LncRNA NEAT1 Promotes the Progression of Gastric Cancer Through Modifying the miR-1224-5p/RSF1 Signaling Axis.

INTRODUCTION: The therapy of patients with advanced phase gastric cancer is still a huge threat, with extremely imperfect therapies authorized. Even if the amassed indications have validated the significance of lncRNA in gastric cancer, few understandings are stated concerning nuclear paraspeckle assembly transcript 1 (NEAT1) practical functions and molecular mechanisms. METHODS: In this research, the expression of NEAT1 and miR-1224-5p in gastric cancer tissues was measured by qRT-PCR analysis, and the expression of remodeling and spacing factor 1 (RSF1) was measured by IHC assay. Then, the bioinformatics prediction software ENCORI was applied to envisage the assumed binding sites. The monitoring roles of NEAT1 or miR-1224-5p on the cell proliferation and migration capacity were verified by CCK-8, wound healing and transwell assay, correspondingly. The interactions among NEAT1, miR-1224-5p and RSF1 were investigated via luciferase analysis. RESULTS: Our findings revealed high expression levels of NEAT1, RSF1 and a decreased expression level of miR-1224-5p in gastric cancer. Upregulation of NEAT1 or knockdown of miR-1224-5p elevated gastric cancer cell proliferation, and migration. Bioinformatics and luciferase analyses simplified that NEAT1 directly cooperated with miR-1224-5p to weaken miR-1224-5p binding to the RSF1 3'-UTR region. Likewise, the mechanical inquiries ratified that initiation of the miR-1224-5p/RSF1 regulatory loop by miR-1224-5p knockdown or overexpressed RSF1 validated the functions of NEAT1 in endorsing gastric cancer cell malignancy. DISCUSSION: Our research initially validated that NEAT1 may regulate the expression of RSF1 competitive sponge to miR-1224-5p, contributed to the supervision of gastric cancer evolution, which exposed new brightness for diagnosis and therapy of gastric cancer.

Clinicopathological characteristics and survival in colorectal signet ring cell carcinoma: a population-based study.

We aimed to reveal clinicopathological features and explore survival-related factors of colorectal signet ring cell carcinoma (SRCC). A population-based study was carried out to investigate colorectal SRCC by using data extracted from the surveillance, epidemiology and end results (SEER) database between 2004 and 2015. In total, 3,278 patients with colorectal SRCC were identified, with a median age of 63 (12-103) years old. The lesions of most patients (60.49%) were located in the cecum-transverse colon. In addition, 81.27% patients had advanced clinical stage (stage III/IV), and 76.69% patients had high pathological grade. The 3-, 5-year cancer-specific survival and overall survival rate was 35.76%, 29.32% and 32.32%, 25.14%. Multivariate analysis revealed that primary site in cecum-transverse colon, married, received surgery, lymph node dissections ≥ 4 regional lymph nodes were independent favorable prognostic. Meanwhile, aged ≥ 65 years, higher grade, tumor size ˃5 cm and advanced AJCC stage were associated with poor prognosis. Patient age, tumor grade, marital status, tumor size, primary tumor location, AJCC stage, surgery and number of dissected lymph node had significant correlation with prognosis of colorectal SRCC.

GRIM-19 over-expression represses the proliferation and invasion of orthotopically implanted hepatocarcinoma tumors associated with downregulation of Stat3 signaling.

The retinoid-interferon-induced mortality-19 (GRIM-19) gene has been identified as a negative regulator associated with tumor development. The current study created a model of an orthotopically implanted hepatocarcinoma tumor to verify the inhibitory effect of GRIM-19 in vivo. After treatment with GRIM-19 carried by attenuated Salmonella, transplanted tumors were measured with an Imaging System. The expression of GRIM-19, Stat3/p-Stat3, cyclinD1, CDK4, PCNA, Bax/Bcl-2, cleaved caspase-9/3, VEGF, and MMP-2/9 was determined using immunohistochemistry and Western blot analysis. The cell cycle was assessed using flow cytometry (FCM). Apoptosis was determined using FCM and a TUNEL assay. Results indicated that GRIM-19 overexpression resulted in inhibition of peritoneal metastasis, induction of cell cycle arrest, and apoptosis in vivo. In addition, the expression of Stat3/p-Stat3 was down-regulated by GRIM-19. These results suggest that GRIM-19 overexpression could suppress the growth of orthotopically implanted hepatocarcinoma tumors by reversing the regulation of the Stat3 signaling pathway. This approach could potentially be a powerful treatment for hepatocarcinoma.

