Y-box-binding protein 1 mediates sorafenib resistance via the extracellular signal regulated-protein kinase pathway in hepatoma cells / 中华肝脏病杂志

Objective: To investigate the effect and possible mechanism of Y-box-binding protein 1 (YB-1) on sorafenib resistance in hepatoma cells. Methods: Lentiviral vectors with YB-1 overexpression and knockdown were constructed, respectively, to stimulate human hepatoma cell lines (HepG2 and Huh7) alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups: overexpression control group (Lv-NC), YB-1 overexpression group (Lv-YB-1), overexpression control combined with sorafenib resistance group (Lv-NC+sorafenib), YB-1 overexpression combined with sorafenib resistance group (Lv-YB-1 + sorafenib). The knockdown part of the experiment was also divided into four groups: knockdown control group (Lv-shNC), YB-1 knockdown group (Lv-shYB-1), knockdown control combined with sorafenib resistance group (Lv-shNC + sorafenib), YB-1 knockdown combined with sorafenib resistance group (Lv-shYB-1 + sorafenib). The occurrence of cell apoptosis was detected by TUNEL. The protein expression levels of phosphorylated (p)-ERK and ERK, key proteins in the extracellular regulatory protein kinase (ERK) signaling pathway, were detected by Western blot and quantified by ImageJ software. Subcutaneous tumorigenesis experiments were performed in nude mice. The effect of YB-1 on the efficacy of sorafenib was verified in vivo. The comparison between the two sets of data was carried out by an independent sample t-test. One-way ANOVA was used for comparisons between the three groups of data above. Results: Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells, while YB-1 overexpression had inhibited cell apoptosis, and at the same time also inhibited the apoptosis-accelerating impact of sorafenib. On the contrary, YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis. Furthermore, sorafenib resistance had down-regulated p-ERK levels (HepG2: Lv-NC 0.685 ± 0.143, Lv-NC + sorafenib 0.315 ± 0.168, P < 0.05; Huh7: Lv-NC 0.576 ± 0.078, Lv-NC + sorafenib 0.150 ± 0.131, P < 0.01), whereas YB-1 overexpression had inhibited sorafenib resistance p-ERK reduction (HepG2: Lv-NC + sorafenib 0.315 ± 0.168, Lv-YB-1 + sorafenib 0.688 ± 0.042, P < 0.05; Huh7: Lv-NC + sorafenib 0.150 ± 0.131, Lv-YB-1 + sorafenib 0.553 ± 0.041, P < 0.05). YB-1 knockdown further increased sorafenib-induced p-ERK downregulation (HepG2: Lv-shNC + sorafenib 0.911 ± 0.252, Lv-shYB-1 + sorafenib 0.500 ± 0.201, P < 0.05; Huh7: Lv-shNC + sorafenib 0.577 ± 0.082, Lv-shYB-1 + sorafenib 0.350 ± 0.143, P < 0.05), which was further verified in naked mice (Lv-shNC + sorafenib 0.812 ± 0.279, Lv-shYB-1 + sorafenib 0.352 ± 0.109, P < 0.05). Conclusion: YB-1 mediates the occurrence of sorafenib resistance via the ERK signaling pathway in hepatoma cells.

Role of MAPK signaling pathway in osteopontin-mediated ossification of the ligamentum flavum / 中国组织工程研究

BACKGROUND:Mitogen-activated protein kinase (MAPK) signaling pathway is an important signal transduction system.Our precious animal experiments have shown that osteopontin can mediate the ossification of the ligamentum flavum.OBJECTIVE:To investigate the role of MAPK signaling pathway in osteopontin-induced ossification of the ligamentum flavum.METHODS:The ligamentum flavum specimens obtained from 16 cases undergoing thoracic/lumbar posterior decompression surgery were divided into ossification and non-ossification groups (n=8 per group).The expression of osteopontin and its receptors CD44 and integrin was observed by immunohistochemical staining.The activation of phosphorylation in MAPK signaling pathway was detected by western blot assay.The MAPK signaling pathway was blocked by SB203580 or U0126 blocker alone to observe the induction of osteopontin.RESULTS AND CONCLUSION:Osteopontin and its receptor CD44 were expressed in the ossification group,but not in the non-ossififcation group.However,the expression of integrin was not detected in the ossification group.The expression levels of alkaline phosphatase and osteocalcin in the ligamentum flavum were significnatly increased under the induction of osteopontin (P ＜ 0.05),and osteopontin could activate the phosphorylation of P38 and ERK1/2 in the MAPK signaling pathway (P ＜ 0.05),but the phosphorylation of JNK was not obvious.p38 phosphorylation blocked with SB203580 blocker could significantly inhibit the osteopontin-induced osteoblast differentiation of ligament flavum cells (P ＜ 0.05),while U0126 blocker had no obvious effect.These results indicate that p38 in MAPK signaling pathway is a key molecule in osteopontin-mediated ossification of the ligamentum flavum.

Effects of CDH1 gene promoter methylation on expression of E-cadherin and beta-catenin and its clinicopathological significance in colon carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the relationship between methylation of the CDH1 gene promoter on the expression of E-cadherin and β-catenin, and to evaluate the correlation with clinicopathological characteristics of the colonic carcinoma.</p><p><b>METHODS</b>Methylation specific PCR (MSP) was used to detect CDH1 gene promoter methylation in the cancer tissue, adjacent tissues and normal tissues in 68 patients. The expression of E-cadherin and β-catenin was determined by immunohistochemistry staining.</p><p><b>RESULTS</b>The positive rate of CDH1 gene promoter methylation was 32.4% in adjacent tissues and 57.4% in cancer tissue, while no detectable methylation was found in all the normal tissues. The difference was statistically significant. The positive rate of E-cadherin was 92.6% in the normal tissues, 66.2% in the adjacent tissues and 44.1% in the cancer tissues. In all normal tissues, β-catenin was expressed only at the cellular membrane but not in the cytosol or nucleus, while the expression of β-catenin was present in the cytosol or nucleus in 29.4% of the adjacent tissues and 50.0% of the cancer tissues. The positive rate of CDH1 gene promoter methylation was negatively correlated with E-cadherin expression(r=-0.312, P=0.01) and positively correlated with β-catenin cytosolic/nucleus expression(r=0.309, P=0.018). The differentiation and metastasis of colonic carcinoma were associated with the aberrant expression of E-cadherin, β-catenin, and methylation of CDH1 promoter (P<0.05).</p><p><b>CONCLUSION</b>CDH1 gene promoter methylation may lead to aberrant expression of E-cadherin and β-catenin in colonic carcinoma, and may play an important role in promoting the invasion of tumor.</p>