Cancer-associated fibroblasts induce sorafenib resistance of hepatocellular carcinoma cells through CXCL12/FOLR1.

BACKGROUND: Due to the high drug resistance of hepatocellular carcinoma (HCC), sorafenib has limited efficacy in the treatment of advanced HCC. Cancer-associated fibroblasts (CAFs) play an important regulatory role in the induction of chemoresistance. This study aimed to clarify the mechanism underlying CAF-mediated resistance to sorafenib in HCC. METHODS: Immunohistochemistry and immunofluorescence showed that the activation of CAFs was enhanced in HCC tissues. CAFs and paracancerous normal fibroblasts (NFs) were isolated from the cancer and paracancerous tissues of HCC, respectively. Cell cloning assays, ELISAs, and flow cytometry were used to detect whether CAFs induced sorafenib resistance in HCC cells via CXCL12. Western blotting and qPCR showed that CXCL12 induces sorafenib resistance in HCC cells by upregulating FOLR1. We investigated whether FOLR1 was the target molecule of CAFs regulating sorafenib resistance in HCC cells by querying gene expression data for human HCC specimens from the GEO database. RESULTS: High levels of activated CAFs were present in HCC tissues but not in paracancerous tissues. CAFs decreased the sensitivity of HCC cells to sorafenib. We found that CAFs secrete CXCL12, which upregulates FOLR1 in HCC cells to induce sorafenib resistance. CONCLUSIONS: CAFs induce sorafenib resistance in HCC cells through CXCL12/FOLR1.

NIR-II fluorescence-guided liver cancer surgery by a small molecular HDAC6 targeting probe.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and ranks third in terms of both mortality and incidence rates. Surgical resection holds potential as a curative approach for HCC. However, the residual disease contributes to a high 5-year recurrence rate of 70%. Due to their excellent specificity and optical properties, fluorescence-targeted probes are deemed effective auxiliary tools for addressing residual lesions, enabling precise surgical diagnosis and treatment. Research indicates histone deacetylase 6 (HDAC6) overexpression in HCC cells, making it a potential imaging biomarker. This study designed a targeted small-molecule fluorescent probe, SeCF3-IRDye800cw (SeCF3-IRD800), operating within the Second near-infrared window (NIR-II, 1000-1700 nm). The study confirms the biocompatibility of SeCF3-IRD800 and proceeds to demonstrate its applications in imaging in vivo, fluorescence-guided surgery (FGS) for liver cancer, liver fibrosis imaging, and clinical samples incubation, thereby preliminarily validating its utility in liver cancer. METHODS: SeCF3-IRD800 was synthesized by combining the near-infrared fluorescent dye IRDye800cw-NHS with an improved HDAC6 inhibitor. Initially, a HepG2-Luc subcutaneous tumor model (n = 12) was constructed to investigate the metabolic differences between SeCF3-IRD800 and ICG in vivo. Subsequently, HepG2-Luc (n = 12) and HCCLM3-Luc (n = 6) subcutaneous xenograft mouse models were used to assess in vivo targeting by SeCF3-IRD800. The HepG2-Luc orthotopic liver cancer model (n = 6) was employed to showcase the application of SeCF3-IRD800 in FGS. Liver fibrosis (n = 6) and HepG2-Luc orthotopic (n = 6) model imaging results were used to evaluate the impact of different pathological backgrounds on SeCF3-IRD800 imaging. Three groups of fresh HCC and normal liver samples from patients with liver cancer were utilized for SeCF3-IRD800 incubation ex vivo, while preclinical experiments illustrated its potential for clinical application. FINDINGS: The HDAC6 inhibitor 6 (SeCF3) modified with trifluoromethyl was labeled with IRDy800CW-NHS to synthesize the small-molecule targeted probe SeCF3-IRD800, with NIR-II fluorescence signals. SeCF3-IRD800 was rapidly metabolized by the kidneys and exhibited excellent biocompatibility. In vivo validation demonstrated that SeCF3-IRD800 achieved optimal imaging within 8 h, displaying high tumor fluorescence intensity (7658.41 ± 933.34) and high tumor-to-background ratio (5.20 ± 1.04). Imaging experiments with various expression levels revealed its capacity for HDAC6-specific targeting across multiple HCC tumor models, suitable for NIR-II intraoperative imaging. Fluorescence-guided surgery experiments were found feasible and capable of detecting sub-visible 2 mm tumor lesions under white light, aiding surgical decision-making. Further imaging of liver fibrosis mice showed that SeCF3-IRD800's imaging efficacy remained unaffected by liver pathological conditions. Correlations were observed between HDAC6 expression levels and corresponding fluorescence intensity (R2 = 0.8124) among normal liver, liver fibrosis, and HCC tissues. SeCF3-IRD800 identified HDAC6-positive samples from patients with HCC, holding advantages for perspective intraoperative identification in liver cancer. Thus, the rapidly metabolized HDAC6-targeted small-molecule NIR-II fluorescence probe SeCF3-IRD800 holds significant clinical translational value. INTERPRETATION: The successful application of NIR-II fluorescence-guided surgery in liver cancer indicates that SeCF3-IRD800 has great potential to improve the clinical diagnosis and treatment of liver cancer, and could be used as an auxiliary tool for surgical treatment of liver cancer without being affected by liver pathology. FUNDING: This paper is supported by the National Natural Science Foundation of China (NSFC) (92,059,207, 62,027,901, 81,930,053, 81,227,901, 82,272,105, U21A20386 and 81,971,773), CAS Youth Interdisciplinary Team (JCTD-2021-08), the Zhuhai High-level Health Personnel Team Project (Zhuhai HLHPTP201703), and Guangdong Basic and Applied Basic Research Foundation under Grant No. 2022A1515011244.

