The Correlation Research Of Clonorchis Sinensis Severin In The Apoptosis, Migration And Invasion Of HCC/CCA Cells

Clonorchiosis is a food-borne parasitic diseases which effect on human health seriously caused by Clonorchis sinensis(C. sinensis,Cs).After chronic infections recuurent, the adult of C. sinensis cause mechanical obstruction in the bile duct, the mechanical stimulation and the excretory/secretory products of C. sinensis can cause varities of immune pathological reactions such as secondary bacterial infectionand toxic effect. It is also cause hepatic duct disease,such as biliary obstruction,cholangitis, lithogenesis, liver abscess and liver fibrosis, lead to hepatocellular carcinoma(HCC) and cholangiocarcinoma(CCA) eventually.Although the relationship between clonorchiosis and HCC/CCA has been attention during the 20 th century,the molecular mechanism of the HCC/CCA caused by C. sinensis remains unclear.The present study suggested that HCC/CCA caused by C. sinensis mainly related to the mechanical stimulation of C. sinensis,the excretory/secretory products(ESP),inflammatory response and gene modification.Our laboratory previous study had showed that Csseverin is a component of Cs ESPs, Csseverin could cause apoptotic inhibition in human HCC cell line PLC cellswhich provided a new clue for the molecular mechanism of the HCC/CCA caused by C. sinensis.Thus we need to further investigate the effects of Csseverin on development of HCC/CCA caused by C. sinensison the basis of the previous studies.In this research, on the one hand, we create stably Csseverin-Overexpressing PLC Cells, our findings indicated that Csseverin can inhibit the mitochondrial-mediated pathway such as the MPTP opening and the release of Cyt c,and the pathway also associates with the Ca2+-binding function of Csseverin; On the other hand, we use the prokaryotic expression to obtain purifiedr Csseverin protein,different dosages of r Csseverinincubation with CCA cells, the experiment confirmed that Csseverin can regulate the formation of cell pseudopodia, further promote the migration and invasion ability of CCA cells.To investigate the effect of Csseverin in the apoptosis?migration and invasion of HCC/ CCA cells, can lay a foundation for further study to thepathogenesis of C.sinensis.ObjectivesTo explore the effects of Csseverin in the process of HCC/CCA caused by Clonorchis sinensis.Methods1. Use the lentiviral expression vector, create stably Csseverin-Overexpressing HCC Cells(PEZ-LV203-Cssevein PLC) and control HCC Cells(PEZ-LV203PLC).The p EZ-LV203-Csseverin and PEZ-LV203 vector were cotransfected into 293 T cells with the Lentivirus packaging plasmid and Endo Fectin TM Lenti transfection reagent,wecollected the virus after 48 h cultivate at 37 �C, 5% CO2.Then we use the virus infected PLC cells and screening transfectants by 3?g/mlpuromycin for 1-2weeks. The GFP green fluorescence in the cells were observed by fluorescence microscope after selected the monoclone and amplificate.2.Verify the expression of intracellular Csseverin geneThe p EZ-LV203-Cssevein PLC and p EZ-LV203 PLC were assayed for Csseverin protein expression by western blotting assay.3.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis :detect the related indicators of apoptosisIX p EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control, the early apoptosis rate was assessed by flow cytometry using Annexin PE/7-AAD; the changes in the activities of initiator caspase(caspase-9) and effector caspase(caspase-3) were detected by Western blotting analysis; we fractionated the cytosol and mitochondria fractions, the shift in Cyt c from the mitochondria and Bax from the cytosol were assessed by Western blotting analysis.4.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis : measure the intracellular Ca2+p EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control, the intracellular Ca2+concentration was estimated by co-incubating the cells with Rhod-2/AM and evaluated by laser scanning confocal microscope.5.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis : assess mitochondrial permeability transition pore(MPTP) changes p EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control,quantitative changes of MPTP were measured by flow cytometry and fluorescence microscope with TMRM probe.6.Prokaryotic expression and purification of r CsseverinAfter identified the Csseverin-p ET28a(+) by sequencing, the recombinant plasmid was transformed into E.coli BL21 and induced by induced by IPTG. Then we collected the supernatant of r Csseverin and detected the protein bands by 12 %SDS-PAGE, the recombinant protein was purified with His Bind Purification kit.7.The effect of r Csseverin on the CCA cellsAfter remover the endotoxin of the recombinant protein(r Csseverin), different concentrations(1, 2, 4 ?g/ml) of r Csseverin were incubated with CCA cells(FRH-0201 and RBE), PBS was used as control. We explore the effect of r Csseverin on the migration and invasion of CCA cells through the wound healing assay and transwell chamber.8.The effect of r Csseverin on the formation of CCA cell invadedopodiaOptimal concentration of r Csseverin were incubated with FRH-0201 and RBE as base with the matrigel, PBS was used as control. We explore the effect of