N6-Methyladenosine Modification of ANLN Enhances Hepatocellular Carcinoma Bone Metastasis.

Bones are categorized as the second most prevalent location of extra-hepatic metastasis in Hepatocellular Carcinoma (HCC), which is linked to an extremely poor prognosis due to limited therapeutic options. N6-methyladenosine (m6A) is a prominent modification involved in HCC, but the exact mechanisms on how m6A modifications induce HCC bone metastases (BM) remain unclear. The key modulators responsible for the abundant m6A RNA modification-induced HCC BM was found to be the METTL3 and YTHDF1. The expression of Anillin actin-binding protein (ANLN) was dramatically higher in HCC with BM tissues, and its messenger RNA (mRNA) stability was enhanced via m6A epitranscriptomic regulation by METTL3 and YTHDF1. High METTL3 and YTHDF1 expression along with nuclear ANLN protein was clinically correlated with BM in HCC patients. Furthermore, HCC BM was attributed to over-expression of nuclear ANLN forming a transcriptional complex with SP1 which enhanced KIF2C transcriptional activity to activate the mTORC1 pathway, therefore increased the expression of RANKL and disproportionated RANKL-OPG expression in bone microenvironment leading to malignant neoplasms invade bone tissue. In addition, inhibition of ANLN m6A modification by DZNeP attenuated HCC BM. This data provides meaningful understanding of the modulation and association of m6A epitranscriptomic-regulated BM in HCC, and moreover, defines potentially valuable therapeutic targets.

One- versus two-stage partial hepatectomy for large resectable solitary hepatocellular carcinomas determined preoperatively to have a narrow resection margin: a propensity score matching analysis.

Background: For patients with a large but resectable solitary hepatocellular carcinoma (HCC) of >5 cm in diameter, it is often difficult to achieve a sufficient resection margin. There is still no study on whether a two-stage hepatectomy to increase a narrow resection margin would be beneficial. Methods: From August 2014 to February 2017, patients with a large but resectable solitary HCC of >5 cm and a preoperative estimated resection margin of <1.0 cm were retrospectively studied. They were divided into one- and two-stage resection groups. A retrospective analysis was performed, followed by propensity score matching (PSM) analysis. Disease recurrence, survival, intraoperative and postoperative data were compared. Results: Before PSM, the 1-, 2-, 3-and 4-year recurrence-free survival rates for the one- and two-stage groups were 44.3%, 31.7%, 24.3%, 19.2% versus 60.6%, 45.4%, 43.5%, 32.3%, respectively (P=0.007). The corresponding OS rates were 61.0%, 45.2%, 43.8%, 38.4% versus 69.6%, 62.5%, 60.7%, 57.3%, respectively (P=0.029). After PSM, the 1-, 2-, 3-and 4-year recurrence-free survival rates for the one- and two-stage groups were 44.0%, 31.5%, 27.3%, 21.0% versus 60.6%, 45.4%, 43.5%, 32.3%, respectively (P=0.013). The corresponding OS rates were 62.5%, 41.1%, 41.1%, 37.5% versus 69.6%, 62.5%, 60.7%, 57.3%, respectively (P=0.038). Differences in the resection margins between the one- and two-stage groups before [0.3 (0-0.5) versus 1.2 (0.8-2.2) cm] and after [0.2 (0-0.5) versus 1.2 (0.8-2.2) cm] PSM were also significant. Conclusions: Two-stage hepatectomy allowed a wider resection margin for patients with a resectable but solitary HCC of >5 cm, and resulted in significantly better long-term survival outcomes after partial hepatectomy.

Increased m6A modification of lncRNA DBH-AS1 suppresses pancreatic cancer growth and gemcitabine resistance via the miR-3163/USP44 axis.

