Long noncoding RNA Gas5 induces cell apoptosis and inhibits tumor growth via activating the CHOP-dependent endoplasmic reticulum stress pathway in human hepatoblastoma HepG2 cells.

In recent years, long noncoding RNAs (lncRNAs) have been demonstrated to be important tumor-associated regulatory factors. LncRNA growth arrest-specific transcript 5 (Gas5) acts as an anti-oncogene in most cancers. Whether Gas5 acts as an oncogene or anti-oncogene in hepatocellular carcinoma (HCC) remains unclear. In the present study, the expression and role of Gas5 in HCC were investigated in vitro and in vivo. Lower expression levels of Gas5 were determined in HCC tissues and cells by quantitative reverse transcription-polymerase chain reaction. Overexpressed Gas 5 lentiviral vectors were constructed to analyze their influence on cell viability, migration, invasion, and apoptosis. Fluorescence in situ hybridization was used to identify the subcellular localization of Gas5. Protein complexes that bound to Gas5 were isolated from HepG2 cells through pull-down experiments and analyzed by mass spectrometry. A series of novel Gas5-interacting proteins were identified and bioinformatics analysis was carried out. These included ribosomal proteins, proteins involved in protein folding, sorting, and transportation in the ER, some nucleases and protein enzymes involved in gene transcription, translation, and other proteins with various functions.78 kDa glucose-regulated protein (GRP78) was identified as a direct target of Gas5 by Rip-qPCR and Western blot analysis assay. Gas5 inhibited HepG2 cell growth and induced cell apoptosis via upregulating CHOP to activate the ER stress signaling pathway. Further studies indicated that the knockdown of CHOP by shRNA partially reversed Gas5-mediated apoptosis in HepG2 cells. Magnetic resonance imaging showed that the ectopic expression of Gas5 inhibited the growth of HCC in nude mice. These findings suggest that Gas5 functions as a tumor suppressor and induces apoptosis through activation of ER stress by targeting the CHOP signal pathway in HCC.

LncRNA MEG3 contributes to adenosine-induced cytotoxicity in hepatoma HepG2 cells by downregulated ILF3 and autophagy inhibition via regulation PI3K-AKT-mTOR and beclin-1 signaling pathway.

Adenosine is a promising cytotoxic reagent for tumors, long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been indicated to play critical roles in tumorigenesis, ILF3 has been recognized as a MEG3-binding protein, however, the roles of adenosine and MEG3 on hepatoma are still ambiguous. To clarify the effects of MEG3 on the adenosine-induced cytotoxicity in hepatoma, MEG3 and ILF3 lentivirus were transduced into human hepatoma HepG2 cells to stimulate overexpression of MEG3 (OE MEG3) and overexpression of ILF3 (OE ILF3), furthermore, ILF3 small interfering RNA (siRNA) was also applied to downregulate the expression of ILF3. In this study, autophagy was markedly inhibited by low concentration of adenosine, which present by not only inhibited transformation from LC3-I to LC3-II and autophagosomes formation, but also the elevation of mTOR and reduction of beclin-1 proteins. Furthermore, low concentration of adenosine also exerted marked cytotoxicity representing induced cell apoptosis together with reductions of cell viability and migration, which were also markedly enhanced by OE MEG3. Novelly and excitingly, adenosine markedly stimulated MEG3 expression, OE MEG3 markedly decreased the ILF3 expression in HepG2 cells, and the adenosine-induced autophagy inhibition, together with the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were also boosted by OE MEG3. More interestingly, OE ILF3 increased autophagy, whereas downregulated ILF3, especially in the case of adenosine, led to marked autophagy inhibition by decreasing beclin-1. The present study demonstrates autophagy inhibition is involved in the adenosine-induced cytotoxicity in HepG2 cells, the cytotoxicity can be synergized by OE MEG3 via downregulated ILF3 to activate PI3K/Akt/mTOR and inactivate the beclin-1 signaling pathway. In conclusion, MEG3 and inhibition of autophagy might be potential targets for augmenting adenosine-induced cytotoxicity in hepatoma.

Long non­coding RNA MEG3 suppresses epithelial­to­mesenchymal transition by inhibiting the PSAT1­dependent GSK­3ß/Snail signaling pathway in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer in China, and the prognosis of patients remains poor mainly due to the occurrence of lymph node and distant metastasis. The long non­coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been shown to have tumor­suppressive properties and to play an important role in epithelial­to­mesenchymal transition (EMT) in some solid tumors. However, whether MEG3 is involved in EMT in ESCC remains unclear. In the present study, the MEG3 expression level and its association with tumorigenesis were determined in 43 tumor tissues of patients with ESCC and in ESCC cells using reverse transcription­quantitative PCR analysis. Gene microarray analysis was performed to detect differentially expressed genes (DEGs). Based on the functional annotation results, the effects of ectopic expression of MEG3 on cell growth, migration, invasion and EMT were assessed. MEG3 expression level was found to be markedly lower in tumor tissues and cells. Statistical analysis revealed that MEG3 expression was significantly negatively associated with lymph node metastasis and TNM stage in ESCC. Fluorescence in situ hybridization assay demonstrated that MEG3 was expressed mainly in the nucleus. Ectopic expression of MEG3 inhibited cell proliferation, migration, invasion and cell cycle progression in EC109 cells. Gene microarray results demonstrated that 177 genes were differentially expressed ≥2.0 fold in MEG3­overexpressing cells, including 23 upregulated and 154 downregulated genes. Functional annotation revealed that the DEGs were mainly involved in amino acid biosynthetic process, mitogen­activated protein kinase signaling, and serine and glycine metabolism. Further experiments indicated that the ectopic expression of MEG3 significantly suppressed cell proliferation, migration, invasion and EMT by downregulating phosphoserine aminotransferase 1 (PSAT1). In pathological tissues, PSAT1 and MEG3 were significantly negatively correlated, and high expression of PSAT1 predicted poor survival. Taken together, these results suggest that MEG3 may be a useful prognostic biomarker and may suppress EMT by inhibiting the PSAT1­dependent glycogen synthase kinase­3ß/Snail signaling pathway in ESCC.

Long non-coding RNA MEG3 induces cell apoptosis in esophageal cancer through endoplasmic reticulum stress.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. Although downregulation of lncRNA MEG3 has been identified in several cancers, little is known about its role in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to detect MEG3 expression in clinical ESCC tissues, investigate its biological functions and the endoplasmic reticulum (ER) stress-relative mechanism. MEG3 expression levels were detected by qRT-PCR in both tumor tissues and adjacent non-tumor tissues from 28 ESCC patients. PcDNA3.1-MEG3 recombinant plasmids were constructed and transfected to EC109 cells. Cell growth was analyzed by CCK-8 assay. Cell apoptosis was analyzed by fluorescence microscope and Annexin V/PI assay. The protein expression was determined by western blot analysis. The results showed that MEG3 decreased significantly in ESCC tissues relative to adjacent normal tissues. PcDNA3.1-MEG3 plasmids were successfully constructed and the expression level of MEG3 significantly increased after MEG3 transfection to EC109 cells. Ectopic expression of MEG3 inhibited EC109 cell proliferation and induced apoptosis in vitro. MEG3 overexpression increased the expression of ER stress­related proteins (GRP78, IRE1, PERK, ATF6, CHOP and cleaved­caspase-3). Our results first demonstrate that MEG3 is downregulated in ESCC tissues. MEG3 was able to inhibit cell growth and induced apoptosis in EC109 cells, most probably via activation of the ER stress pathway.