Highly expressed miR-144-3p promotes the proliferation, migration and invasion of colon carcinoma cells by activating the Wnt/ß-catenin signaling pathway through targeting SFRP1.

Background: To investigate the influence of miR-144-3p on the proliferation, migration and invasion of colon carcinoma by targeting secreted frizzled-related protein 1 (SFRP1) as well as of the Wnt/ß-catenin signaling pathway. Methods: Based on the TCGA database, the association between the expression of miR-144-3p and the clinical information and prognosis of patients with colon carcinoma was examined, and SFRP1 was selected as the target gene for further studies based on bioinformatics prediction tools. CCK8 assay, wound healing assay and transwell invasion assay were employed to examine the impact of miR-144-3p on colon carcinoma cells. The regulation of SFRP1 by miR-144-3p was investigated using a dual-luciferase reporter system, and a rescue experiment was conducted to further elucidate whether miR-144-3p promotes the migration of colon carcinoma cells through targeting SFRP1 or not. The Wnt/ß-catenin signaling pathway-mediated effect of miR-144-3p in colon carcinoma was finally validated through the targeting of SFRP1. Results: The bioinformatics analysis showed that the miR-144 expression levels were substantially greater in colon carcinoma tissue than in para-carcinoma tissue and were closely with clinical stage and prognosis. The findings obtained from the trial indicated that miR-144-3p substantially expressed in colon carcinoma tissue sample and the colon carcinoma cells, and the overexpressed miR-144-3p boosted the colon carcinoma cells' proliferation, migration and invasion. The results of dual-luciferase reporter gene assay revealed that miR-144-3p targeted SFRP1, and rescue experiment was carried out and its results indicated that miR-144-3p increased colon carcinoma cells' migration through targeting SFRP1. In addition, the molecular axis of miR-144-3p/SFRP1 may over-activate the Wnt/ß-catenin signaling pathway. Conclusions: The present study has identified a novel malignant biological behavior, namely the ability of miR-144-3p to enhance the proliferation, migration and invasion of colon carcinoma cells by targeting SFRP1 and activating the Wnt/ß-catenin signaling pathway. Consequently, miR-144-3p emerges as a promising diagnostic and therapeutic target for colon carcinoma.

Knockout of the Cannabinoid Receptor 2 Gene Promotes Inflammation and Hepatic Stellate Cell Activation by Promoting A20/Nuclear Factor-κB (NF-κB) Expression in Mice with Carbon Tetrachloride-Induced Liver Fibrosis.

BACKGROUND This study aimed to investigate the effect of deleting the cannabinoid receptor 2 (CB2) gene on the development of hepatic fibrosis induced by carbon tetrachloride (CCl4) in mice via regulating inflammation. MATERIAL AND METHODS The DNA was extracted from the tails of mice to identify whether the cannabinoid receptor 2 gene was successfully knocked out. A liver fibrosis model was established by an intraperitoneal injection of CCl4 into mice. Hepatic damage and hepatic fibrosis were evaluated by detecting serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and staining paraffin sections of liver tissue with hematoxylin-eosin (HE). The secretion and distribution of collagen in liver tissue were observed by Masson staining. Western blot analysis was performed to detect the expression of a-smooth muscle actin (alpha-SMA), transforming growth factor-ß1 (TGF-ß1), tumor necrosis factor alpha-induced protein 3 (A20), phosphorylated nuclear factor-kB p65 (p-NF-kappaB p65), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) in liver tissue. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-6 and TNF-alpha mRNA in liver tissue. RESULTS Compared with the control mice, the mice with CB2 knockout that were exposed to CCl4 exhibited increased liver damage, liver fibrosis, and upregulated alpha-SMA, TGF-ß1, A20, and p-NF-kappaB p65 protein levels. IL-6 and TNF-alpha protein levels and mRNA levels were upregulated. CONCLUSIONS The deletion of the CB2 gene promoted the activation of hepatic stellate cells in mice with liver fibrosis and aggravated liver fibrosis by up-regulating the protein expression of A20 and p-NF-kappaB p65 and inducing inflammatory response, potentially providing new insight into the treatment of liver fibrosis.

The protective effect of cannabinoid type II receptor agonist AM1241 on ConA-induced liver injury in mice via mitogen-activated protein kinase signalling pathway.

