For this reason, several of the miRNAs previ ously identified as being associat

Characteristic down regulation of E cadherin is regarded as the key step to EMT. HCCs with EMT features consistently exhibit more venous invasion, metastases, and a poorer prognosis than those without EMT characteristics. Whether insufficient RFA directly induces the EMT of residual HCC cells and further promotes the metastasis remains unclear. In the present study, we investigated the Amuvatinib 分子量 morpho logical changes, cell growth, migration and invasion of HCC cell lines after insufficient RFA in vitro. Furthermore, we analyzed the changes of epithelial and mesenchymal markers, and Akt and ERK12 signaling pathways involved in the process in HCC cells after insufficient RFA. We also performed in vivo experiments to study the growth and metastasis of HCC cells after insufficient RFA in a BALBc nunu mice model.<br><br> Methods Cell culture Established human HCC cell lines, SMMC7721 and Huh7 were from the American Type AT-406 chemical 構造 Culture Collection. All cells were maintained in high glucose Dulbeccos modified Eagle medium supplement with 10% fetal bovine serum, 100 Uml penicillin and 100 ugml streptomycin in a humidi fied atmosphere of 5% CO2 at 37 C. Chemicals and antibodies LY294002 and PD98059 were purchased from Beyotime. Antibodies with specificity for the phos phorylated forms of Akt and ERK12 were purchased from Cell signaling. Antibodies recognizing E cadherin, N cadherin, vimentin, snail and SMA were bought from Abcam. Antibodies recognizing B actin, MMP 2 and MMP 9 antibodies were obtained from Santa Cruz. Heat treatment Insufficient RFA was simulated in vitro as described be fore.<br><br> Briefly, SMMC7721 or Huh7 cells were seeded into the 6 well plates. After 24 h, the plates were sealed and submerged in a water bath AG-490 分子量 set to 47 C for 5 min. Thereafter, cells were allowed to recover, and when the surviving populations reached 80% conflu ence, cells were propagated into the 6 well plates and exposed to above heat treatment for 10 min. Then the process was repeated and cells were sequentially exposed to above heat treatment for 15 min, 20 min and 25 min. Cells survived from the treatment were designated as SMMC7721 H and Huh7 H respectively. The morpho logical characteristics of HCC cells were observed by microscopy. Proliferation assay Cell proliferation was analyzed using the 3 2, 5 diphenyltetrazolium bromide assay.<br><br> Briefly, HCC cells were cultured in 96 well plates at a concentration of 3 103 cellswell, and incu bated for 24 h, 48 h, or 72 h. MTT solution was added to each well at a final concentration of 0. 5 mgml and incubated for 4 h. At the end of incubation, formazan crystals resulting from MTT reduction were dissolved by addition of 150 ul dimethyl sulfoxide per well. The ab sorbance was measured at 570 nm using an automated ELISA plate reader. Colony formation assay HCC cells were seeded into 6 well dishes at a concen tration of 1 103 cellswell and allowed to grow in complete medium for 2 weeks. The colonies obtained were washed with PBS and fixed in 4% paraformalde hyde for 20 min at room temperature and then washed with PBS followed by staining with crystal violet.