Overexpressed lncRNA CDKN2B-AS1 is an independent prognostic factor for liver cancer and promotes its proliferation.

PURPOSE: To study the expression of long non-coding RNA (lncRNA) CDKN2B-AS1 in the liver tissue of patients with liver cancer and its effect on the proliferation of liver cancer cell line QGY-7703. METHODS: The expression of differentially expressed genes in liver cancer tissues and normal liver tissues was analyzed by bioinformatics. The expressions of differentially expressed genes in clinical samples were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to analyze whether there is a significant difference of CDKN2B-AS1 expression in clinical features of patients with liver cancer. The clinical data were analyzed to find out the correlation between the expressions of differentially expressed genes and the overall survival, tumor size and TNM staging. The Gene Set Enrichment Analysis (GSEA) enrichment analysis was used to predict the function of CDKN2B-AS1. The cell proliferation was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was measured by flow cytometry. Expressions of proteins were detected by Western blotting. RESULTS: The expression of CDKN2B-AS1 in liver cancer was significantly higher compared to normal tissues. The results of qRT-PCR were consistent with the results of the Cancer Genome Atlas (TGCA). The data of CDKN2B-AS1 showed that the expression of CDKN2B-AS1 was associated with the total survival, tumor size and TNM staging. GSEA results showed that genes are enriched in cell cycle sets and others. Compared with the control, the proliferation of QGY-7703 cells overexpressing CDKN2B-AS1 was significantly increased (p<0.001). Western blotting results showed that the expressions of CDK2 and CDK4 were up-regulated and the expression of P16 was decreased after CDKN2B-AS1 was overexpressed. CONCLUSIONS: LncRNA CDKN2B-AS1 was highly expressed in liver cancer, and its expression was positively correlated with the overall survival, tumor size and TNM stage. LncRNA CDKN2B-AS1 promoted the proliferation and expression of liver cancer cells.

Long noncoding RNA linc-UBC1 promotes tumor invasion and metastasis by regulating EZH2 and repressing E-cadherin in esophageal squamous cell carcinoma.

PURPOSE: Recent reports have shown that long noncoding (lnc) RNAs are critical during tumorigenesis. This study focused on the influence of linc-UBC1 on the metastasis of esophageal squamous cell carcinoma (ESCC) and the underlying mechanism. METHODS: In 50 ESCC tissues and 5 ESCC cell lines, linc- UBC1 expression was detected by RT-qPCR. Moreover, correlation analysis was conducted between linc-UBC1 expression level and clinicopathological features. Overall survival of these ESCC patients was analyzed by Kaplan-Meier method. In addition, wound healing assay and cell invasion assay were utilized to identify whether linc-UBC1 could affect the migration and invasion ability of ESCC cells. Western blotting and luciferase assay were used to explore the potential mechanism. RESULTS: In ESCC tissues, linc-UBC1 expression level was significantly higher and was remarkably related with clinical features such as TNM stage and nodal metastasis. Meanwhile, overall survival of ESCC patients with high expression of linc-UBC1 was significantly worse than that of patients with low expression. Besides, the migration and invasion ability of ESCC cells was inhibited via knockdown of linc-UBC1. Further study showed that knockdown of linc-UBC1 could suppress the protein level of EZH2 and promote the protein level of E-cadherin. CONCLUSIONS: The results indicate that linc-UBC1 is a novel oncogene in tumorigenesis and could promote the metastasis via EZH2 and E-cadherin, which may offer a possible therapeutic target in ESCC.

CCL2/CCR2 axis induces hepatocellular carcinoma invasion and epithelial-mesenchymal transition in vitro through activation of the Hedgehog pathway.