Background: Gemcitabine is among the most commonly utilized chemotherapeutic agents for treating pancreatic cancer (PC), yet patients ultimately develop chemoresistance and thus exhibit a poor prognosis. Long noncoding RNAs (lncRNAs) can function as key regulators of PC progression and may serve as prognostic biomarkers in individuals with gemcitabine-resistant PC. This study sought to explore the role of the lncRNA DBH-AS1 in this oncogenic setting. Methods: Based on public databases and qRT-PCR analyses the expression of lncRNA DBH-AS1 in PC tissues and cell lines. The effects of lncRNA DBH-AS1 on proliferation and gemcitabine resistance were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA DBH-AS1, miR-3163 and USP44. Results: We found that PC tissues exhibited DBH-AS1 downregulation that was particularly pronounced in gemcitabine-resistant PC tissues and cells. This DBH-AS1 downregulation was negatively correlated with the malignancy of PC tumors and with patient survival outcomes. Additionally, decreased DBH-AS1 expression in PC was found to be linked to the METTL3-dependent m6A methylation of the lncRNA, with functional analyses revealing that DBH-AS1 was able to suppress the growth of PC cells. Mechanistically, DBH-AS1 was able to increase PC cell sensitivity to gemcitabine by sequestering miR-3163 and thus upregulating USP44 in these tumor cells. Clinically, patient-derived PC tumor xenografts exhibiting high levels of DBH-AS1 expression were found to be responsive to gemcitabine treatment. Conclusions: Overall, these data underscore a key role for DBH-AS1 as a regulator of PC tumor growth and a promising therapeutic target capable of predicting PC patient responsiveness to gemcitabine treatment.

A nomogram for predicting the risk of postoperative recurrence of hepatitis B virus-related hepatocellular carcinoma in patients with high preoperative serum glutamyl transpeptidase.

Background: Recurrence is a major risk factor affecting the postoperative survival of patients with hepatocellular carcinoma (HCC), especially those with high preoperative serum γ-glutamyl transpeptidase (GGT) levels. This study had the aim of developing a personalized predictive tool to accurately determine the risk of postoperative recurrence of hepatitis B-virus (HBV)-related HCC in patients with high preoperative serum GGT levels. Methods: Patients who underwent curative liver resection of HBV-related HCC and had high preoperative GGT levels were consecutively enrolled between 2008 and 2011. Prognostic indicators for recurrence were determined using Cox regression analysis. A nomogram was then developed and assessed by integrating the independent risk factors into the model. Results: A total of 603 eligible patients were included. The final nomogram for predicting HCC recurrence in patients with high preoperative GGT levels consisted of five independent prognostic factors: α-fetoprotein (AFP), HBV-DNA, satellite nodules, microvascular invasion, and tumor grade. The C-index of the nomogram for predicting recurrence was 0.759, and validation showed high accuracy and discriminatory. Conclusions: The predictive nomogram developed and validated in this study performs well in predicting postoperative recurrence of HBV-related HCC in patients with high preoperative GGT levels. It can provide personalized assessments to inform the development of surveillance strategies and allows patients with a high risk of recurrence to be selected for further adjuvant treatment.

miR-515-5p suppresses HCC migration and invasion via targeting IL6/JAK/STAT3 pathway.

MicroRNAs (miRNAs) have been identified as critical modulators of cell migration and invasion, which are the major causes of cancer progression including hepatocellular carcinoma (HCC). However, the accurate role of miR-515-5p in HCC is still uncertain. Here, we report that miR-515-5p expression is down-regulated in HCC tissues and cell lines, and associated with absence of capsule formation (p = 0.015)﹑microvascular invasion(p = 0.003)﹑and advantange TNM stage (II-III) (p = 0.014) in HCC patients. Overexpression of miR-515-5p inhibited migration and invasion of HCC cells in vitro and in vivo, while miR-515-5p knockdown has the inverse effect. Moreover, using miRNA databases and dual-luciferase report assay, we find miR-515-5p directly binds to the 3'-untranslated region (3'-UTR) of interleukin 6 (IL6). In addition, the regulatory association between miR-515-5p and the IL-6/Janus kinase (JNK)/signal transducer and activator of transcription-3 (STAT3) signaling pathway was explored. Furthermore, overexpression of miR-515-5p inhibited the activation of the JAK/STAT3 signaling pathway, which was rescued by overexpression of IL-6. The results of the current study indicate that miR-515-5p overexpression may serve an important role in inhibiting migration and invasion of HCC cells via suppression of IL-6/JAK/STAT3 signaling pathway activation. MiR-515-5p may serve as a potential therapeutic target for HCC.

Hypoxia Activates SOX5/Wnt/ß-Catenin Signaling by Suppressing MiR-338-3p in Gastric Cancer.

MicroRNAs are known to be important in a variety of cancer types. The specific expression and roles of miR-338-3p in the context of gastric cancer, however, remains largely unknown. In this study, we found that miR-338-3p was expressed significantly lower in established/primary human gastric cancer cells than that in human gastric epithelial cells; miR-338-3p is also decreased in human gastric cancer tissues and was positively associated with the worse prognosis of patients with gastric cancer. Enforced expression of miR-338-3p could inhibit cell growth, survival, and proliferation, while inducing cell apoptosis. In addition, miR-338-3p negatively regulated SOX5 expression through directly binding to the 3'-untranslated region of SOX5, and an inverse correlation was found between miR-338-3p and SOX5 messenger RNA expression in gastric cancer tissues. Furthermore, miR-338-3p-induced inactivation of Wnt/ß-catenin signaling was greatly abrogated by SOX5 upregulation. Finally, we found that hypoxic conditions were linked with reduced miR-338-3p expression in the context of gastric cancer. In conclusion, miR-338-3p acts as a tumor suppressor in gastric cancer, possibly by directly targeting SOX5 and blocking Wnt/ß-catenin signaling. These findings might provide novel therapeutic targets for gastric cancer.

Downregulation of miR-196-5p Induced by Hypoxia Drives Tumorigenesis and Metastasis in Hepatocellular Carcinoma.

In hepatocellular carcinoma (HCC), the hypoxic tumor microenvironment can drive enhance tumor malignancy and recurrence. The microRNA (miRNA) miR-196-5p has been shown to modulate the progression of several cancer types, but its roles in HCC remain uncertain. In the present report we observed significant miR-196-5p downregulation in HCC tissues and cells, and we found that the expression of this miRNA significantly impaired the proliferation and metastatic potential of HCC in vitro and in vivo. We identified high-mobility group AT-hook 2 (HMGA2) as a miR-196-5p target gene that was associated with the ability of miR-196-5p to modulate the progression of HCC. Expression of miR-196-5p and HMGA2 were correlated with the clinical characteristics and poor outcomes in patients with HCC. Finally, we found that hypoxic conditions were linked with reduced miR-196-5p expression in the context of HCC. Together these results highlight the role for miR-196-5p as an inhibitor of the proliferation and metastasis of HCC via the targeting of HMGA2, with this novel hypoxia/miR-196-5p/HMGA2 pathway serving as a potential target for future therapeutic intervention.

miR-455-5p suppresses hepatocellular carcinoma cell growth and invasion via IGF-1R/AKT/GLUT1 pathway by targeting IGF-1R.

Hepatocellular carcinoma (HCC) is among the most frequently observed forms of cancer. MicroRNAs (miRNAs) are increasingly thought to play a key role in regulating the onset and progression of a wide range of cancer types. In the present report, we found that miR-455-5p expression was significantly decreased in both HCC patient tumor tissues and cell lines, and that this reduction in expression was linked to poorer patient outcomes. When we overexpressed miR-455-5p in HCC cell lines (Huh7 and HepG2), this was linked with impaired proliferation, colony formation, migration, and invasion. We further found that this miRNA was able to directly bind the insulin growth factor receptor (IGF-1R) 3'-untranslated region, thereby suppressing IGF-1R expression in HCC cells. Consistent with this, miR-455-5p overexpression was associated with reduced glucose transporter (GLUT) 1 expression, which in turn inhibited HCC cell uptake of glucose, production of lactate, and generation of ATP. Together these results thus indicate that mIR-455-5p is able to suppress tumor functionality via impairing glycolysis in HCC cells, highlighting this miRNA as a potential target for anti-cancer therapeutic interventions.

