The overexpression of GRASP might inhibit cell proliferation and invasion in hepatocellular carcinoma.

This study aimed to validate the methylation of key genes in hepatocellular carcinoma (HCC) screened by bioinformatics analysis and explore whether they affected HCC cell proliferation, migration, and invasion. Using The Cancer Genome Atlas (TCGA) database, HCC-related differentially methylated positions (DMPs) were screened, genes corresponding to DMPs were selected, and prognosis-related genes were identified. A representative DMP was used to divide the DMPs into hyper- and hypomethylated groups. Expression of key genes in cell lines was detected using quantitative real-time polymerase chain reaction and western blot analysis. After treatment of HepG2 cells with 5-Aza-2'-deoxycytidine (5-Aza-DC), gene expression was observed. Bisulfite sequencing PCR assay was used to detect methylation frequency. Overexpressed GRASP lentiviral vectors were constructed to analyze their influence on cell proliferation, migration, and invasion using cell counting kit-8 and transwell assays. Forty-three HCC prognosis-related genes were screened using the TCGA database. cg00249511 (SCT) was used to divide the DMPs into hyper- and hypomethylated groups, distinguishing between high- and low-risk samples. The prognosis survival model constructed using 12 genes revealed the prognosis type. GRASP messenger RNA was downregulated in HepG2 and upregulated after 5-Aza-DC treatment. In HCC tissues, methylation frequency of GRASP was upregulated. GRASP overexpression inhibited HepG2 cell proliferation, invasion, and G-CSFR expression. Thus, GRASP might be a prognosis-related gene controlled by methylation.

Mechanisms of Huangqi Decoction Granules () on Hepatitis B Cirrhosis Patients Based on RNA-Sequencing.

OBJECTIVE: To explore the action mechanisms of Huangqi Decoction Granules (, HQDG) on hepatitis B cirrhosis. METHODS: A total of 85 patients with hepatitis B cirrhosis were randomly divided into HQDG group (42 cases) and control group (43 cases) by a random number table and were treated with HQDG or placebo for 48 weeks (6 g per times and orally for 3 times a day), respectively. After RNA-sequencing of serum samples extracted from the patients, the differentially expressed genes (DEGs) in HQDG and control groups before and after treatment were separately screened. The DEGs were then performed pathway enrichment analysis and proteinprotein interaction (PPI) network analysis. The expression levels of key genes were detected by quantitative realtime polymerase chain reaction (qRT-PCR). RESULTS: After the investigation, 4 and 3 cases were respectively excluded from HQD and control groups because of the incomplete data. Additionally, 3 and 5 cases were lost to follow up in HQD and control groups respectively. Finally, a total of 70 cases with good compliance were included for further DEGs analysis. A total of 1,070 DEGs (including 455 up-regulated genes and 615 down-regulated genes) in HQDG group and 227 DEGs (including 164 up-regulated genes and 63 down-regulated genes) in the control group were identified after treatment. Compared with the control group, 1,043 DEGs were specific in HQDG group. Besides, 1 up-regulated transcription factor (TF, such as GLI family zinc finger 1, GLI1) and 25 down-regulated TFs (such as drosophila mothers against decapentaplegic proteinfamily member 2, SMAD2) were identified. Pathway enrichment analysis showed that down-regulated Ras homolog gene family member A (RHOA) was enriched in pathogenic Escherichia coli infection. In the PPI network, up-regulated epidermal growth factor receptor (EGFR), and down-regulated cell division cycle 42 (CDC42) as well as v-akt murine thymoma viral oncogene homolog 1 (AKT1) had higher degrees. Moreover, long non-coding RNAs (lncRNA) growth arrest-specific 5 (GAS5) was involved in the lncRNA-target regulatory network. Furthermore, qRT-PCR revealed that expression levels of CDC42 and GLI1 had significant differences in HQDG group before and after treatment (P<0.05). CONCLUSIONS: CDC42 and GLI1 may be the targets of HQDG in patients with hepatitis B cirrhosis. Additionally, SMAD2, EGFR, AKT1, RHOA and GAS5 might be associated with the curative effect of HQDG on hepatitis B cirrhosis patients.

LMO3 promotes hepatocellular carcinoma invasion, metastasis and anoikis inhibition by directly interacting with LATS1 and suppressing Hippo signaling.

BACKGROUND: In this research, we aimed to investigate the biological functions of LIM domain only 3 (LMO3) in hepatocellular carcinoma (HCC) and uncover the underlying molecular mechanism in it. METHODS: HCC tissue microarray (n = 180) was used to analyze the correlation between LMO3 expression and clinicopathological findings. In vitro transwell matrigel invasion assay and annexin V anoikis assay in HCC cells were conducted to investigate LMO3 related biological functions. In vivo intrahepatic and lung metastasis models were used to determine the role of LMO3 in HCC metastasis. Quantitative real-time PCR, western blotting and immunohistochemical staining were performed to investigate the expression and mechanism of LMO3 in HCC. RESULTS: We found that the expression of LMO3 was significantly upregulated in HCC tissues, and it was closely related to clinicopathological findings and patient prognoses. Knockdown of LMO3 suppressed the invasion and anoikis inhibition of HCC cells in vitro. Meanwhile, the metastasis of SMMC-7721 cells was also suppressed by LMO3 knockdown in vivo. Furthermore, we found that LMO3 knockdown increased the phosphorylation of YAP and LATS1, and decrease Rho GTPases activities. LMO3 directly interacted with LATS1, and thus suppressed Hippo signaling. Recombinant LMO3 (rLMO3) protein administration decreased the phosphorylation of YAP and LATS1, and increased Rho GTPases activities. The inhibitors of the Hippo pathway abrogated rLMO3 protein-induced HCC cell invasion and anoikis inhibition. CONCLUSIONS: These results suggest that LMO3 promotes HCC cell invasion and anoikis inhibition by interacting with LATS1 and suppressing Hippo signaling. LMO3 may serve as a potential therapeutic target for HCC in future.