Modified Laparoscopic Anatomic Hepatectomy: Two-Surgeon Technique Combined with the Simple Extracorporeal Pringle Maneuver.

Laparoscopic anatomic hepatectomy (LAH) has become increasingly prevalent worldwide in recent years. However, LAH remains a challenging procedure due to the anatomical characteristics of the liver, with intraoperative hemorrhage being a primary concern. Intraoperative blood loss is the leading cause of conversion to open surgery; therefore, effective management of bleeding and hemostasis is crucial for a successful LAH. The two-surgeon technique is proposed as an alternative to the traditional single-surgeon approach, with potential benefits in reducing intraoperative bleeding during laparoscopic hepatectomy. However, there remains a lack of evidence to determine which mode of the two-surgeon technique yields superior patient outcomes. Besides, to our knowledge, the LAH technique, which involves the use of a cavitron ultrasonic surgical aspirator (CUSA) by the primary surgeon while an ultrasonic dissector by the second surgeon, has been rarely reported before. Herein, we present a modified, two-surgeon LAH technique, wherein one surgeon employs a CUSA while the other uses an ultrasonic dissector. This technique is combined with a simple extracorporeal Pringle maneuver and low central venous pressure (CVP) approach. In this modified technique, the primary and secondary surgeons utilize a laparoscopic CUSA and an ultrasonic dissectorconcurrently to achieve precise and expeditious hepatectomy. A simple extracorporeal Pringle maneuver, combined with the maintenance of low CVP, is employed to regulate the hepatic inflow and outflow in order to minimize intraoperative bleeding. This approach facilitates the attainment of a dry and clean operative field, which allows for the precise ligation and dissection of blood vessels and bile ducts. The modified LAH procedure is simpler and safer due to its effective control over bleeding as well as the seamless transition between the roles of primary and secondary surgeons. It holds great promise for future clinical applications.

Magnesium Supplementation Stimulates Autophagy to Reduce Lipid Accumulation in Hepatocytes via the AMPK/mTOR Pathway.

Metabolic-associated fatty liver disease (MAFLD) (previously known as nonalcoholic fatty liver disease (NAFLD)) is a disease with high worldwide prevalence, but with limited available therapeutic interventions. Autophagy is a cell survival mechanism for clearing excess lipids in hepatocytes and affects the occurrence and development of MAFLD. In addition, some studies have shown that magnesium deficiency is common in patients with obesity and metabolic syndrome. Magnesium supplementation can effectively improve metabolism-related diseases such as obesity and fatty liver. Our study successfully constructed a cellular model of MAFLD by 1 mM free fatty acid (FFA) intervention in LO2 cells for 24 h, and there was an increase in lipid accumulation in hepatocytes after FFA intervention. Magnesium supplementation was shown to reduce lipid deposition in hepatocytes induced by FFA, and Western blotting (WB) analysis showed that magnesium supplementation could downregulate the expression of Fasn and SREBP1 and increase the expression of LPL, suggesting that magnesium can reduce lipid accumulation by reducing lipid synthesis and increasing lipid oxidation. Magnesium supplementation could affect cellular lipid metabolism by activating the AMPK/mTOR pathway to stimulate autophagy. Our results identified a relationship between magnesium and lipid accumulation in hepatocytes and showed that magnesium supplementation reduced lipid deposition in hepatocytes by activating autophagy by activating the AMPK-mTOR pathway.

A novel DNA damage repair-related signature for predicting prognositc and treatment response in non-small lung cancer.

