Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis.

OBJECTIVE: This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1 (PCED1B-AS1) in the development of hepatocellular carcinoma (HCC). METHODS: A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients. The interactions of PCED1B-AS1 and microRNA-34a (miR-34a) were detected by dual luciferase activity assay and RNA pull-down assay. The RNA expression levels of PCED1B-AS1, miR-34a and CD44 were detected by RT-qPCR, and the protein expression level of CD44 was determined by Western blotting. The cell proliferation was detected by cell proliferation assay, and the cell invasion and migration by transwell invasion assay. The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study. RESULTS: PCED1B-AS1 was highly expressed in HCC tissues, which was associated with poor survival of HCC patients. Furthermore, PCED1B-AS1 interacted with miR-34a in HCC cells, but they did not regulate the expression of each other. Additionally, PCED1B-AS1 increased the expression level of CD44, which was targeted by miR-34a. The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro, while CD44 exhibited the opposite effects. Furthermore, PCED1B-AS1 suppressed the role of miR-34a. Moreover, the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo. CONCLUSION: PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

MicroRNA-181a-5p suppresses cell proliferation by targeting Egr1 and inhibiting Egr1/TGF-ß/Smad pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the leading cause of cancer mortality worldwide. Early growth response factor 1 (Egr1) plays a crucial role in cancer progression. However, its precise role in HCC has not been clear. Here, we identified the aggravating role of Egr1 in cell proliferation of HCC firstly. The expression of Egr1 was significantly increased in HCC tissues. Functionally, overexpression of Egr1 enhanced, whereas silenced Egr1 expression attenuated HCC cells proliferation in vitro. Mechanistically, up-regulated Egr1 induced cell proliferation through activating Transforming growth factor (TGF)-ß1/Smad signaling pathway concomitantly with upregulation of p-Smad2 and p-Smad3. Secondly, miR-181a-5p was down-regulated in clinical HCC specimens and its expression was inversely correlated with Egr1 expression. Functionally, overexpression of miR-181a-5p inhibited, whereas decreased expression of miR-181a-5p promoted HCC cells proliferation in vitro. Furthermore, we demonstrated that miR-181a-5p overexpression directly suppressed Egr1, resulting in a down-regulated TGF-ß1/Smad pathway. Besides, the silenced Egr1 expression could rescue the enhanced cell proliferation induced by miR-181a-5p inhibitor. Thus, we concluded that miR-181a-5p is a negative regulator of Egr1 that can suppress tumor proliferation in HCC through targeting Egr1/TGF-ß1/Smad pathway, which may be a potential therapeutic approach of HCC.

CRISPR/Cas9­mediated hypoxia inducible factor­1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma.

Transarterial embolization (TAE) is a palliative option commonly used for the treatment of advanced, unresectable hepatocellular carcinoma (HCC). However, patient prognosis in regards to overall survival has not improved with this method, mainly due to hypoxia­inducible factor­1α (HIF­1α)­induced angiogenesis and invasiveness. Thus, it is hypothesized that HIF­1α may be an ideal knockout target for the treatment of HCC in combination with TAE. Thus, in the present study, HIF­1α knockout was conducted in human liver cancer SMMC­7721 cells and a xenograft HCC model was established using a lentivirus­mediated CRISPR/Cas system (LV­Cas) with small guide RNA­721 (LV­H721). Furthermore, hepatic artery ligation (HAL) was used to mimic human transarterial chemoembolization in mice. The results revealed that HIF­1α was highly expressed in both HCC patient tissues and SMMC­7721­induced tumor tissues. The HIF­1α knockout in SMMC­7721 cells significantly suppressed cell invasiveness and migration, and induced cell apoptosis under CoCl2­mimicking hypoxic conditions. Compared with the control groups, HAL + LV­H721 inhibited SMMC­7721 tumor growth in orthotopic HCC and markedly prolonged the survival of HCC­bearing mice, which was accompanied by a lower CD31 expression (tumor angiogenesis) and increased apoptosis in the tumor cells. These findings demonstrated a valuable antitumor synergism in combining CRISPR/Cas9­mediated HIF­1α knockout with TAE in mice and highlighted the possibility that HIF­1α may be an effective therapeutic knockout target in combination with TAE for HCC treatment.

Hepatitis B virus upregulates GP73 expression by activating the HIF-2α signaling pathway.

