Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism / 中华肿瘤杂志

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

The mechanisms of inhibitory effect of adenovirus-mediated wild-type PTEN gene on the proliferation in activated hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Using an adenoviral vector, the wild-type PTEN gene was transduced into activated hepatic stellate cell (HSC) cultured in vitro and cell cycle markers and were detect. Thereby, the potential mechanisms of inhibitory effect of the wild-type PTEN overexpression on the proliferation in activated HSC was investigated.</p><p><b>METHODS</b>The wild type PTEN gene was transduced into activated HSC (HSC-T6 ) cultured in vitro mediated by adenoviral vector. PTEN expression in HSC was measured by Western blot and Real-time fluorescent quantitation PCR. Flow cytometry (FCM) was then used to detect cell cycle phase of activated HSC. And the expressions of cyclinD1 and cyclin dependent kinase 4 (CDK4) in HSC were determined by Western blot.</p><p><b>RESULTS</b>The data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC cultured in vitro. The over-expression of wild type PTEN resulted in the increased number of HSC at G0/G1 phase ( P less than 0.01), and the number of HSC at S phase and G2/M phase were decreased significantly, P less than 0.01. Furthermore, there were decreased cyclinD1 and CDK4 expression in HSC infected with Ad-PTEN, P less than 0.01.</p><p><b>CONCLUSION</b>The over-expression of wild type PTEN inhibit transition of activated HSC in vitro from G1 to S phase and arrest cell cycle of them at G0/G1 phase via the down-regulated expressions of cyclinD1 and CDK4, and then inhibit HSC proliferation.</p>

The role of extracellular signal-regulated kinase in induction of apoptosis with salvia miltiorrhiza monomer IH764-3 in hepatic stellate cells / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effect of Salvia miltiorrhiza monomer IH764-3 on apoptosis in hydrogen peroxide (H2O2)-stimulated hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSCs were cultured in medium with different IH764-3 doses (10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L) and without IH764-3. Direct cell count, 3H-thymidine incorporation, Annexin-V/Propidium Iodide double-labeled flow cytometry, TUNEL and transmission electron microscopy were employed to estimate the influence of IH764-3 on proliferation and apoptosis of HSCs. The expression of extracellular signal-regulated kinase 1 (ERK1) mRNA and protein in HSCs were detected using RT-PCR and Western blot respectively.</p><p><b>RESULTS</b>It was showed that H2O2 could promote HSC proliferation. In contrast, IH764-3 at concentrations of 10 mg/L, 20 mg/L, 30 mg/L and 40 mg/L inhibited its proliferation. The inhibition rates were 7.13%, 28.36%, 53.80% and 73.10% (P < 0.01). And the inhibition rates of IH764-3 at concentrations of 30 mg/L at 12 h, 24 h and 48 h were 22.24%, 40.51% and 61.65%. Furthermore, IH764-3 could also induce the HSC apoptosis in dose-dependent an dtime-dependent manners (P < 0.01). In addition, after exposed of HSCs to IH764-3 for 24 h, ERK production decreased and ERK1 mRNA was down-regulated earlier about 2 h after exposure to IH764-3.</p><p><b>CONCLUSION</b>IH764-3 may inhibit the proliferation and induce apoptosis of HSCs in both dose-dependent and time-dependent manners, which may be related to down-regulation of ERK expression.</p>

