However, current research investigating CSCs is impeded by the difficulty of isolating pure CSCs and maintaining them in vitro. In the present study, the synergistic inhibition of glycogen synthase kinase 3 and mitogen­activated protein kinase kinase using small molecules, CHIR99021 and PD184352, efficiently generated CSCs from immortalized human mammary epithelial cells (HMLEs) and resulted in the acquisition of mesenchymal traits and the expression of epithelial­mesenchymal transition markers. The cell proliferation, invasion and migration of HMLE cells were significantly promoted by CHIR99021 and PD184352 (P<0.05). Furthermore, the cell cycle was shifted from the G0/G1 phase to the G2/M phase, and the apoptotic rate was suppressed in HMLE cells following treatment with CHIR99021 and PD184352. Compared with control group, the stimulated cells exhibited an increased ability to form mammospheres and regenerate a tumor. In addition to these properties, the induced cells also exhibited notable chemotherapy resistance. In vivo, the treatment of cells with CHIR99021 and PD184352 promoted the growth of HMLE­engrafted tumor types. These results provide a practical strategy for the generation of CSCs using small molecules in vitro, which provides a cell resource that may be used for drug screening. Additionally, the present results additionally highlighted the synergistic functions of Wnt and mitogen­activated protein kinase kinase signaling pathways in tumorigenesis.

Polyethylene Glycol Electrolyte Lavage Solution versus Colonic Hydrotherapy for Bowel Preparation before Colonoscopy: A Single Center, Randomized, and Controlled Study.

This single center, randomized, and controlled study aimed to compare the effectiveness and safety of polyethylene glycol electrolyte lavage (PEG-EL) solution and colonic hydrotherapy (CHT) for bowel preparation before colonoscopy. A total of 196 eligible outpatients scheduled for diagnostic colonoscopy were randomly assigned to the PEG-EL (n = 102) or CHT (n = 94) groups. Primary outcome measures included colonic cleanliness and adverse effects. Secondary outcome measures were patient satisfaction and preference, colonoscopic findings, ileocecal arrival rate, examiner satisfaction, and cecal intubation time. The results show that PEG-EL group was associated with significantly better colonic cleanliness than CHT group, fewer adverse effects, and increased examiner satisfaction. However, the CHT group had higher patient satisfaction and higher diverticulosis detection rates. Moreover, the results showed the same ileocecal arrival rate and patient preference between the two groups (P > 0.05). These findings indicate that PEG-EL is the preferred option in patients who followed the preparation instructions completely.