The RNA binding protein neuro-oncological ventral antigen 1 (NOVA1) regulates IL-6 mRNA stability to enhance JAK2-STAT3 signaling in CRC.

The molecular mechanisms governing the metastasis of colorectal cancer (CRC) are incompletely understood. In the present study, we found NOVA1 to be expressed at higher levels in CRC cell lines and tissue samples, and this upregulation was positively correlated with TNM stage (p = 0.034), poor differentiation (p = 0.001), and lymph node metastasis (p = 0.008). Both overall survival (OS) and relapse-free survival (RFS) were both significantly decreased in patients with high NOVA1 expression relative to those with low expression. Through a multivariate analysis, we determined that NOVA1 independently predicted poor outcomes in those with CRC. In further functional studies, we found that NOVA1 expression controlled the proliferation and invasive characteristics of CRC cells via a mechanism wherein NOVA1 bound and stabilized the IL6 mRNA, enhancing IL-6/JAK2/STAT3 signaling to in turn upregulate matrix metalloproteinases (MMPs) 2, 7, and 9. NOVA1 therefore plays key functional roles in regulating CRC progression, and our results further indicate that it serve as a valuable prognostic biomarker and potentially a target for therapeutic treatment in individuals with CRC.

MicroRNA-212-3p inhibits the Proliferation and Invasion of Human Hepatocellular Carcinoma Cells by Suppressing CTGF expression.

MicroRNA-212-3p inhibits several human cancers but its effects on hepatocellular carcinoma (HCC) remain unclear. In this study, we show that miR-212-3p is down-regulated in HCC cell lines and tissues, and correlates with vascular invasion (p = 0.001), and the absence of capsule formation (p = 0.009). We found that miR-212-3p influenced the epithelial to mesenchymal transition (EMT) of HCCLM3 and Huh7 cells. Mechanistically, miR-212-3p repressed cell invasion through the suppression of connective tissue growth factor (CTGF). We therefore validate the anti-HCC effects of miR-212-3p through its ability to suppress CTGF and subsequent EMT.

MicroRNA-197-3p acts as a prognostic marker and inhibits cell invasion in hepatocellular carcinoma.

MicroRNAs (miRNAs) serve an important regulatory role in carcinogenesis and cancer progression. Aberrant expression of miR-197-3p has been reported in various human malignancies. However, the role of miR-197-3p in the progression and prognosis of hepatocellular carcinoma (HCC) remains unknown. The present study demonstrated that miR-197-3p was downregulated in HCC tissues and that the low level of miR-197-3p expression in HCC tumours correlated with aggressive clinicopathological characteristics; thus, miR-197-3p may serve as a predictor for poor prognosis in patients with HCC. Additionally, miR-197-3p markedly inhibited the metastasis of HCC cells in vitro and in vivo. Bioinformatics analysis further identified zinc finger protein interacted with K protein 1 (ZIK1) as a novel target of miR-197-3p in HCC cells. These findings suggest that miR-197-3p may regulate the survival of HCC cells, partially through the downregulation of ZIK1. Therefore, the miR-197-3p/ZIK1 axis may serve as a novel therapeutic target in patients with HCC.

CBX6 overexpression contributes to tumor progression and is predictive of a poor prognosis in hepatocellular carcinoma.

Aberrant chromobox (CBX) family protein expression has been reported in a variety of human malignancies. However, the role of CBX6 in hepatocellular carcinoma (HCC) progression and patient prognosis remains unknown. In this study, we found that CBX6 was frequently up-regulated in HCC clinical samples and HCC cell lines and that CBX6 expression was significantly correlated with larger tumor sizes (≥ 5 cm, p = 0.011) and multiple tumors (n ≥ 2, p = 0.018). Survival analyses indicated that patients with higher CBX6 expression levels had significantly shorter recurrence-free survival (RFS) and overall survival (OS) than patients with lower CBX6 expression levels, and multivariate analyses confirmed that increased CBX6 expression was an independent unfavorable prognostic factor for HCC patients. Functional study demonstrated that CBX6 profoundly promoted HCC cell growth both in vitro and in vivo, and mechanistic investigation revealed that the S100A9/NF-κB/MAPK pathway was essential for mediating CBX6 function. In conclusion, our results represent the first evidence that CBX6 contributes to tumor progression and indicate that the protein may serve as a novel prognostic biomarker for HCC and as a therapeutic target in the treatment of the disease.

