The PDGF-D/miR-106a/Twist1 Pathway Orchestrates Epithelial-mesenchymal Transition In Gemcitabine Resistance Hepatoma Cells

Background and objective:Hepatocellular carcinoma(HCC) is one of the most common gastrointestinal malignances of the clinical.HCC always has high mortality with the nature of insidious onset, rapid progression and poor prognosis.Systemic chemotherapy is the important treatment method of advanced hepatocellular carcinoma, but it always failure part due to development of acquired drug resistance.In view of present situation of advanced hepatocellular carcinoma, it is urgent to investigate the molecular mechanism of drug resistance in HCC. To explore the underlying mechanism of drug resistance in HCC, we developed gemcitabine-resistant(GR) HCC cells(“Hep G2 GR”,“Huh-7 GR”).With culture the drug-resistant cells, we observed that gemcitabine-resistant cells acquired epithelial-mesenchymal transition(EMT) phenotype.The aberrant activation of platelet-derivedgrowth factor D(PDGF-D) pathway has been involved in the development of EMT.Further study demonstrate that small RNA may be involved in the regulation of PDGF-D mediated EMT. Based on the cell models built by use of gene overexpression and gene silencing technology, mi R-106 a was primarily determined to act on the PDGF-D pathway by using bio informatics analysis on mi RNA chips.The down-regulation of mi R-106 a could significantly up-regulated the expression of several target genes related to EMT. Bio informatics analysis indication that mi R-106 a target Twist1 genes mediate EMT in gemcitabine resistance of hepatoma cells.Based on these interesting facts, a hypothesis was proposed that thedown-regulation of mi R-106 a by PDGF-D contributes to the EMT in gemcitabine-resistant hepatocellular carcinoma cells.The regulating effects of PDGF-D on the expression of mi R-106 a and subsequently,mi R-106 a on the expression of its target genes and the EMT phenotype in gemcitabine-resistant cells were investigated. Moreover, the association between mi R-106 target genes and the EMT phenotype in drug resistant cells was also revealed.Methods:1.In parent and gemcitabine resistant of hepatoma cells, we ensure the results of mi RNAs chip is correct and to find the key mi RNA involve in PDGF-D-mediated EMT:Based on our previous study,we intend to further complete the model of the gemcitabine resistant cells.We use gene transfection and RNA interference methods to introduct PDGF-D over-expressing or silencing in hepatoma cells.We use a mi RNA microarray to screen the differentially expressed mi RNAs and we demonstrate mi R-106 a is the key mi RNA which may be involved in PDGF-D signal pathway.We select mi R-106 a for the subsequent research and biological information analysis testified that Twist1 may be a target gene of mi R-106 a.2.In parent and gemcitabine resistant of hepatoma cells,we investigate the the inherent relationship among PDGF-D,mi R-106 a and Twist1:The expression of mi R-106 a is measured by q RT-PCR assay and the results reveal that mi R-106 a is widely down-regulate in HCC GR cells.we use q RT-PCR and Western-Blot assay to measure the expression of PDGFD and Twist1. We analyze its inherent relationship among PDGF-D,mi R-106 aand Twist1.3.In parental hepatoma cells, we investigate the effect of PDGF-D over-expression on mi R-106 a and it,s target gene:In parental hepatoma cells, we transfect PDGF-D plasmid into HCC cells to upregulate the expression of PDGF-D.The expression level of mi R-106 a is measured by q RT-PCR method.We investigate whether PDGF-D over-expressing could downregulate the expression of mi R-106 a protein.we use q RT-PCR and Western-Blotassays to detect the expression of Twist1 and explore the effects of PDGF-D over-expression on Twist1.4.When the expression of PDGF-D is downregulate, we investigate the effects on mi R-106 a and the target gene in HCC GR cells:we transfect PDGF-D si RNA into HCC GR cells to downregulate the expression of PDGF-D. We mesaure the expression of mi R-106 a by q RT-PCR assay. When the expression of PDGF-D is down-regulate,we research wheather PDGF-D could upregulate the expression of mi R-106 a. Finally,We measure