DNA damage repair (DDR) is essential for maintaining genome integrity and modulating cancer risk, progression, and therapeutic response. DDR defects are common among non-small lung cancer (NSCLC), resulting in new challenge and promise for NSCLC treatment. Thus, a thorough understanding of the molecular characteristics of DDR in NSCLC is helpful for NSCLC treatment and management. Here, we systematically analyzed the relationship between DDR alterations and NSCLC prognosis, and successfully established and validated a six-DDR gene prognostic model via LASSO Cox regression analysis based on the expression of prognostic related DDR genes, CDC25C, NEIL3, H2AFX, NBN, XRCC5, RAD1. According to this model, NSCLC patients were classified into high-risk subtype and low-risk subtype, each of which has significant differences between the two subtypes in clinical features, molecular features, immune cell components, gene mutations, DDR pathway activation status and clinical outcomes. The high-risk patients was characterized with worse prognosis, lower proportion and number of DDR mutations, unique immune profile and responsive to immunetherapy. And the low-risk patients tend to have superior survival, while being less responsive to immunotherapy and more sensitive to treatment with DNA-damaging chemotherapy drugs. Overall, this molecular classification based on DDR expression profile enables hierarchical management of patients and personalized clinical treatment, and provides potential therapeutic targets for NSCLC.

Follistatin Alleviates Hepatic Steatosis in NAFLD via the mTOR Dependent Pathway.

Purpose: In this study, we aimed to investigate the effect of follistatin (FST) on hepatic steatosis in NAFLD and the underlying mechanism, which has rarely been reported before. Methods: Liver samples from NAFLD patients and normal liver samples (from liver donors) were collected to investigate hepatic FST expression in humans. Additionally, human liver cells (LO2) were treated with free fatty acid (FFA) to induce lipid accumulation. Furthermore, lentivirus with FST overexpression or knockdown vectors were used to generate stable cell lines, which were subsequently treated with FFA or FFA and rapamycin. In the animal experiments, male C57BL/6J mice were fed with a high-fat diet (HFD) to induce NAFLD, after which the adeno-associated virus (AAV) gene vectors for FST overexpression were administered. In both cell culture and mice, we evaluated morphological changes and the protein expression of sterol regulatory element-binding protein1 (SREBP1), acetyl-CoA carboxylase1 (ACC1), carbohydrate-responsive element-binding protein (ChREBP), fatty acid synthase (FASN), and Akt/mTOR signaling. The body weight and serum parameters of the mice were also measured. Results: Hepatic FST expression level was higher in NAFLD patients compared to normal samples. In LO2 cells, FST overexpression alleviated lipid accumulation and lipogenesis, whereas FST knockdown aggravated hepatic steatosis. FST could regulate Akt/mTOR signaling, and the mTOR inhibitor rapamycin abolished the effect of FST knockdown on hepatic de novo lipogenesis (DNL). Furthermore, FST expression was increased in HFD mice compared to the corresponding controls. FST overexpression in mice reduced body weight gain, hyperlipidemia, hepatic DNL, and suppressed Akt/mTOR signaling. Conclusion: Hepatic FST expression increases in NAFLD and plays a protective role in hepatic steatosis. FST overexpression gene therapy alleviates hepatic steatosis via the mTOR pathway.Therefore, gene therapy for FST is a promising treatment in NAFLD.

Hsa-microRNA-370-3p targeting Snail and Twist1 suppresses IL-8/STAT3-driven hepatocellular carcinoma metastasis.

The pro-inflammatory factor interleukin-8 (IL-8) is related to poor prognosis in hepatocellular carcinoma (HCC) patients. Interleukin-8 enhanced HCC invasion by upregulating Snail and Twist1, whether this modulation relies on microRNAs (miR) is unclear. In this study, hsa-miR-370-3p was screened as candidate miRNA targeting Snail and Twist1, and its expression was downregulated by IL-8. Luciferase assays and RNA electrophoretic mobility shift assays were used to evaluate the interaction between miR-370-3p and targeted mRNAs. Coimmunoprecipitation, luciferase, and ChIP assays were undertaken to investigate the mechanisms underlying IL-8-mediated modification of miR-370-3p. Gain- and loss-of-function studies, Transwell assays, and a xenograft nude mouse model were used to investigate pro- and antitumor activities. Interleukin-8 and miR-370-3p levels were analyzed for clinical relevance in HCC patients. Our results showed that HCC patients with high levels of IL-8 experienced more metastasis and shorter survival. Interleukin-8 induced epithelial-mesenchymal transition and promoted liver cancer cell migration, invasion, and metastasis both in vitro and in vivo. MicroRNA-370-3p interacted with its cognate mRNA within the 3'-UTR regions of Twist1 and Snail mRNA directly and specifically and attenuated IL-8 protumoral effects on liver cancer cells. Interleukin-8 negatively modulated miR-370-3p through signal transducer and activator of transcription 3 (STAT3) activation by recruiting histone deacetylase 1 (HDAC1) to miR-370-3p promoter. The STAT3 and HDAC antagonists inhibited liver cancer cell migration and invasion. Patients with high miR-370-3p and low IL-8 levels had longer overall survival. In conclusion, our study elucidated a novel axis IL-8/STAT3/miR-370-3p/Twist1 and Snail relying on HDAC1 recruitment, which showed both diagnostic and therapeutic potentials of miR-370-3p in HCC metastasis.