Differences in the expression profiles of claudin proteins in human gastric carcinoma compared with non­neoplastic mucosa.

Numerous genetic alterations associated with cancer progression have the potential to serve as biomarkers for the early diagnosis of cancer. Numerous studies have suggested that claudin proteins, which are the primary components of tight junction structures, are associated with the regulation of cell polarity and cell differentiation. To investigate the expression profiles of the tight junction proteins claudin­2, ­5, ­7 and ­8 in gastric carcinoma, immunohistochemical analysis, western blotting and reverse transcription­quantitative polymerase chain reaction analysis was used to detect the expression profiles of these claudin proteins in gastric carcinoma tissues and in homologous non­neoplastic mucosal tissues. According to the present study, the expression levels of claudin­7 and claudin­8 were downregulated, while the expression of claudin­5 was upregulated in gastric carcinoma tissues compared with in non­neoplastic mucosal tissues. Additionally, no notable difference was observed between claudin­2 expression in gastric carcinoma tissues and non­neoplastic mucosae. Correlations between claudin­7 and ­8 expression and lymphatic metastasis in gastric carcinoma tissues were additionally reported. In summary, the present study revealed the distinct expression profiles of claudin­5, ­7 and ­8 in non­neoplastic mucosal tissues and gastric carcinoma tissues. Furthermore, the expression of these claudin proteins was highly associated with metastatic progression and prognosis in patients with gastric carcinoma, and had predictive value for the metastasis and survival of patients with gastric carcinoma.

An LC-MS/MS method for quantification of demethylzeylasteral, a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study.

In this study, a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC-MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.

Knockdown of RIPK1 Markedly Exacerbates Murine Immune-Mediated Liver Injury through Massive Apoptosis of Hepatocytes, Independent of Necroptosis and Inhibition of NF-κB.

Receptor-interacting protein kinase (RIPK)1 has an essential role in the signaling pathways triggered by death receptors through activation of NF-κB and regulation of caspase-dependent apoptosis and RIPK3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. We examined the effect of RIPK1 antisense knockdown on immune-mediated liver injury in C57BL/6 mice caused by α-galactosylceramide (αGalCer), a specific activator for invariant NKT cells. We found that knockdown of RIPK1 markedly exacerbated αGalCer-mediated liver injury and induced lethality. This was associated with increased hepatic inflammation and massive apoptotic death of hepatocytes, as indicated by TUNEL staining and caspase-3 activation. Pretreatment with zVAD.fmk, a pan-caspase inhibitor, or neutralizing Abs against TNF, almost completely protected against the exacerbated liver injury and lethality. Primary hepatocytes isolated from RIPK1-knockdown mice were sensitized to TNF-induced cell death that was completely inhibited by adding zVAD.fmk. The exacerbated liver injury was not due to impaired hepatic NF-κB activation in terms of IκBα phosphorylation and degradation in in vivo and in vitro studies. Lack of RIPK1 kinase activity by pretreatment with necrostatin-1, a RIPK1 kinase inhibitor, or in the RIPK1 kinase-dead knock-in (RIPK1D138N) mice did not exacerbate αGalCer-mediated liver injury. Furthermore, RIPK3-knockout and MLKL-knockout mice behaved similarly as wild-type control mice in response to αGalCer, with or without knockdown of RIPK1, excluding a switch to RIPK3/MLKL-mediated necroptosis. Our findings reveal a critical kinase-independent platform role for RIPK1 in protecting against TNF/caspase-dependent apoptosis of hepatocytes in immune-mediated liver injury.