the expression of Twist1 by q RT-PCR and Western-Blot assays to explore the effects of PDGF-D-silencing on Twist1 transient.5.The effects of mi R-106 a on the expression of target gene and the biological characteristics of EMT in HCC and HCC GR cells :We trasfect mi R-106 a inhibits into HCC cells and to measure the expression of Twist1 by q RT-PCR and Western-Blot assays.we survey whether mi R-106 a could upregulate Twist1,s expression.We trasfect mi R-106 a minics into HCC GR cells and to measure the expression of Twist1 by q RT-PCR and Western-Blot assays.we survey whether mi R-106 a could downregulate Twist1,s expression.We trasfect mi R-106 a minics in HCC GR cells for24 hours and to explore The effects of mi R-106 a over-expression on the biological characteristics of EMT-cells by the morphology observation, migration or invasion assay, scratch assay, detachment and attachment assays.6. the target gene of mi R-106 a for the influence of hepatocellular carcinoma cells on it,s molecules phenotype and biological characteristics of EMT:We transfect Twist1 si RNA into HCC GR cells to down-regulate the expression of Twist1.we messure the expressions of the molecule makers of EMT,which inclusive E-caherin and Vimentin.We trasfect Twist1 si RNA into HCC GR cells for 24 h and to explore the effects of the biological characteristics of EMT cells by the morphology observation, migration or invasion assay, scratch assay,detachment and attachment assays.Results:1.HCC GR cells have EMT characteristics:We developed HCC GR(Hep G2 GR and Huh-7 GR)cells using the method of increasing concentration gradient. We use MTT,q RT-PCR,Wetern-Blot assays to identify the property of drug resistance and EMT in gemcitabine resistant hepatocellular carcinoma cells. To detect the expressions of mi RNAs and to further select differentially expressed mi RNA in HCC and HCC GR cells by a mi RNA microarray and q RT-PCR assay was used to verify the accuracy of mi RNA chips,result.2.gemcitabine resistance of hepatoma cells emerge the morphological characteristics of EMT involved in the abnormal activation of PDGF-D signaling pathway:The expressions of PDGF-D and PDGFR βare upregulated in HCC GR cells compare to HCC cells. When inhibit the expression of PDGF-D,we could overcome the molecule phenotype and biological characteristics of EMT in HCC GR cells.3.mi R-106 a orchestrates epithelial-mesenchymal transition in gemcitabine resistance hepatoma cells:mi R-106 a is extensive down-regulation in HCC GR cells compare to HCC cells.PDGF-D negatively regulates mi R-106 a,sexpression, while upregulated Twist1,s expression.4. mi R-106 a target Twist1 gene influence the EMT biological characteristics of EMT in gemcitabine resistance of hepatoma cells:mi R-106 a negatively regulates the expression of Twist1.we trasfect mi R-106 a minics to HCC GR cells and observe the reversion of biological characteristics of EMT in HCC GR cells. We observed that HCC GR Cells shape transfer to recovery of polarity, with pseudopod disappeared and composed of spindle shaped fibroblast like morphology into polygon. The migration and invasive ability of HCC GR cells are decline when we trasfect mi R-106 a mincs to it.5.Down-regulation of Twist1 reverses EMT to MET in HCC GR cells:We transfect Twist1 si RNA into HCC GR cells. Down-regulation of Twist1 reverses EMT to MET in HCC GR cells and the results from western blot and q RT-PCR showed that the expressions of epithelial markers such as E-cadherin is higher in HCC GR cells;the expression of mesenchymal markers such as Vimentin are lower in HCC GR cells. Down-regulation of Twist1 inhibit cell migratory and invasive activity in HCC GR cells.Conclusion:1.PDGF-D down-regulates the expression of mi R-106 and mi R-106 a orchestrates epithelial-mesenchymal transition in gemcitabine resistance of hepatoma cells.2. mi R-106 a target Twist1 gene influence the biological characteristics of EMT in gemcitabine resistance of hepatoma cells.3.Down-regulation of Twist1 reverses EMT to MET in HCC GR cells. Twist1mediated-EMT on the phenotype and molecular biological characteristics through the downstream gene of EMT in gemcitabine resistance of hepatoma cells.