Dynamic changes of alpha-AR, beta1-AR and beta2-AR expression during hepatic fibrogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes of a-AR, b1-AR and b2-AR expression in hepatic fibrosis.</p><p><b>METHODS</b>Rat hepatic fibrosis model was established by bile duct ligation (BDL). HE and Masson staining were used to determine hepatic fibrosis levels. Immunohistochemistry was applied to detect alpha -smooth muscle actin (alpha -SMA), a marker of hepatic stellate cell (HSC) activation; Western blot and real-time RT-PCR were used to measure the dynamic changes of alpha -AR, beta(1)-AR, beta(2)-AR expression on protein and mRNA levels, respectively, during the development of hepatic fibrosis.</p><p><b>RESULTS</b>(1) HE and Masson trichrome staining showed that the liver fibrosis models were established successfully. (2) At 1, 2, 3, 4 wk after BDL, alpha -SMA positive area density of the model group (10.58% +/- 1.75%, 24.14% +/- 2.02%, 29.74% +/- 2.59%, 34.28% +/- 2.01%) was significantly higher than that of the sham operation group (4.12% +/- 1.51%), P less than 0.01. (3) The expression of alpha -AR, beta(1)-AR, beta(2)-AR protein and mRNA was increased with the development of the hepatic fibrosis (P less than 0.05). (4) alpha -SMA expression was positively associated with alpha -AR, beta(1)-AR, beta(2)-AR, r values were 0.564, 0.753 and 0.606, respectively.</p><p><b>CONCLUSION</b>The expression of alpha -SMA is increased dramatically during the fibrosis, and is positively associated with the expression of alpha -AR, beta(1)-AR and beta(2)-AR.</p>

The influence of down-regulation of focal adhesion kinase by RNA interference on the adhesion and migration of rat hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of focal adhesion kinase (FAK) in adhesion and migration of hepatic stellate cells (HSC).</p><p><b>METHODS</b>Two recombinant plasmids expressing short hairpin RNAs (shRNAs) targeting FAK were constructed and one plasmid substantially suppressing FAK expression in HSC was selected. Real-time PCR and Western blot were used to detect the knockdown effects of FAK gene. After 48-hour treatment with FAK shRNA, toluidine blue colorimetric assay was used to detect the cell adhesion. Wound-healing assay and improved Boyden double-chamber were used to detect the cell migration induced by FN.</p><p><b>RESULTS</b>The recombinant plasmid expressing FAK shRNA was successfully constructed and transfected into HSC. Compared with the controls, the expression of FAK mRNA and protein in HSC treated with FAK shRNA was markedly down-regulated by 76.82% and 72.53%, respectively. The expression of p-FAK (Tyr397) protein was also decreased by 62.71% 48 h posttransfection. The adhesion of HSC was inhibited by 58.69% at 48 h after shRNA transfection. FAK gene silencing could also dramatically inhibit FN-stimulated HSC migration, and the cell migration distance and the cell number of crossing membrane were decreased by 58.27% and 83.70%, respectively.</p><p><b>CONCLUSIONS</b>FAK gene silencing suppresses adhesion and migration of HSC, and FAK may be a potential target for novel anti-fibrosis therapies.</p>

The effects of FRNK on expressions of MMP-2 mRNA and TIMP-2 mRNA in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effects of FAK-related non-kinase (FRNK) on expressions of type I collagen and matrix metalloproteinase-2 (MMP-2) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in rat hepatic stellate cells (HSC).</p><p><b>METHODS</b>Using in vitro cell culture technique, FRNK plasmids were transfected into HSC mediated by cationic liposome. Type I collagen synthesis capability in HSC was examined by 3H-Pro incorporation assay. The levels of FRNK in HSC were assayed by Western blot, and the expressions of MMP-2 and TIMP-2 were assayed by RT-PCR on mRNA levels.</p><p><b>RESULTS</b>The exposure of HSC to FRNK caused the expression of FRNK protein to be up-regulated, and the FRNK protein contents reached the highest point at 48 h after the transfection, P less than 0.05. The expressions of MMP-2 mRNA were up-regulated by FRNK; the expressions of TIMP-2 mRNA were down-regulated by FRNK; the ratios of MMP-2 mRNA/TIMP-2 mRNA were enhanced by FRNK.</p><p><b>CONCLUSION</b>After FRNK was transfected, the capability of type I collagen synthesis in HSC was inhibited, which may be related to the up-regulation of MMP-2 mRNA/TIMP-2 mRNA.</p>