Golgi Protein 73 (GP73) is a newly identified diagnostic and prognostic marker for liver cancer. GP73 is highly expressed in liver cancer tissues, however, the mechanism of its overexpression in tumors remains unknown. In the present study, the effect of hepatitis B virus (HBV) on GP73 expression was investigated in HepG2 cells, which are negative for HBV, and in HepG2.2.12 cells, which are integrated with HBV, using reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, the cells were transfected with plasmid constructs overexpressing hepatitis B virus protein X (HBx), hypoxia-inducible factor (HIF)-1α, or HIF-2α in order to examine their roles in GP73 expression. The results demonstrated that HBV upregulated the expression of GP73 and HIF-2α in liver cancer cells. HIF-2α induced the expression of GP73 in HepG2 cells and was positively correlated with GP73 expression in liver cancer tissues. By contrast, HBx and HIF-1α did not induce GP73 expression in liver cancer cells. In summary, HBV may upregulate the expression of GP73 by activating the HIF-2α signaling pathway. The present results may illuminate the mechanism by which GP73 is overexpressed in liver cancer tissues.

miR-107-mediated decrease of HMGCS2 indicates poor outcomes and promotes cell migration in hepatocellular carcinoma.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) has been implicated in human cancers, but its role and clinical significance in hepatocellular carcinoma (HCC) remain unknown. Here, we show that HMGCS2 is downregulated and exhibits antimetastatic potential in HCC. Low expression of HMGCS2 was associated with poor tumor differentiation, vascular invasion and worse overall and disease-free survivals in two independent cohorts consisting of 743 cases. In vitro data demonstrated HMGCS2 overexpression suppressed, whereas HMGCS2 silence promoted HCC cell migration via Epithelial-Mesenchymal Transition (EMT) process and the activation of ERK/c-Jun signaling pathway. Inhibition of ERK phosphorylation by PD098059 markedly attenuated the malignant phenotypes mediated by HMGCS2 siRNA. Furthermore, miR-107 was identified as an upstream regulator of HMGCS2 via directly targeting the 3'-UTR of HMGCS2 mRNA. Collectively, our findings suggest HMGCS2 serve as a promising prognostic biomarker and exert anti-tumor activity towards HCC, and therefore provide a potential target for HCC clinical intervention.

UBE2T promotes hepatocellular carcinoma cell growth via ubiquitination of p53.

Deregulation of Ubiquitin-conjugating enzyme E2T (UBE2T) contributes to the progression of human cancers. However, its clinical significance and role in hepatocellular carcinoma (HCC) remain unclear. Here, we show that UBE2T is up-regulated in HCC and exerts oncogenic activities via ubiquitination of p53. High UBE2T expression was correlated with higher pathological grade, advanced TNM stage, tumor vascular invasion, and poor overall and disease-free survivals in two independent cohorts containing 827 patients with HCC. UBE2T was further identified as an independent factor for overall survival by multivariate analyses. Luciferase reporter assays confirmed that UBE2T was directly targeted by miR-543 which was down-regulated in HCC. In vitro experiments demonstrated that UBE2T overexpression promoted, whereas UBE2T knockdown inhibited HCC cell growth. Ectopic expression of UBE2T resulted in the decreases of p53, p21 and Noxa. Further studies revealed that UBE2T facilitated the degradation of p53 protein via enhancing its ubiquitination. Collectively, our findings suggest UBE2T serves as a promising prognostic factor for HCC and functions as an oncogene. The newly identified miR-543/UBE2T/p53 axis may represent a new potential therapeutic target for HCC intervention.

Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis.

BACKGROUND AND AIM: MicroRNAs (miRNAs) are non-coding RNA molecules of 21-24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases including cancer. METHODS: We analyzed the miRNA expression profiles in 10 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) from 10 non-viral hepatitis patients, using a mammalian miRNA microarray containing whole human mature and precursor miRNA sequences. RESULTS: A total of 15 miRNAs exhibited higher expression in the HCC samples than that in the NT samples, and one miRNA demonstrated lower expression in the HCC samples than in the NT samples. A total of 18 miRNAs identified valid expression only in HCC samples, with six only in NT samples. The chip results were confirmed by Northern blot analysis. CONCLUSION: Our study may help clarify the molecular mechanisms involved in the pathogenesis of HCC, and miRNAs potentially serve as a novel diagnostic tool of HCC.