Antiviral activities of atractylon from Atractylodis Rhizoma.

Atractylodis Rhizoma is a traditional medicinal herb, which has antibacterial, antiviral, anti­inflammatory and anti­allergic, anticancer, gastroprotective and neuroprotective activities. It is widely used for treating fever, cold, phlegm, edema and arthralgia syndrome in South­East Asian nations. In this study, 6 chemical compositions of Atractylodis Rhizoma were characterized by spectral analysis and their antiviral activities were evaluated in vitro and in vivo. Among them, atractylon showed most significant antiviral activities. Atractylon treatment at doses of 10­40 mg/kg for 5 days attenuated influenza A virus (IAV)­induced pulmonary injury and decreased the serum levels of interleukin (IL)­6, tumor necrosis factor­α and IL­1ß, but increased interferon­ß (IFN­ß) levels. Atractylon treatment upregulated the expression of Τoll­like receptor 7 (TLR7), MyD88, tumor necrosis factor receptor­associated factor 6 and IFN­ß mRNA but downregulated nuclear factor­κB p65 protein expression in the lung tissues of IAV­infected mice. These results demonstrated that atractylon significantly alleviated IAV­induced lung injury via regulating the TLR7 signaling pathway, and may warrant further evaluation as a possible agent for IAV treatment.

MicroRNA-421 induces hepatic mitochondrial dysfunction in non-alcoholic fatty liver disease mice by inhibiting sirtuin 3.

BACKGROUND AND AIM: Mitochondrial dysfunction plays a major role in critical initiating or propagating events in nonalcoholic fatty liver disease (NAFLD), but its pathogenesis remains obscure. Recently, microRNAs have been found to affect oxidant stress and lipid metabolism. In this study, we elucidated the functions of microRNA-421 in the development of NAFLD and identified its potential targets. METHODS: An experimental model for the study of NAFLD was constructed by feeding a high fat diet to C57BL/6J mice. Differentially expressed miRNA in livers of NAFLD mice compared with controls were identified by high-throughput sequencing. Relative repression of luciferase expression standardized to a transfection control was analyzed by luciferase reporter assays. RESULTS: The microRNA profiling presented that microRNA-421 expression was significantly upregulated in hepatic tissues of NAFLD model mouse. The sirtuin 3 was identified as a functionally relevant target of microRNA-421. The microRNA-421 acts upstream of SIRT3/FOXO3 pathway in modulation the oxidant stress and lipid metabolism. Overexpression of microRNA-421 decreased SIRT3 and FOXO3 protein levels, and then led to MnSOD and CAT decrease, the downstream targets of SIRT3/FOXO3 pathway. On the contrary, suppression of microRNA-421 had adverse effects on performance of celluar oxidative damage. CONCLUSIONS: Regulating or inhibiting hepatic microRNA-421 could decrease celluar oxidative damage and contribute to therapeutic potential in NAFLD.

Quantitative succinylome analysis in the liver of non-alcoholic fatty liver disease rat model.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a clinical frequent disease. However, its pathogenesis still needs further study, especially the mechanism at the molecular level. The recent identified novel protein post-translational modification, lysine succinylation was reported involved in diverse metabolism and cellular processes. In this study, we performed the quantitative succinylome analysis in the liver of NAFLD model to elucidate the regulatory role of lysine succinylation in NAFLD progression. METHODS: Firstly, experimental model of NAFLD was induced by carbon tetrachloride injection and supplementary high-lipid and low-protein diet. Then series histochemical and biochemical variables were determined. For the quantitative succinylome analysis, tandem mass tags (TMT)-labeling, highly sensitive immune-affinity purification, liquid chromatography-tandem mass spectrometry techniques were applied. Bioinformatics analysis including gene ontology annotation based classification; Wolfpsort based subcellular prediction; function enrichment; protein-protein interaction network construction and conserved succinylation site motifs extraction were performed to decipher the differentially changed succinylated proteins and sites and p-value < 0.05 was selected as threshold. RESULTS: Totally, 815 succinylation sites on 407 proteins were identified, of which 243 succinylation acetylation sites on 178 proteins showed changed succinylation level with the threshold fold change > 1.5. Theses differentially changed succinylated proteins were involved in diverse metabolism pathways and cellular processes including carbon metabolism, amino acid metabolism, fat acid metabolism, binding and catalyzing, anti-oxidation and xenobiotics metabolism. Besides, these differentially changed succinylated proteins were prominently localized to cytoplasm and mitochondria. Moreover, 8 conserved succinylation site motifs were extracted around the succinylation sites. CONCLUSIONS: Protein succinylation was an extensive post-translation modification in rat. The changed succinylation level in diverse proteins may disturb multiple metabolism pathways and promote non-alcoholic fatty liver disease development. This study provided a basis for further characterization of the pathophysiological role of lysine succinylation in NAFLD progression, which laid a foundation for the innovation of novel NAFLD drugs and therapies.