Combined Treatment of Tanshinone I and Epirubicin Revealed Enhanced Inhibition of Hepatocellular Carcinoma by Targeting PI3K/AKT/HIF-1α.

Background: Epirubicin (EADM) is a common chemotherapeutic agent in hepatocellular carcinoma (HCC). The accumulation of hypoxia-inducible factor-1α (HIF-1α) is an important cause of drug resistance to EADM in HCC. Tanshinone I (Tan I) is an agent with promising anti-cancer effects alone or with other drugs. Some tanshinones mediate HIF-1α regulation via PI3K/AKT. However, the role of Tan I combined with EADM to reduce the resistance of HCC to EADM has not been investigated. Therefore, this study aimed to investigate the combined use of Tan I and EADM in HCC and the underlying mechanism of PI3K/AKT/HIF-1α. Methods: HCC cells were treated with Tan I, EADM, or the combined treatment for 48 hrs. Cell transfection was used to construct HIF-1α overexpression HCC stable cells. Cell viability, colony formation, and flow cytometric assays were used to detect the viability, proliferation, and apoptosis in HCC cells. Synergism between Tan I and EADM were tested by calculating the Bliss synergy score, positive excess over bliss additivism (EOBA), and the combination index (CI). Western blotting analyses were used to detect the levels of ß-actin, HIF-1α, PI3K p110α, p-Akt Thr308, Cleaved Caspase-3, and Cleaved Caspase-9. Toxicity parameters were used to evaluate the safety of the combination in mice. The xenograft model of mice was built by HCC stable cell lines, which was administrated with Tan I, EADM, or a combination of them for 8 weeks. Immunohistochemistry staining (IHC) was used to assess tumor apoptosis in mouse models. Results: Hypoxia could upregulate HIF-1α to induce drug resistance in HCC cancer cells. The combination of Tan I and EADM was synergistic. Although Tan I or EADM alone could inhibit HCC cancer cells, the combination of them could further enhance the cytotoxicity and growth inhibition by targeting the PI3K/AKT/HIF-1α signaling pathway. Furthermore, Tan I and EADM synergistically reversed HIF-1α-mediated drug resistance to inhibit HCC. The results of toxicity parameters showed that the combination was safe in mice. Meanwhile, animal models showed that Tan I not only improved the anti-tumor effect of EADM, but also reduced the drug reactions of EADM-induced weight loss. Conclusion: Our results suggested that Tan I could effectively improve the anti-tumor effect of EADM, and synergize EADM to reverse HIF-1α mediated resistance via targeting PI3K/AKT/HIF-1α signaling pathway.

Combined HIF-1α and SHH Up-Regulation Is a Potential Biomarker to Predict Poor Prognosis in Postoperative Hepatocellular Carcinoma.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) or sonic hedgehog (SHH) is associated with hepatocellular carcinoma (HCC) progression. Hypoxia inhibits ferroptosis, which induces cancer cell death. However, the correlation between the combined HIF-1α and SHH up-regulation with prognosis, and the association between SHH and ferroptosis remain unclear. This study aimed to investigate them. METHODS: We detected the expression of HIF-1α and SHH in HCC. Cox regression, clinical data, and Kaplan-Meier analyses were performed. In vitro cell experiments verified the relationship between HIF-1α and SHH, and observed the invasion of hypoxic HCC cells. The correlation between SHH and ferroptosis was also analyzed. RESULTS: HIF-1α and SHH expression levels were significantly correlated with HCC (p < 0.0001). HIF-1α and SHH expression levels were found to be associated with TNM stage (p = 0.0121, p = 0.0078, respectively), vascular invasion (p < 0.0001, p < 0.0001, respectively), and recurrence (p = 0.0212, p = 0.0392, respectively). The combined upregulation of HIF-1α and SHH was an independent factor for predicting the overall survival (OS) of patients with HCC (p = 0.003), who had the shortest OS (p = 0.0009). SHH paralleled the increase in HIF-1α expression, which promotes cancer cell invasion. The upregulation of SHH was related to the inhibition of the expression of ferroptosis-related factors (FANCD2, p < 0.0001 and FTH1, p = 0.0009) in HCC. CONCLUSION: Combined HIF-1α and SHH upregulation is a potentially poor prognosis indicator in patients with HCC because the upregulation of SHH inhibits ferroptosis in hypoxic cancer cells.

Efficacy of Adjuvant Transarterial Chemoembolization after Radical Hepatectomy in Solitary Hepatocellular Carcinoma Patients: A Retrospective Study.