INTRODUCTION: The endocannabinoid system plays an important role in regulating the immune responses in inflammation. At present, there are no good clinical drugs for many immune liver diseases. METHODS: We explored the protective effect of the cannabinoid type II (CB2) receptor agonist AM1241 on the liver of mice with acute liver injury caused by concanavalin from the perspective of inflammation and immunity. Pathological evaluation in hepatic tissue was examined by haematoxylin and eosin (HE) staining and the levels of biochemical parameters in the serum were measured by automatic biochemical analysis. The content of inflammatory factors was measured by enzyme-linked immunosorbent assay and real-time quantitative reverse transcription polymerase chain reaction (real-time PCR). The liver apoptosis-related proteins were observed by immunohistochemistry. The expression of liver injury-related proteins was analysed by Western blot. Immune cells were isolated from the liver of mice and studied in vitro. RESULTS: Reduced levels of alanine transaminase and aspartate transaminase were observed in ConA-induced liver injury mice treated with AM1241, together with attenuated liver damage evidenced by H&E staining. Moreover, AM1241 inhibited the protein and gene expression levels of TNF-α, IL-6 and IFN-γ in the livers of mice. The phosphorylation levels of p38, JNK, ERK1/2, P65 and cAMP response element-binding protein (CREB) in the mouse were significantly reduced in AM1241 pretreatment, while the level of p-JNK increased. In addition, the P/T-P65 and P/T-CREB of the AM1241 pretreatment group were significantly reduced. The results of immunohistochemistry measurement are consistent with those of Western blotting. The CB2-mediated effect is through macrophage-like Kupffer cells. CONCLUSION: Our study suggests that the ConA-induced liver injury model in mice is protected by CB2 agonist AM1241 by modulation of CB2 receptor-rich immune cells, for example, Kupffer cells. Reduced inflammatory responses regulate apoptosis/cell death in the liver particularly hepatocytes and other parenchymal cells.

[Cannabinoid receptor 2 deletion promotes proliferation and activation of hepatic macrophages in mice with acute liver injury induced by concanavalin A].

Objective To investigate the effect of cannabinoid receptor 2 (CB2) gene deletion on liver macrophages in mice with acute liver injury induced by concanavalin A (ConA). Methods The mice with gene deletion were identified by PCR. Twenty 8-week-old wild-type C57BL/6J male mice were randomly divided into control group and model group. Twenty C57BL/6J male mice with the deletion of CB2 were divided into CB2-/- control group and CB2-/- model group. The wild-type and CB2-/- mouse model groups were injected with ConA 20 mg/kg via tail vein to replicate the model of acute liver injury, and the control groups were injected with the same amount of PBS. Nine hours after modeling, the serum was taken for the detection of alanine aminotransferase (ALT); the degree of liver injury in each group was observed by HE staining; the expression levels of CD68 and TNF-alpha proteins related to liver macrophages were detected by Western blotting; and the positive level of F4/80 in the liver tissue was detected by immunohistochemistry. Results Compared with the two control groups, the liver injury degree of mice in the model groups were serious; the serum ALT level significantly increased; the positive expression of F4/80 in the liver tissue; and the expression of CD68 and TNF-alpha proteins were significantly enhanced. Compared with the WT model group, the CB2-/- model group had an increase in the degree and area of liver injury, the serum ALT, the positive expression of F4/80 in the liver tissue, and the expression of CD68 and TNF-alpha proteins. Conclusion The deletion of CB2 gene increases the proliferation and activation of macrophages in the mice with ConA-induced acute liver injury.

Anthocyanins isolated from blueberry ameliorates CCl4 induced liver fibrosis by modulation of oxidative stress, inflammation and stellate cell activation in mice.

To study the mechanism of anthocyanins from blueberry on mice model of hepatic fibrosis. We observed that the levels of serum ALT and AST of 100 mg*kg-1*d-1, 200 mg*kg-1*d-1 anthocyanins group were reduced compared to the CCl4 treated group. Mitochondrial electron chain complex 1 and 2 activities, determined by microplate assays, were reduced in CCl4 treated group and restored by anthocyanin treatment. MDA and protein carbonyl content of liver homogenate were induced by CCl4 and anthocyanin treated group reduced both significantly.Monocyte chemoattractant protein 1 (MCP1), Interleukin 1 beta (IL1ß), macrophage inflammatory protein 2 (MIP-2) were induced by CCl4 were attenuated by anthocyanin. Colagen Ⅲ and α-SMA was significantly increased as determined by histology and anthocyanins decreased their level. The protein levels of MMP-9, TIMP1 and PCNA of liver homogenate was also modulated by anthocyanins. In isolated hepatic stellate cells, activation as determined by fibrotic gene expression was attenuated by anthocyanin. Anthocyanins from blueberry may have protective effects on CCl4 induced hepatic fibrosis. The mechanism may be related to reduce ROS generating sources and associated oxidative damage, decrease the influence of pro-inflammatory cytokines, and suppress the activity of hepatic stellate cells and downregulation TIMP1, PCNA, Col-Ⅲ, α-SMA and up-regulation MMP-9.