Urolithin A mediates p38/MAPK pathway to inhibit osteoclast activity / 中国组织工程研究

BACKGROUND:Overactive osteoclasts disrupt bone homeostasis and play a bad role in the pathological mechanisms of related skeletal diseases,such as osteoporosis,fragility fractures,and osteoarthritis.Studies have confirmed that ellagic acid and ellagtannin have the potential to inhibit osteoclast differentiation.As their natural metabolites,urolithin A has antioxidant,anti-inflammatory,anti-proliferative and anti-cancer effects,but its effect on osteoclast differentiation and its underlying molecular mechanisms remain unclear. OBJECTIVE:To explore the effect of urolithin A on osteoclast differentiation induced by receptor activator for nuclear factor-κB ligand and its mechanism. METHODS:Mouse mononuclear macrophage leukemia cells(RAW264.7)that grew stably were cultured in vitro.Toxicity of urolithin A(0,0.1,0.5,1.5,2.5 μmol/L)to RAW264.7 cells were detected by cytotoxic MTS assay to screen out the safe concentration.Different concentrations of urolithin A were used again to intervene with receptor activator for nuclear factor-κB ligand-induced differentiation of RAW264.7 cells in vitro.Then,tartrate-resistant acid phosphatase staining and F-actin ring and nucleus staining were performed to observe its effect on the formation and function of osteoclasts.Finally,the expressions of urolithin A on upstream and downstream genes and proteins in the MAPK signaling pathway were observed by western blot and RT-qPCR assays. RESULTS AND CONCLUSION:Urolithin A inhibited osteoclast differentiation and F-actin ring formation in a concentration-dependent manner and 2.5 μmol/L had the strongest inhibitory effect.Urolithin A inhibited the mRNA expression of Nfatc1,Ctsk,Mmp9 and Atp6v0d2 and the protein synthesis of Nfatc1 and Ctsk,related to osteoclast formation and bone resorption.Urolithin A inhibited the activity of osteoclasts by downregulating the phosphorylation of p38 protein to inhibit the mitogen-activated protein kinase signaling pathway.

Biological function and molecular mechanism of URI in HepG2 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells.</p><p><b>METHODS</b>The cDNA sequence and shRNA of URI were obtained and sub-cloned into eukaryotic expression vectors. Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line. The cell proliferation and anchor-independent growth in URI-overexpressing and knockdown HepG2 cells were determined by CCK-8 and soft agar colony assay. Flow cytometry was applied to detect the cell cycle and apoptosis of γ-ray irradiated cells. Apoptosis related genes were detected by Western blot.</p><p><b>RESULTS</b>The pCDNA3.1-URI and pGPU6-URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained. Cell proliferation rates of the HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were (588.78 ± 32.12)%, (959.33 ± 58.8)% and (393.93 ± 39.7)%, respectively (P < 0.05). The number of cell clones of HepG2, pCDNA3.1-URI-HepG2 and pGPU6-URIi-HepG2 cells were 43 ± 7, 85 ± 5 and 20 ± 4 (P < 0.05), respectively. After γ-ray irradiation, the URI-overexpressing cell line showed a significantly lower apoptosis rate and G(2)/M phase arrest than those in the URI-depleted cell line (P < 0.05). In the HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.92 ± 0.03, 1.11 ± 0.13 and 0.82 ± 0.01 (P < 0.05). In the pCDNA3.1-URI-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 1.79 ± 0.12, 0.48 ± 0.01 and 2.20 ± 0.30 (P < 0.05), respectively. In the pGPU6-URIi-HepG2 cells, the relative protein expression levels of URI, Bax and Bcl-2 were 0.50 ± 0.04, 1.52 ± 0.20 and 0.38 ± 0.01 (P < 0.05), respectively. The expression of Bax was down-regulated and Bcl-2 was up-regulated in the URI-overexpressing cell line. However, on the contrary, expression of Bax was up-regulated and Bcl-2 was down-regulated in the URI-depleted cell line.</p><p><b>CONCLUSIONS</b>URI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro. After the impairment of URI expression, the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist γ-ray irradiation is reduced. URI may become a potential new target for cancer therapy of hepatocellular carcinoma.</p>

Effects of silencing RPB5-mediating protein (RMP) gene on cell proliferation and migration of liver cancer SMMC-7721 cells / 肿瘤

Objective:To establish the RPB5-mediating protein (RMP)-silenced stable cell lines and study the inhibitory effects of small interfering RNA (siRNA) targeting RMP gene on the proliferation and migration of human hepatoma SMMC-7721 cells. Methods:Three RMPi siRNAs were designed and synthesized in vitro and transfected into SMMC-7721 cells. The inhibitory effect of siRNA on RMP gene expression was measured by RT-PCR to select the best siRNA. The expression vector pGPU6-Neo-RMP-484 was transfected into SMMC-7721 cells by the lipofectamine and the cells stably expressing the siRNA were selected by G418. RT-PCR was used to detect the interference efficacy against RMP gene. Cell proliferation and adhesion were measured by MTT assay. Wound healing test was used to observe the migration ability of cells. Results:The SMMC-7721 cell lines with down-regulated RMP expression were established by using RNA interference technology. Compared with the negative control cells, expression of RMP mRNA was down-regulated by(83.67±2.56)% .The proliferation of stable-transfected cells was inhibited by(74.33±0.58)% . The adhesion capability of stable-transfected cells was enhanced but the migration capacity was decreased compared with the negative control cells. Conclusion:The pGPU6-Neo-RMP-484 cell lines with stable transfection of RMP siRNA recombinant vector are successfully screened,which can be used as a cellular model for studying the molecular mechanism of RMP. Down-regulation of RMP gene expression can effectively inhibit the proliferation, enhance the adhesion, and decrease the migration of SMMC-7721 cells.