Background: More and more studies have suggested that hepatocellular carcinoma (HCC) patients with high-risk recurrence factors can benefit the most from postoperative adjuvant transarterial chemoembolization (PA-TACE) for its potential effect in delaying cancer recurrence. However, it remains unclear if solitary HCC (SHCC) patients particularly those without high-risk recurrence factors should also receive PA-TACE. This study aimed to analyze the efficacy of PA-TACE in them. Methods: Retrospectively, we enrolled 123 SHCC patients who either received radical hepatectomy alone (No TACE group, n = 39) or followed by PA-TACE (PA-TACE group, n = 84) in our institution. Prognostic risk factors, disease-free survival (DFS), and overall survival (OS) were analyzed using the Cox proportional hazard regression model, the Kaplan-Meier method, and the log-rank test. Results: Liver cirrhosis was the only independent risk factor for SHCC patients. Overall, the PA-TACE group had no improved OS (P = 0.977) but worse DFS compared with the No TACE group (P = 0.045). Consistently, in subgroup analysis, SHCC patients with negative microvascular invasion (MVI), tumor size ≤ 5 cm and preoperative alpha-fetoprotein (AFP) < 400 ng/ml had similar OS (P = 0.466, P = 0.864, P = 0.488, respectively) but even worse DFS (P = 0.035, P = 0.040, P = 0.019, respectively) than those in the No TACE group. Besides, there was no significant difference in DFS and OS between the two groups of SHCC patients with liver cirrhosis (P = 0.342, P = 0.941, respectively). Conclusions: PA-TACE may not improve the long-term survival of SHCC patients, but may even potentially promote their postoperative tumor recurrence, especially for those with MVI-negative, tumor size ≤ 5 cm, and preoperative AFP < 400 ng/ml.

Evidence of SARS-CoV-2 infection in gallbladder and aggravating cholecystitis to septic shock: a case report.

Coronavirus disease 2019 (COVID-19) has threatened human health worldwide and could lead to multiple organs injury. However, the impact on the virus infecting the biliary system, especially the gallbladder, has remained unclear and no pathological evidence has been reported yet. A case of SARS-CoV-2 infection in a gallbladder with cholecystitis, which progressed rapidly to sepsis and required an emergency operation was investigated and reported. Clinical specimens of the COVID-19 patient including serum, oropharyngeal swabs, sputum, bile, abdominal drainage fluid, urine, stool, and gallbladder tissue were collected and tested for SARS-CoV-2 RNA using a quantitative polymerase chain reaction (qPCR) assay. Fresh normal gallbladder tissue and gangrenous gallbladder tissue were also collected for further research including hematoxylin and eosin (HE), immunohistochemistry (IHC), and immunofluorescent (IF) staining, and compared with the gallbladder from the COVID-19 patient. The bile, as well as the serum, oropharyngeal swabs, sputum, abdominal drainage fluid, urine, and rectal swabs were consecutively negative for SARS-CoV-2 RNA. The viral host receptor angiotensin-converting enzyme 2 (ACE2) was highly expressed in gallbladder epithelial cells, and viral nucleocapsid protein (NP) was visualized in the cytoplasm of gallbladder epithelial cells. Immune cells including CD2, CD3, CD4, CD8, CD20, CD38, CD68, and MPO were positive in gangrenous gallbladder tissues without SARS-CoV-2 infection, and were relatively downregulated in SARS-CoV-2 infective gallbladder tissue. This study provided evidence of SARS-CoV-2 infection in the gallbladder and verified that the gallbladder was one of the target organs that SARS-CoV-2 could attack and damage using ACE2 as a cell receptor. Due to the immune dysregulation involved, more vigilant management and early assessment is needed for COVID-19 patients with the comorbidity of cholecystitis.

The impact of liver fibrosis on microvascular invasion and prognosis of hepatocellular carcinoma with a solitary nodule: a Surveillance, Epidemiology, and End Results (SEER) database analysis.

BACKGROUND: The pathogenesis of non-cirrhotic hepatocellular carcinoma (HCC) with a high recurrence remains controversial, while microvascular invasion (MVI) is highly suggestive of tumor recurrence. This study aimed to investigate the effects of liver fibrosis on MVI and prognosis in HCC. METHODS: Based on the data of HCC in the Surveillance, Epidemiology, and End Results (SEER) database [2004-2015], multivariate logistic regression was used for correlation analysis. Survival was analyzed by Log-Rank test and Cox regression, and decision curve analysis and receiver operating characteristic curves were established to evaluate alternative diagnostic and prognostic strategies. RESULTS: The study included 1,492 patients with MVI (17.8%) or without MVI (82.2%) for HCC with a solitary nodule. Liver fibrosis was significantly correlated with the occurrence of MVI, and the risk of MVI in patients with a fibrosis score F5-6 was lower than in those with a score of F0-4 (OR =0.651, 95% CI: 0.492-0.860). Combining liver fibrosis could improve the prediction performance of MVI risk models, but liver fibrosis was less associated with survival outcomes in comparison with other tumor characteristics. CONCLUSIONS: Lower liver fibrosis correlated with a higher risk of MVI in HCC with a solitary nodule and was a good indicator for improving the performance of MVI risk models. However, it was not a prognostic sensitive indicator.