r Csseverin on the morphology and degradation to matrix of invadedopodia by scanning electron microscopy; we observe the effects of Csseverin on the morphology of invadedopodia in CCA cell by laser confocal microscope with phalloidin stain.Results1.Use the lentiviral expression vector, create stably Csseverin-Overexpressing HCC Cells(PEZ-LV203-Csseverin PLC) and control HCC Cells(PEZ-LV203PLC).After selected the monoclone and amplificate, we canobservethe green fluorescence protein(GFP) by fluorescent microscope, indicate that PLC cells were infected with virussuccessful.2.Verify the expression of intracellular Csseverin geneWestern blotting showed that compared to the p EZ-LV203 PLC cell, the expression of Csseverin was upregulated in the p EZ-LV203-Csseverin PLC cell, the result indicate that we create stably Csseverin-Overexpressing PLC Cells.3.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis :detect the related indicators of apoptosisp EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control. Flow cytometry showed that the apoptotic ratio ofp EZ-LV203-Csseverin PLC ? p EZ-LV203 PLCand PLC cells were 9.85% ? 32.10%and 34.51%,respectively. Compared to the control cells(PEZ-LV203 PLC and PLC), the apoptotic ratio of experimental cells(p EZ-Lv203-Severin PLC) decreased significantly; Western blotting showed that compared to the control cells(PEZ-LV203 PLC and PLC),the Csseverin overexpression PLC cells(PEZ-LV203-Cssevein PLC) decreased the amounts expression of cleaved caspase-9 and cleaved caspase-3; subcellular fractionation was performed to examine the Baxand Cyt c levels in both cytosolic and mitochondrialcompartments, Western blotting analysis showed that compared PEZ-LV203 PLC and PLC, Csseverin overexpression PLC cells(PEZ-LV203-Cssevein PLC) showed a drastic reduction in the trantslocation of Bax to mitochondria, with a significantly downregulated the release of cytochrome c from the mitochondria.4.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis : measure the intracellular Ca2+p EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control. Cells were stained with fluorescent probe dihydrorhod-2 AM(Rhod-2 AM) for intracellular free calcium.Laser scanning confocal microscope showed that compared with control group(PEZ-LV203 PLC and PLC), overexpression of the Csseverin(PEZ-LV203-Cssevein PLC) signifiantly reduced the raise of intracellular free Ca2+.5.p EZ-LV203-Csseverin PLC, p EZ-LV203 PLC and PLC were induced spontaneous apoptosis: assess mitochondrial permeability transition pore(MPTP) changesp EZ-LV203-Cssevein PLC cells were pretreated by serum-starved for 48 h,PEZ-LV203 PLC and PLC cells were used as control. Cells were stained with TMRMin the dark. Flow cytometry andfluorescence microscope showed that compared with control group(PEZ-LV203 PLC and PLC), red fluorescence intensity was significantly enhanced in Csseverin overexpression PLC cells(PEZ-LV203-Csseverin PLC), suggesting decreased MPTP opening6.Prokaryotic expression and purification of r CsseverinAfter identified the Csseverin-p ET28a(+) by sequencing, the purifiedr Csseverininduced by IPTG and purified with His Bind Purification kit. The purified recombinant protein showed a single band around43 k Dain 12 % SDS-PAGE.7.The effect of r Csseverin on the CCA cellsDifferent concentrations(1, 2, 4 ?g/ml) of r Csseverin were incubated with CCA cells(FRH-0201 and RBE), PBS was used as control. The wound healing assay showed that, at 0h, the distance of groups were almost equal, at 24 h and 48 h,compared with PBS group, with the increase of r Csseverin protein concentration, the distanceof cells were narrowed, the migration rate of cells were increased gradually.Moreover, transwell migration and invasion experiment results show that compared with PBS group, with the increase of r Csseverin protein concentration, the perforated cell number of cells wereincreased significantly.8.The effect of r Csseverin on the formation of CCA cell invadedopodiaOptimal concentration of r Csseverin were incubated with FRH-0201 and RBE as base with the matrigel, PBS was used as control. Scanning electron microscopy showed that compared with PBS group, the invadedopodia of r Csseverin group cells were greater, and the degradation to matrix were obvious. Laser confocal microscope with phalloidin stain observed that compared with PBS group, the invadedopodia of r Csseverin group cells were gathered obviously, extended outward.Conclusions1. Our study is the first to have examinedthe potential mechanism by which Csseverindecrease the intracellular Ca2+ level of HCC PLC cell result in the suppression ofMPTP opening. Moreover, there is a drastic reduction in the mitochondrialtranslocate of Bax and release of Cyt c from mitochondrial to cytoplasmic.Furthermore, Csseverin inhibit the downstream caspase cascade to protect fromapoptosis.2. After incubated with CCA cells(FRH-0201 and RBE),wedemonstrated thatCsseverin could promote the migration and invasion ability of CCA cells throughregulating the formation of cell pseudopodia.In conclusion, our present findings suggest that Csseverin can regulate the apoptosis?migration and invasion of HCC/ CCA cells. Csseverin might be involved in the process of HCC patients by C. sinensis infestation.