ABSTRACT
Objective To investigate the effects of IL-1 β on proliferation and migration of gallbladder cancer cells.Methods The secretion of IL-1 β in tissues of gallbladder cancer, chronic cholecystitis and normal gallbladder as well as in supernatant of gallbladder cancer cell lines (GBC-SD, SGC996) and HIBEpic cells was determined by enzyme-linked immunosorbent assay (ELISA) method.The levels of IL-1 β mRNA in GBC-SD, SGC996 and HIBEpic cells were measured by RT-PCR assay.The effects of exogenous IL-1 β on the proliferation of GBC-SD and SGC996 cells in vitro and in vivo were evaluated using WST-1 assay and xenograft tumor model, respectively.The effects of exogenous IL-1 β on the migration of GBC-SD and SGC996 cells in vitro were measured by Tranwell assay.The levels of Twist protein in GBC-SD and SGC996 ceils were examined by western blot assay after treatment with exogenous IL-1 β.In addition, the proliferation and migration of GBC-SD and SGC996 cells after gene silencing of Twist by Twist-siRNA were also evaluated.Results The level of IL-1β protein in normal gallbladder was low (66.4 ± 35.0)pg/ml,while it was significantly increased in gallbladder cancer and chronic cholecystitis [(616.4 ± 95.7) pg/ml and (422.3 ± 48.9) pg/ml, P ＜ 0.05].The levels of IL-1 βin GBC-SD and SGC996 cell culture medium [(587.4 ± 99.8) pg/ml and (657.2 ± 76.6) pg/ml] were much higher than those in the HIBEpic cells [(38.4± 12.1)pg/ml, P ＜ 0.05].Exogenous IL-1β promoted the proliferation of GBC-SD and SGC996 cells both in vitro and in vivo as well as migration in vitro (P ＜ 0.05).The level of Twist protein was significantly increased after treatment with exogenous IL-1 β.In addition, gene silencing of Twist blocked IL-1 β-induced proliferation and migration of GBC-SD and SGC996 cells.Conclusion IL-1 β promoted proliferation and migration of gallbladder cancer cells via Twist activation.
ABSTRACT
There are multiple genes involved in the progress of evolution in breast cancer.Recent study showed that Twist gene may play key role in promoting tumor growth,invasion and metastasis through the epithelial-mesenchymal transition (EMT).Other studies also demonstrated that Twist gene expression maybe associated with patient outcomes and guiding clinical tailored therapies.
ABSTRACT
In order to investigate the role of Twist gene in the metastasis of hepatocellular carcinoma (HCC), total RNA was respectively extracted from three HCC cell strains with different metastatic potentials, HepG2, MHCC-97L and MHCC-97H. The first strand eDNA was synthesized by reverse transcription, which was then used as template to perform fluorescent quantitative polymerase chain reaction (FQ-PCR). The quantity of Twist gene expression was normalized by that of the housekeeping gene, GAPDH for each sample. ANOVA was used to estimate the relationship between Twist gene and metastasis potential of HCC. The results showed that the normalized initial eDNA concen- trations of Twist gene in HepG2, MHCC-97L and MHCC-97H were (9.45±0.25)×104, (1.82±0.41)×10-3, (3.06±0.62)×10-3, respectively. FQ-PCR revealed significant differences in the ex- pression level of Twist among HCC cell strains with different metastatic potentials. It was concluded that high expression level of Twist was closely associated with more aggressive behaviors of HCC. Twist provides a novel indicator for HCC metastasis.
ABSTRACT
Objective To construct a plasmid expression vector coding for the short hairpin RNA(shRNA) targeting Twist mRNA.Methods Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed.The recombinant plasmids were identified by PCR,and then transfected separately into bladder cancer cell line T24.The Twist gene silencing effect was detected by RT-PCR and Western blotting.Results The expected band of 400 bp was amplified from the plasmids coding for shRNA by PCR.By DNA sequencing,it was the same with the insertion element as with the shRNA of synthetic.Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in inhibition of Twist mRNA and protein expressions by 90% and 86%,respectively.The shRNA1 had the most obvious effect in Two types of plasmids interference.Conclusion The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.