eIF5B increases ASAP1 expression to promote HCC proliferation and invasion.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. Despite the therapeutic advances that have been achieved during the past decade, the molecular pathogenesis underlying HCC remains poorly understood. In this study, we discovered that increased expression eukaryotic translation initiation factor 5B (eIF5B) was significantly correlated with aggressive characteristics and associated with shorter recurrence-free survival (RFS) and overall survival (OS) in a large cohort. We also found that eIF5B promoted HCC cell proliferation and migration in vitro and in vivo partly through increasing ASAP1 expression. Our findings strongly suggested that eIF5B could promote HCC progression and be considered a prognostic biomarker for HCC.

Low CDX1 expression predicts a poor prognosis for hepatocellular carcinoma patients after hepatectomy.

The caudal-type homeobox 1 (CDX1) transcription factor is a member of the caudal-related homeobox transcription factor gene family and has been reported to be down-regulated in a variety of cancers. However, the expression status and significance of CDX1 in hepatocellular carcinoma (HCC) is still controversial, and little is known about the role of CDX1 in HCC·In our previous study, we investigated the expression and clinical significance of CDX1 in HCC samples from 313 HCC patients. We found CDX1 was strikingly down-regulated in HCC samples. CDX1 expression was associated with poor differentiation (P = 0.002), and patients with low CDX1 expression had a significantly poorer prognosis. A subgroup analysis revealed a difference in prognosis between groups with low and high CDX1 expression among patients who had tumors <5 cm in size and who were alpha-fetoprotein (AFP) negative. Moreover, low expression was more frequently observed in the early recurrence group (within 2 years, P = 0.002). In addition, multivariate Cox regression analysis indicated that the CDX1 expression level, tumor size, presence of hepatitis B e antigen (HBeAg), vascular invasion, and presence of hepatitis B surface antigen (HBsAg) were independent risk factors for HCC recurrence, and the CDX1 expression level, tumor size, tumor number, and presence of HBsAg were independent predictor of overall survival of HCC patients. In conclusion, the downregulation of CDX1 is associated with poor prognosis; and it may serve as a novel predictor of the prognosis of HCC patients after curative resection.

Ligand-directed stearic acid grafted chitosan micelles to increase therapeutic efficacy in hepatic cancer.

Targeted delivery system would be an interesting platform to enhance the therapeutic effect and to reduce the side effects of anticancer drugs. In this study, we have developed lactobionic acid (LA)-modified chitosan-stearic acid (CS-SA) (CSS-LA) to deliver doxorubicin (DOX) to hepatic cancer cells. The average particle size of CSS-LA/DOX was ∼100 nm with a high entrapment efficiency of >95%. Drug release studies showed that DOX release from pH-sensitive micelles is significantly faster at pH 5.0 than at pH 7.4. The LA conjugated micelles showed enhanced cellular uptake in HepG2 and BEL-7402 liver cancer cells than free drug and unconjugated micelles. Consistently, CSS-LA/DOX showed enhanced cell cytotoxicity in these two cell lines. Annexin-V/FITC and PI based apoptosis assay showed that the number of living cells greatly reduced in this group with marked presence of necrotic and apoptotic cells. LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies began to appear. In vivo, CSS-LA/DOX showed an excellent tumor regression profile with no toxic side effects. The active targeting moiety, long circulation profile, and EPR effect contributed to its superior anticancer effect in HepG2 based tumor. Our results showed that polymeric micelles conjugated with LA increased the therapeutic availability of DOX in the liver cancer cell based solid tumor without any toxic side effects. The active targeting ligand conjugated nanoparticulate system could be a promising therapeutic strategy in the treatment of hepatic cancers.

