Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Promotes Hepatic Stellate Cells Migration via Canonical NF-κB/MMP9 Pathway.

In the liver, the signal and function of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) have mainly been assessed in association with liver regeneration. However, the effects of TWEAK on liver fibrosis have not been fully elucidated. To investigate the effects of TWEAK on human hepatic stellate cells (HSCs) and to explore the relevant potential mechanisms, human HSCs line-LX-2 were cultured with TWEAK. Cell migration was detected by transwell assay; cell viability was evaluated by Cell Counting Kit-8; the expression of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13 gene was identified by quantitative real-time polymerase chain reaction and western blotting; the activity of matrix metalloproteinases (MMPs) was tested by enzyme-linked immuno sorbent assay; small interfering RNA transfection was applied for depletion of MMP9 and p65. The result of transwell assay revealed that TWEAK promoted LX-2 migration. Subsequently, our data testified that the expression and activity of MMP9 was induced by TWEAK in LX-2 cells, which enhanced the migration. Furthermore, our findings showed that TWEAK upregulated the phosphorylation of IκBα and p65 protein to increase MMP9 expression in LX-2 cells. Meanwhile, the alpha-smooth muscle actin, vimentin and desmin expression were upregulated following TWEAK treatment. The results in the present study revealed that TWEAK promotes HSCs migration via canonical NF-κB/MMP9 pathway, which possibly provides a molecular basis targeting TWEAK for the therapy of liver fibrosis

Regulation of CD44v6 expression in gastric carcinoma by the IL-6/STAT3 signaling pathway and its clinical significance

As a cancer stem cell marker, CD44 variant 6 (CD44v6) has been implicated in carcinogenesis, tumor progression, and metastasis in a variety of human carcinomas. However, little is known about the expression of CD44v6 in Gastric Carcinoma (GC). Therefore we investigated CD44v6 expression in clinical specimen and further explore the underlying molecular mechanisms. In this study, we systemically investigated CD44v6 expression by immunohistochemistry in normal, premalignant gastric mucosa (low and high grade intraepithelial neoplasia), and GC at various stages. The correlation of CD44v6 expression with clinicopathological characteristics, and prognosis in GC was also analyzed. Next, we investigated cell proliferation, migration and invasion in GC cell lines. Furthermore, we explored a novel mechanism by which CD44V6 was upregulated in GC cell. The immunohistochemistry results showed that enhanced expression of CD44v6 was closely associated with tumor differentiation, lymph node metastasis, TNM stage and poor prognosis in GC patients. In gastric cancer cell lines, CD44v6 involved in cell proliferation, invasion and metastasis in Next, report on a novel mechanism by which interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling up-regulates expression of CD44v6. RNA interference silencing of STAT3 resulted in decrease of CD44v6 levels. We also found that STAT3 inhibitor AG490 decrease expression of CD44v6 by blocking activation of STAT3, even in the presence of IL-6. Targeting STAT3-mediated CD44v6 up-regulation may represent a novel, effective treatment by eradicating the stomach tumor microenvironment

TWEAK induced canonical NF-κB pathway activation then augmented MMP9 expression in LX-2 cells.

<p>(A) Whole cell extracts were prepared and analyzed by western blotting for the p-IκBα, IκBα and p-p65, p65 in LX-2 cells treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. β-actin served as an internal control. (B) The expression of p65 in LX-2 cells transfected with scrambled RNA (20 nM) or siRNA specific for p65 (20 nM) was examined by qRT-PCR (B) and western blotting (C). β-actin served as an internal control. (D) LX-2 cells were transfected with control siRNA (20nM) or siRNA specific for p65 (20nM) for 48 h and further incubated with TWEAK (100 ng/ml) for 24 h. The protein of p65, p-p65 and MMP9 in cell lysates were measured by western blotting. β-actin was used as a loading control. All data are represented as mean ± SD of three independent experiments. ****<i>P<</i>0.0001.</p

TWEAK significantly increased the expression of MMP7, MMP8, MMP9, MMP13 and the activity of MMP9 in LX-2 cells.

<p>(A) The mRNA expression of MMP7, MMP8, MMP9 and MMP13 were measured by qRT-PCR in LX-2 cells treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. β-actin served as an internal control. (B) The mRNA of MMP1, MMP2, MMP3, MMP10, MMP11 and MMP12 was examined by qRT-PCR in LX-2 cells treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. β-actin served as an internal control. (C) Western blotting to examine the expression of MMP7, MMP8, MMP9 and MMP13 in LX-2 cells treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. β-actin was used as a loading control. (D) Activated MMP9 expression in LX-2 cells culture medium was investigated by Elisa after being treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. (E) Activated MMP7, MMP8 and MMP13 in LX-2 cells culture medium were tested by Elisa after being treated with 40 ng/ml and 100 ng/ml TWEAK for 24 h. Results are expressed as the mean ± SD of three independent experiments. *<i>P<</i>0.05, **<i>P<</i>0.01,****<i>P<</i>0.0001.</p

List of primer sequences.

<p>List of primer sequences.</p

LX-2 cells migration was enhanced by TWEAK.

<p>(A) LX-2 cells were treated with vehicle (PBS) or 20 ng/ml, 40 ng/ml and 100 ng/ml TWEAK for 24 h, then the transwell assay was performed. The data shown here are from three independent experiments with similar results. Original magnification 200×. (B) The number of migrated cells were displayed as histogram, compared with the control group. The data were displayed as the mean value of cells in five fields based on three independent experiments. (C) LX-2 cells were treated with vehicle (PBS) or vary concentrations of TWEAK for 24 h or 48 h and assayed by CCK-8. Results are expressed as the mean ± SD of three independent experiments. **<i>P<</i>0.05, ***<i>P<</i>0.01 when compared with the control group.</p