MiR-940 promotes malignant progression of breast cancer by regulating FOXO3.

Breast cancer (BC) is a common cancer with poor survival. The present study aimed to explore the effect of miR-940 on the process of BC cells and its target gene FOXO3. The expression of miR-940 was assessed in BC tissues and cells using qRT-PCR. Furthermore, the correlation between miR-940 and prognosis of BC patients from the TCGA database was analyzed. CCK8 assays and colony formation assays were used to explore the effect of miR-940 on BC cell proliferation. The invasion abilities were detected by transwell assays. Luciferase reporter assay was performed to scrutinize the relationship between miR-940 and FOXO3. Finally, rescue experiments were performed through FOXO3 down-regulation and miR-940 inhibitors by using CCK8 assays, colony formation assays and transwell assays. miR-940 was significantly up-regulated in BC cells and tissues. In addition, the high level of miR-940 correlated with poor survival of BC patients (P=0.023). CCK8 assays, colony formation assays and transwell assays indicated that miR-940 promoted the proliferation and invasion abilities of BC cells. The luciferase reporter assay suggested that miR-940 directly targeted FOXO3. Moreover, we found that the effect of si-FOXO3 was rescued by miR-940 inhibitors in BC cells. miR-940 may promote the proliferation and invasion abilities of BC cells by targeting FOXO3. Our study suggested that miR-940 could be a novel molecular target for therapies against BC.

A novel protein encoded by circFNDC3B inhibits tumor progression and EMT through regulating Snail in colon cancer.

BACKGROUND: Colon cancer (CC) is a common malignant cancer. Recently, circFNDC3B was found to exert biological function in multiple cancers. However, it was unclear whether the potential protein encoded by circFNDC3B is involved in carcinogenesis of CC. METHODS: We used Sanger sequence and RNase R digestion assay to confirm the existence of circFNDC3B, and quantitative real-time PCR was used to evaluate the circRNA's expression. Then fluorescence in situ hybridization (FISH) was performed to study location of circFNDC3B. The identification of protein encoded by circFNDC3B was performed using LC-MS/MS. The function of circFNDC3B-218aa on proliferation, invasion and migration were assessed by CCK8 assays, colony formation assays, transwell assays, wound-healing assays and animal experiments. RNA-sequencing and western blot were used to identify the gene regulated by circFNDC3B-218aa. Finally, glucose metabolism-related assays were performed to further investigate function of circFNDC3B-218aa. RESULTS: CircFNDC3B was localized mostly in the cytoplasm, and was decreased in CC cell lines and tissues. The patients with low circFNDC3B expression had a shorter OS (P = 0.0014) than patients with high expression. Moreover, circFNDC3B inhibited the proliferation, invasion and migration of CC cells. Next, we identified that circFNDC3B could encode a novel protein circFNDC3B-218aa. Furthermore, circFNDC3B-218aa, not circFNDC3B, inhibited the proliferation, invasion and migration of CC. Additionally, the in vivo experiments implied that up-regulated circFNDC3B-218aa exhibited an inhibitory effect on CC progression. By RNA-sequencing, western blot and glucose metabolism-related assays, we found that circFNDC3B-218aa inhibited the expression of Snail, and subsequently promoted the tumor-suppressive effect of FBP1 in CC. CONCLUSIONS: The novel circFNDC3B-218aa may serve as a tumor suppressive factor and potential biomarker which may supply the potential therapeutic target for CC.

Development of a Novel Six-miRNA-Based Model to Predict Overall Survival Among Colon Adenocarcinoma Patients.

Introduction: Colon carcinoma is a common malignant tumor worldwide. Accurately predicting prognosis of colon adenocarcinoma (CA) patients may facilitate clinical individual decision-making. Many studies have reported that microRNAs (miRNAs) were associated with prognosis for patients with colon carcinoma. This study aimed to identify the prognosis-related miRNAs for predicting the overall survival (OS) of CA patients. Methods: Firstly, we analyzed the CA datasets from the Cancer Genome Atlas (TCGA), and looked for the prognosis-related miRNAs. Then, we developed a novel prediction model based on these miRNAs and the clinical characteristics. Time-dependent receiver operating characteristics (ROC) curves and calibration plots were used to evaluate the discrimination and accuracy of the signature and model. Finally, cell function assays and bioinformatics analyses were performed to evaluate the role of these selected miRNAs in modulating biological process in CA. Results: Six prognosis-related miRNAs were included in the miRNA-based signature, and it could effectively distinguish low-risk patients and high-risk patients. Furthermore, we established a prognostic model incorporating the six-miRNA-based signature and clinical characteristics. Areas under curves (AUCs) indicated that the six-miRNA-based model has a better predictive ability than TNM stage (AUC: 0.805 vs. 0.694). The calibration plots suggested close agreement between model predictions and actual observations. GO analysis showed that the target genes of these miRNAs are mainly involved in enrichment in protein binding and regulation of transcript and cytosol. KEGG pathway enrichment analysis indicated that these genes were mainly enriched in PI3K-Akt signaling pathway. Finally, we found that the five miRNAs except miR-152 were upregulated in tumor tissues and CA cells. The functional experiments revealed that miR-1245a, miR-3682, miR-33b, and miR-5683 promoted the migratory abilities and proliferation of CA cell, whereas miR-152 showed opposite effects. However, miR-4444-2 did not influence the migratory ability and proliferation of CA cell. Conclusions: In conclusion, we developed a novel six-miRNA-based model to predict 5-year survival probabilities for CA patients. This model has the potential to facilitate individualized treatment decisions.