A prospective randomized controlled trial to compare pringle manoeuvre with hemi-hepatic vascular inflow occlusion in liver resection for hepatocellular carcinoma with cirrhosis.

BACKGROUND: The duration of hepatic vascular inflow occlusion and the amount of intraoperative blood loss have significant negative impacts on postoperative morbidity, mortality and long-term survival outcomes of patients who receive partial hepatectomy for hepatocellular carcinoma (HCC) with cirrhosis. AIM: This study aimed to compare the perioperative outcomes of partial hepatectomy for HCC superimposed on hepatitis B-related cirrhosis using two different occlusion techniques. METHODS: A randomized controlled trial was carried out to evaluate the impact of two different vascular inflow occlusion techniques. The postoperative short-term results were compared. RESULTS: During the study period, 252 patients received partial hepatectomy for HCC with cirrhosis. Of these patients, 120 were randomized equally into two groups: the Pringle manoeuvre group (n = 60) and the hemi-hepatic vascular inflow occlusion group (n = 60). The number of patients who had poor liver function on postoperative day 5 with ISLGS grade B or worse was 24 and 13, respectively (P = 0.030). The postoperative complication rate was significantly higher in the Pringle manoeuvre group (40 versus 22 %, P = 0.030). However, the Pringle manoeuvre group had significantly shorter operating time (116 versus 136 min, P = 0.012) although there was no significant difference in intraoperative blood loss between the two groups [200 ml (range 10-5,000 ml) versus 300 ml (range 100-1,000 ml); P = 0.511]. There was no perioperative mortality. CONCLUSIONS: The results indicated that for patients with HCC with cirrhosis, hemi-hepatic vascular inflow occlusion was a better inflow occlusion method than Pringle manoeuvre.

[Effect of CD8(+)CD28(-) suppressor T cells induced by dendritic cell with major histocompatibility complex 1 expression RNA interference on immune tolerance in rat intestinal transplantation].

OBJECTIVE: To investigate the effect of CD8(+)CD28(-) suppressor T cells(Ts) induced by dendritic cell(DC) with major histocompatibility complex 1(MHC-1) expression RNA interference on immune tolerance in rat intestinal transplantation. METHODS: The expression level of CD8(+)CD28(-)Ts were successfully induced by DC with MHC-1 expression interfered by RNA interference technique under the stimulator of allograft antigen. Orthotopic intestinal transplantation was performed in 36 rats by modified three cuffs method. The recipients were randomly divided into three groups(12 rats in each group):group A was experimental group with CD8(+)CD28(-) Ts being administrated, mixed T cells were injected in group B, while in group C, NS were administrated. On the first day and the seventh day posttransplant, the 36 rats of the 3 groups were administrated through vena dorsalis penis respectively. Six rats were selected randomly from each group and the animals were sacrificed on the 14 th day postoperatively, serum levels of TGF-beta, IFN-gamma and the values of Na(+)-K(+)-ATPase activity of the intestinal graft were assayed and the intestinal pathologic morphology, intestinal allograft survival were observed concerning the remainders. RESULTS: On the 14 th day after operation, the expression levels of TGF-beta and IFN-gamma in group A were significantly up-regulated as compared with those in group B and group C(P<0.05). Na(+)-K(+)-ATPase activity in group A was(6.3+/-1.0) kU/g, much higher than the levels of group B(3.6+/-0.9)kU/g and group C(2.9+/-1.3) kU/g and the differences were significant(P<0.05). The data suggested preliminarily that pathological scores of intestinal graft in group A were lower than those in group B and group C. The survival time of the recipients in group A was 32.0 days, much longer than that in group B (17.5 days, P<0.05) and group C(21.0 days, P<0.05). CONCLUSION: CD8(+)CD28(-) Ts induced by DC with MHC-1 expression RNA interference can alleviate acute rejection and lead to immune tolerance in rat intestinal transplantation.