LINC00313 facilitates osteosarcoma carcinogenesis and metastasis through enhancing EZH2 mRNA stability and EZH2-mediated silence of PTEN expression.

BACKGROUND: Osteosarcoma is one of the five leading causes of cancer death among all pediatric malignancies. Recent advances in non-coding RNAs suggested that many long noncoding RNAs (lncRNAs) are dysregulated in cancer tissues and play important roles in carcinogenesis. We aimed to further explore the mechanisms of Long Intergenic Non-Protein Coding RNA 313 (LINC00313)-promoted malignant phenotypes of osteosarcoma. METHODS: The mRNA expressions were determined by quantitative real-time PCR. Protein levels were detected using Western blotting or immunohistochemistry staining. Protein binding to genomic DNA and RNA were measured using chromatin and RNA immunoprecipitation assay, respectively. CCK-8 and EdU incorporation assay were adopted to detect cell proliferation. Transwell assay was employed to assess the capacity of cell migration and invasion. The roles of LINC00313 and its target genes in tumorigenesis and metastasis of osteosarcoma were evaluated using subcutaneous xenograft models and tail vein inoculation models. RESULTS: LINC00313 was elevated in osteosarcoma tissues compared with adjacent normal tissues. Higher LINC00313 was associated with advanced grades of osteosarcoma. LINC00313 promoted cell proliferation, migration, invasion in vitro and tumor growth as well as metastasis in vivo through inhibiting PTEN expression to promote AKT phosphorylation. Mechanistically, LINC00313 favored the interaction between FUS and EZH2, leading to the prolonged half-life of EZH2 mRNA, thereby in turn up-regulating EZH2 proteins and increasing EZH2-mediated epigenetic silence of PTEN. CONCLUSION: LINC00313 exerted oncogene-like actions through increasing EZH2 mRNA stability, leading to PTEN deficiency in osteosarcoma.

CADM1 regulates the G1/S transition and represses tumorigenicity through the Rb-E2F pathway in hepatocellular carcinoma.

BACKGROUND: Increasing evidence indicates that downregulation of cell adhesion molecule 1 (CADM1) contributes to tumorigenesis in various cancers. The present study was undertaken to investigate the CADM1 expression pattern in human hepatocellular carcinoma (HCC), and to elucidate the mechanism underlying CADM1-mediated tumor suppression. METHODS: CADM1 expression in HCC cell lines was measured by quantitative real-time PCR. The function of CADM1 in the context of tumor suppression in HCC cells was determined using proliferation assays, cell cycle analysis, EdU incorporation assays, in vitro colony formation analysis, and in vivo tumorigenicity assays. The mechanism by which CADM1 acts as a tumor suppressor gene in HCC was investigated using Western blotting analysis. RESULTS: Downregulation of CADM1 expression is frequently detected in both HCC cells and clinical samples. Restoration of CADM1 expression in HCC cell lines significantly inhibits cell growth and negatively regulates the G1/S transition. CADM1 overexpression can inhibit the tumorigenicity of HCC cells both in vitro and in vivo. Western blotting analysis revealed that ectopic expression of CADM1 in HCC cells is associated with increased expression of Retinoblastoma (Rb) protein. CONCLUSIONS: Our results showed that suppression of tumorigenesis by CADM1 may be mediated by the Rb-E2F pathway, involving upregulation of Rb protein levels. This pathway could therefore represent an attractive target for HCC therapy.

Oncogenic role of microRNA-423-5p in hepatocellular carcinoma.

BACKGROUND: It has been found that microRNA-423-5p (miR423-5p) is an oncogenic factor and frequently upregulated in gastric carcinoma. However, the involvement of miR423-5p in hepatocellular carcinoma (HCC) has been rarely reported. The aim of this study was to assess whether miR423-5p is aberrantly expressed in HCC tissues, and to characterize its roles in the cancerous biology of HCC. METHODS: HCC and corresponding nonmalignant tissues were obtained from 115 patients during liver transplantation to detect the expression level of miR423-5p. The miR423-5p mimic and inhibitor were transfected into LM3 cell line. Cell viability assay, cell cycle analysis, transwell invasion and migration experiments were used to evaluate the oncogenic role of miR423-5p. RESULTS: miR423-5p was significantly upregulated in HCC compared with nonmalignant tissues, and this upregulation was negatively associated with recurrence-free survival. For patients beyond the Milan criteria, low expression of miR423-5p was correlated with better prognosis. Functional analysis showed that miR423-5p enhanced the proliferative, invasive and migratory capacity of HCC cells. CONCLUSIONS: miR423-5p contributed to the tumorigenesis and progression of HCC. It could be a new predictor in HCC patients beyond the Milan criteria and would help to improve patient outcomes and enlarge recipient pools of liver transplantation.

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx.

BACKGROUND: Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication. METHODS: HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA. RESULTS: Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting. CONCLUSION: The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.

Tri-iodothyronine enhances liver regeneration after living donor liver transplantation in rats.

BACKGROUND: Tri-iodothyronine (T3) has been shown to be a hepatic mitogen. The aim of this study was to evaluate the effect of T3 on liver regeneration after 50% partial liver transplantation (pLT) in rats. METHODS: Immediately after pLT, a single dose of T3 (4 mg/kg body weight) was administered. Liver/body weight ratio (LBWR), hepatocyte proliferation (Ki-67), biochemical parameters, and changes in cell cycle related proteins were evaluated. RESULTS: T3 promoted liver regeneration as shown by an increased liver/body weight ratio and Ki-67 proliferation index after pLT. On the transcriptional level, T3-treated rats had an increased expression of cyclin D1 and cyclin A as demonstrated by real time RT-PCR and Western blot. CONCLUSIONS: Exogenous administration of T3 significantly improved liver regeneration after pLT, and therefore it may represent a promising strategy to improve the clinical outcome after living donor liver transplantation in the future.

The impact of miR-34a on protein output in hepatocellular carcinoma HepG2 cells.

MicroRNAs are small non-coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR-34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR-34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR-34a. Transfection of miR-34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2-DE method, 34 proteins were successfully identified by MALDI-TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR-34a. Bioinformatics analysis produced a protein-protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR-34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR-34a. In conclusion, abrogation of miR-34a function could cause downstream molecules to switch on or off, leading to HCC development.