C14orf166 Is a Biomarker for Predicting Hepatocellular Carcinoma Recurrence.

Aim: Chromosome 14 open reading frame 166 (C14orf166) acts as a transcriptional repressor and is correlated with centrosome architecture manipulation. Nevertheless, the function of C14orf166 in hepatocellular carcinoma (HCC) progression remains unclear. We aimed to investigate the role C14orf166 plays in HCC and further compared the prognostic value of C14orf166 with that of clinicopathological features. Methods: C14orf166 expression was evaluated in a human liver cell line, HCC cell lines, HCC tissues and adjacent noncancerous liver tissues with qRT-PCR, western blot and immunohistochemistry. Patients were divided into two different groups according to C14orf166 level. The relationship between C14orf166 expression and clinicopathological features was assessed by Pearson chi-squared test and receiver operating characteristic curves. Cumulative disease-free survival (DFS) and overall survival (OS) curves were evaluated using the Kaplan-Meier method. Results: C14orf166 mRNA and protein expression is upregulated in HCC cell lines and tissues. The level of C14orf166 was correlated with serum alpha-fetoprotein level, lymph node metastasis, tumor size and recurrence, with high C14orf166 expression correlating with high HCC recurrence risk. The poor OS and DFS of HCC patients are partly due to the persistently high HCC recurrence risk. When combined with serum alpha-fetoprotein level, the predictive accuracy of C14orf166 for HCC recurrence was enhanced (AUC = 0.712, 95% CI 0.603-0.821; p = 0.001). Conclusions: This study demonstrated that C14orf166 is a high-risk biomarker and predictive factor for HCC recurrence, providing information for the selection of appropriate treatment strategies.

HIF-2α upregulation mediated by hypoxia promotes NAFLD-HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway.

Non-alcoholic fatty liver disease (NAFLD) is a relevant risk factor for developing hepatocellular carcinoma (HCC). Steatohepatitic HCC (SH-HCC), characterized by HCC with steatosis, is influenced by lipid metabolism disorders. A hypoxic microenvironment is common in HCC and affects lipid metabolism. However, whether hypoxia-induced HIF-2α upregulation exacerbates lipid accumulation to contribute to SH-HCC progression remains unclear. In this study, we demonstrated that HIF-2α was elevated in tissues from NAFLD-HCC patients and was associated with survival. Under hypoxic conditions, upregulated HIF-2α was accompanied by lipid accumulation and PI3K-AKT-mTOR pathway activation. HIF-2α knockdown (KD) in steatotic HCC ameliorated triglyceride accumulation and steatosis. HIF-2α-KD steatotic HCC showed minimal lipid synthesis in a hypoxic environment, which contributes to a reduction in malignant behaviours. However, treatment with MHY1485 restored these behaviours. STAM mice, a mouse model that develops NAFLD-HCC, exhibit more rapid tumour progression upon exposure to hypoxia. STAM mice treated with INK-128 presented abrogated mTOR expression and tumour progression under hypoxic conditions with lower triglycerides and steatosis. In conclusion, in a hypoxic microenvironment, HIF-2α upregulation promotes steatotic HCC progression by activating lipid synthesis via the PI3K-AKT-mTOR pathway. Therefore, HIF-2α can be a biomarker and target in developing specific therapeutic measures for NAFLD-HCC patients.

Potential Five-MicroRNA Signature Model for the Prediction of Prognosis in Patients with Wilms Tumor.

BACKGROUND Wilms tumor (WT) is the most common type of pediatric renal malignancy, and is associated with poor prognosis. The aim of the present study was to identify microRNA (miRNA) signatures which might predict prognosis and categorize WTs into high- and low-risk subgroups. MATERIAL AND METHODS The miRNA expression profiles of WT patients and normal samples were obtained from the Therapeutically Applicable Research to Generate Effective Treatment database. Differentially expressed miRNAs between WT patients and normal samples were identified using the EdgeR package. Subsequently, correlations between differentially expressed miRNAs and the prognosis of overall survival were analyzed. Enrichment analyses for the targeted mRNAs were conducted via the Database for Annotation, Visualization, and Integration Discovery. RESULTS A total of 154 miRNAs were identified as differentially expressed in WT. Of those, 18 miRNAs were associated with overall survival (P<0.05). A prognostic signature of 5 differentially expressed miRNAs (i.e., has-mir-149, has-mir-7112, has-mir-940, has-mir-1248, and has-mir-490) was constructed to classify the patients into high- and low-risk subgroups. The targeted mRNAs of these prognostic miRNAs were primarily enriched in Gene Ontology terms (i.e., protein autophosphorylation, protein dephosphorylation, and stress-activated MAPK cascade) and the Kyoto Encyclopedia of Genes and Genomes signaling pathways (i.e., MAPK, AMPK, and PI3K-Akt). CONCLUSIONS The 5-miRNA signature model might be useful in determining the prognosis of WT patients. As a promising prediction tool, this prognosis signature might serve as a potential biomarker for WT patients.

Nuclear GAPDH is vital for hypoxia-induced hepatic stellate cell apoptosis and is indicative of aggressive hepatocellular carcinoma behavior.

Background/aim: Hepatic stellate cells (HSCs) are critical determinants of liver tumor behavior such as vascular invasion, cell proliferation and migration. The apoptosis of HSCs can inhibit tumor growth and contribute to repressing hepatocellular carcinoma (HCC) progression. Our study aims to investigate the impact of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on HSCs under hypoxic conditions and the association of nuclear GAPDH with HCC patient outcomes and tumor progression. Patients and methods: Following stable cell passage, 0.3% O2 was used to induce hypoxia. Cell proliferation and apoptosis were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays and flow cytometry, respectively. Proteins expression were detected by extracting nuclear and cytoplasmic proteins and performing Western blots. GAPDH nuclear translocation was blocked by the agent deprenyl. Immunohistochemical staining for GAPDH was investigated in 137 HCC tissue samples from our center. An analysis of the clinicopathological features, Kaplan-Meier analysis and Cox proportional hazards regression analysis were applied. Results: MTT assays and flow cytometry analyses showed that the nuclear accumulation of GAPDH led to the apoptotic death of HSCs, while blockade of this process with deprenyl significantly decreased apoptosis. Western blots revealed that deprenyl inhibited the nuclear translocation of GAPDH. An analysis of the immunohistochemical staining of HSCs in HCC tissue samples (137) revealed that nuclear GAPDH expression was significantly positively correlated with HIF-1α expression. Overall survival (OS) and time-to-recurrence (TTR) estimated by Kaplan-Meier analyses showed that patients with high HIF-1α or low nuclear GAPDH levels in HSCs had significantly poorer prognosis compared with patients with low HIF-1α or high nuclear GAPDH expression in HSCs. Moreover, patients with combined high HIF-1α/low nuclear GAPDH expression in HSCs had the worst prognosis. The Cox regression analysis revealed that the combination of nuclear GAPDH/HIF-1α expression in HSCs was an independent prognostic factor for OS and TTR in HCC patients. Conclusions: These findings provide a novel mechanism underlying the involvement of intranuclear GAPDH in hypoxia-induced HSCs apoptosis and a correlation between nuclear GAPDH levels and the clinical prognosis, which may prompt the development of a novel therapeutic strategy for HCC.

Lactobacillus rhamnosus GG Ameliorates Liver Injury and Hypoxic Hepatitis in Rat Model of CLP-Induced Sepsis.

BACKGROUND: Probiotic use to prevent gastrointestinal infections in critical care has shown great promise in recent clinical trials. Although well-documented benefits of probiotic use in intestinal disorders, the potential for probiotic treatment to ameliorate liver injury and hypoxic hepatitis following sepsis has not been well explored. METHODS: In order to evaluate, if Lactobacillus rhamnosus GG (LGG) treatment in septic rats will protect against liver injury, this study used 20-22-week-old Sprague-Dawley rats which were subjected to cecal ligation and puncture to establish sepsis model and examine mRNA and protein levels of IL-1ß, NLRP3, IL-6, TNF-a, VEGF, MCP1, NF-kB and HIF-1α in the liver via real-time PCR, Elisa and Western blot. RESULTS: This study showed that LGG treatment significantly ameliorated liver injury following experimental infection and sepsis. Liver mRNA and protein levels of IL-1ß, NLRP3, IL-6, TNF-a, VEGF, MCP1, NF-kB and HIF-1α were significantly reduced in rats receiving LGG. CONCLUSIONS: Thus, our study demonstrated that LGG treatment can reduce liver injury following experimental infection and sepsis and is associated with improved hypoxic hepatitis. Probiotic therapy may be a promising intervention to ameliorate clinical liver injury and hypoxic hepatitis following systemic infection and sepsis.