Small cell type neuroendocrine carcinoma colliding with squamous cell carcinoma at esophagus.

Collision tumor is an extremely rare tumor which defined as the concrescence of two distinct primaries neoplasms. We report here a case of collision tumor at lower third esophagus composed of small cell type neuroendocrine carcinoma (NEC), which is an very rare, highly aggressive and poorly prognostic carcinoma and squamous cell carcinoma (SqCC). In our case, pathologically, the small cell carcinoma display the characteristic of small, round, ovoid or spindle-shaped tumor cells with scant cytoplasm, which colliding with a moderately differentiated squamous cell carcinoma. Immunohistochemical staining demonstrated positive activities for CD56, synaptophysin, 34ßE12, CK 5/6, ki-67 (70%-80%), but negative for CD99, chromogranin A, and TTF-1. Accurate diagnosis was made base on these findings.

Overexpression of GRIM-19, a mitochondrial respiratory chain complex I protein, suppresses hepatocellular carcinoma growth.

GRIM-19 has been demonstrated as an important regulator for the normal tissue development. Recently, more evidences regarded GRIM-19 as the new tumor suppressor. However, the possible mechanisms underlying GRIM-19 suppressing cancer growth are unclear. In the present study, Paired hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues were obtained from 54 patients who underwent primary surgical HCC tissue resection. GRIM-19 protein expression in HCC tissues was performed by immunohistochemistry. Cells were transfected by lentiviruses plasmid expressing GRIM-19. RT-PCR and Western blot analyses were performed to confirm the expression of GRIM-19 mRNA or protein. Cell proliferation was assessed by MTT and FCM analyses. Mitochondrial membrane potential and apoptosis were respectively determined by using fluorescence microscopy and FCM analyses. AKT1, pAKT1, cyclinD1, CDK4, PCNA, Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3, and cytochrome C were detected by Western blot and immunofluorescence. GRIM-19 protein expression was markedly lower in HCC than in paired adjacent non-tumor liver tissues. GRIM-19 overexpression in HCC cells significantly induced cell cycle arrest and enhanced apoptosis. We also found that AKT1 expression and phosphorylation were regulated by the expression of GRIM-19. Collectively, our study demonstrated that GRIM-19 overexpression suppressed HCC growth and downregulated AKT1 expression, suggesting that GRIM-19 might play a crucial role in hepatocarcinogenesis through negatively regulating the PI3K/AKT signaling pathway.

The effect of cell cycle and expression of cyclin B1 and cyclin C protein in hepatocellular carcinoma cell line HepG2 and SMMC-7721 after of silencing ß-catenin gene.

BACKGROUND/AIMS: Abnormalities in cell cycle regulation are reported to be strongly associated with tumorigenesis and progression of tumors. Wnt/ß-catenin signaling pathway and cell cycle play key roles during the genesis and development of hepatocellular carcinoma (HCC). Current studies indicated that expressions of cyclin A, E and D1 were affected after silencing of ß-catenin gene in HCC, but it is unclear if other cyclins are affected. METHODOLOGY: To determine the relation, small interference RNA (siRNA) against ß-catenin was transfected into HCC cell lines HepG2 and SMMC-7721, and cell cycle and cyclin B1 and cyclin C protein expression were detected. RESULTS: Cell cycle was arrested in G0/G1 at 72h after transfection and the cell cycle began to transfer from G0/G1 to G2/M through S and had a trend to revert at 96h. In addition, ß-catenin protein expression was decreased at both 72 and 96h, although the level was slightly higher at 96h than that at 72h. However, cyclin B1 expression decreased at 72h and increased at 96h, cyclin C expression increased at 72h and decreased at 96h. CONCLUSIONS: These findings suggest that silencing ß-catenin gene may induce the changes of cell cycle and cyclin B1 and cyclin C protein expression. Wnt/ß-catenin signaling pathway probably takes part in the genesis and development of HCC through regulating cell cycle and the expression of cyclin B1 and cyclin C.