Clinical characteristics of remote Zeus robot-assisted laparoscopic cholecystectomy: a report of 40 cases.

AIM: To summarize the performing essentials and analyze the characteristics of remote Zeus robot-assisted laparoscopic cholecystectomy. METHODS: Robot-assisted laparoscopic cholecystectomy was performed in 40 patients between May 2004 and July 2005. The operating procedures and a variety of clinical parameters were recorded and analyzed. RESULTS: Forty laparoscopic cholecystectomy procedures were successfully completed with Zeus robotic system. And there were no post-operative complications. Total operating time, system setup time and performing time were 100.3 +/- 18.5 min, 27.7 +/- 8.8 min and 65.6 +/- 18.3 min, respectively. The blood loss and post-operative hospital stay were 30.6 +/- 10.2 mL and 2.8 +/- 0.8 d, respectively. Camera clearing times and time used for operative field adjustment were 1.1+/- 1.0 min and 2.0 +/- 0.8 min, respectively. The operative error was 7.5%. CONCLUSION: Robot-assisted laparoscopic cholecystectomy following the principles of laparoscopic operation has specific performing essentials. It preserves the benefits of minimally invasive surgery and offers enhanced ability of controlling operation field, precise and stable operative manipulations.

Zeus robot-assisted laparoscopic cholecystectomy in comparison with conventional laparoscopic cholecystectomy.

BACKGROUND: The robotic surgical system overcomes many technological obstacles of conventional laparoscopic surgery, and possesses enormous clinical applied potential. The aim of this study was to compare the efficacy of Zeus robot-assisted laparoscopic cholecystectomy with conventional laparoscopic cholecystectomy. METHODS: Forty patients undergoing elective cholecystectomy were randomly divided into two groups. Patients in group A (n=20) underwent Zeus robot-assisted laparoscopic cholecystectomy, and patients in group B (n=20) received conventional laparoscopic cholecystectomy. The parameters on operative field, operative time, the number of actions, the rate of operative errors and minimal trauma were evaluated and compared between the two groups. RESULTS: The number of clearing camera (1.1+/-1.0 times) and the time of adjusting the operative field (2.2+/-0.7 minutes) in group A were significantly less than those (4.5+/-1.5 times) and (7.5+/-1.2 minutes) in group B. The number of dissection actions (337+/-86 times) and the rate of operative errors (10%) in group A were less than those (389+/-94 times), (25%) in group B. The total operation time (104.9+/-20.5 minutes) and setup time (29.5+/-9.8 minutes) in group A were significantly longer than those (78.6+/-17.1 minutes), (12.6+/-2.5 minutes) in group B. Blood loss and postoperative hospitalization were similar. No postoperative complications occurred in both groups, and open cholecystectomy was performed in each group. CONCLUSIONS: Zeus robot-assisted cholecystectomy inherits the benefits of minimally invasive surgery. The Zeus robotic surgical system is better than conventional laparoscopic technique in controlling the operative field and can be manipulated precisely and stably though it requires more operative time.

[Laparoscopic cholecystectomy with remote zeus surgical robotic system: report of 16 cases].

OBJECTIVE: To explore the methodology and operative essentials of laparoscopic cholecystectomy with remote Zeus surgical robotic system. METHODS: Based on strict training and successful experiment in animal model of swine, laparoscopic cholecystectomy using Zeus robotic system was performed on 16 patients with biliary diseases, including choledocholithiasis, cholelithiasis, polyposis of gallbladder, and chronic cholecystitis, 10 males and 16 females, aged 33 (14 approximately 27), 26 April to 31 August 2004. The general data, preoperative preparation time, operation time, amount of bleeding, complications, and hospitalization time were analyzed. RESULTS: All operations were performed without event. Along with the accumulation of experience the preoperative preparation time was shortened from 90 min to 30 min with an average of 41.7 min, and the operation time from 120 min to 30 min with an average of 64.4 min. The average amount of bleeding was 27.7 ml, and the average postoperative hospitalization time was 2.4 d. A telephone follow-up 30 days after operation showed no abnormality. CONCLUSION: Laparoscopic cholecystectomy with Zeus surgical robotic system is feasible and reliable with the advantages of clearer images in the field of operation, more precise handling, and remote surgery or education.