Chemokine (C-C motif) ligand 2 (CCL2) has been shown to play an important role in the regulation of tumor cell growth, metastasis and host immune response. CCL2 preferentially binds to the C-C chemokine receptor type 2 (CCR2), which is expressed in various tissues. However, the role of the CCL2/CCR2 axis in hepatocellular carcinoma (HCC) invasion and its molecular mechanisms remain unclear. The aim of this study was to elucidate this issue. The human HCC cell line MHCC-97H was treated with CCL2. Cyclopamine, a smoothened (SMO) antagonist, was used to inhibit SMO activity. CCR2 siRNA and Gli-1 siRNA were used to inhibit CCR2 and Gli-1 expression respectively. The effect of CCL2 and Hedgehog (Hh) signaling on cancer cell epithelial-mesenchymal transition (EMT) and invasion was evaluated by quantitative real­time PCR analysis, western blotting and Transwell invasion assay. Our results revealed that CCL2 induced HCC cell invasion and EMT. This effect was accompanied by the activation of Hh signaling, the upregulation of Snail and vimentin and the reduction of E-cadherin. Notably, prior silencing of CCR2 with siRNA abolished CCL2-induced Hh signaling activation, Snail and vimentin upregulation, E-cadherin reduction, as well as HCC cell invasion and EMT. Furthermore, pretreatment with cyclopamine or predepletion of Gli-1 by siRNA also eliminated the changes of Snail, vimentin and E-cadherin, and HCC invasion and EMT caused by CCL2. Collectively, our results revealed that the link between the CCL2/CCR2 axis and the Hh pathway plays an important role in HCC progression. Therefore, the CCL2/CCR2 axis may represent a promising therapeutic target to prevent HCC progression.

Preparation and characterization of the Adriamycin-loaded amphiphilic chitosan nanoparticles and their application in the treatment of liver cancer.

In the present study, two nanoparticles including lactose myristoyl carboxymethyl chitosan (LMCC) and algal polysaccharide myristoyl carboxymethyl chitosan (AMCC), were obtained for hepatic-targeted Adriamycin (ADM) drug delivery systems. ADM was successfully loaded into the LMCC or AMCC nanoparticle by dialysis. The release function and liver targeting of the nanoparticles was explored, and it was revealed that ADM release from the nanoparticles was greatest at acidic pH 5.5. ADM-conjugated nanoparticles were readily taken up by HU7 human hepatocellular carcinoma cells, relative to HT22 mouse hippocampal neuron cells in vitro. In vivo, ADM-loaded nanoparticles had significant antitumor efficacy with a 62.7% inhibition rate, followed by ADM and ADM-AMCC (51.2 and 42.5%, respectively). The tissue distribution study confirmed that ADM-LMCC had an improved liver delivery efficacy, by comparison with ADM. Furthermore, a series of safety studies, including hemolysis, acute toxicity and organ toxicity, revealed that the ADM-loaded LMCC and AMCC nanoparticles had advantages over the commercially available injectable preparation of Adriamycin hydrochloride, in terms of low toxicity levels and increased tolerated dose. These results indicated that LMCC is a promising carrier for injectable ADM nanoparticle and ADM-conjugated nanoparticles may improve the efficacy of ADM by hepatic targeting.

Downregulation of mir-23b in plasma is associated with poor prognosis in patients with colorectal cancer.

MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs have potential as cancer biomarkers. The main objective of the present study was to assess the effect of miRNA-23b (miR-23b) expression in plasma on the diagnosis and prognosis of colorectal cancer (CRC). Reverse transcription-quantitative polymerase chain reaction (PCR) was used to measure miR-23b expression levels, and methylation-specific PCR was used to test the promoter methylation status. Subsequently, the expression level of miR-23b in plasma samples was compared between CRC patients and healthy control individuals. The miR-23b expression levels were significantly lower in CRC cells and primary CRC tissues than in nonmalignant colorectal tissues (P<0.001). It was also shown that miR-23b expression is downregulated by promoter methylation and can be restored by demethylation agent treatment. miR-23b was significantly decreased in plasma samples from CRC patients compared with the healthy control individuals (P<0.001). The value of the area under the receiver operating characteristic curve was 0.842 (sensitivity, 84.38%; specificity, 77.08%; 95% confidence interval, 0.763-0.922). Low plasma miR-23b expression was significantly associated with clinical stage, tumor depth, distant metastasis and tumor recurrence. CRC patients with low miR-23b expression in plasma exhibited a shorter recurrence-free survival time and poorer overall survival rate. The present results suggested that the downregulation of miR-23b in the plasma has the potential to be a diagnostic and prognostic biomarker in CRC.

LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer.

Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cell proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling.