Research On An Effect Of Specific DcR3 SiRNA On Hepatocellular Carcinoma Cell Line

Background and ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors in China and the morbidity is increasing year by year,which severely harms the health of people.Apoptosis is one of the paths to remove the malignat cells,but the tumor can express the decoy receptors of death receptors to get a capacity of anti-apoptosis.Decoy receptor 3(DcR3),which is a newly identified member of the tumor necrosis factor receptor(TNFR)super-family,has a positive role in tumor development by inhibiting the FasL,LIGHT and TL1A's effect on inducing apoptosis and immune regulation.There were some reports that DcR3 was overexpressed in HCC tissue,its overexpression inhibited the cell apoptosis and had a relationship with the TNM staging,infiltration or metastasis of the tumor.RNA interference(RNAi)is the process of sequence-specific, post-transcriptional gene silencing in animals and plants,initiated by double-stranded RNA(dsRNA)which is homologous in sequence to the silenced gene.The mediators of sequence-specific messenger RNA degradation are 21-25 nucleotide small interfering RNAs(siRNAs) generated by dsRNA specific endonuclease(Dicer)cleavage from longer dsRNAs,siRNAs,which are easy to uptake by cells,are more specifical, stable,and their fuctions on gene silencing are more potent than other technologies of gene silencing.Therefore,RNAi technology is widely used for study of gene function.In the present study,DcR3 RNAi technology was used to explore the effects on expression of DcR3, proliferation,apoptosis and movement in human hepatocellular carcinoma cell line HepG2.Materials and Methods1.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression of DcR3 in human hepatocellular carcinoma cell line HepG2,liver cell line of para- tumor tissues QSG-7701 and normal liver cell line HL-7702.2.Lipofectamine^TM2000 mediated transient transfection was used to transmit DcR3 siRNA into HepG2 cells,then detected the transfection efficiency with fluorescence microscope and the flow cytometry.3.Semi-quantitive reverse transcription-polymerase chain reaction was used to select a specific siRNA with the best interfering effect.Immunocyto-chemistry method was used to assess the expression of DcR3 protein.4.The inhibiting rate of Proliferation in HepG2 cells was tested by MTT assay.5.The Cell cycle of cells was analysed by the flow cytometry(FCM).6.The apoptosis was explored by FCM and DNA ladder.7.The transferring ability was investigated by the wound healing test. Results1.Human hepatocellular carcinoma cell lines HepG2 over-expressed DcR3 mRNA,while QSG-7701 and HL-7702 did not.2.Lipofectamine^TM2000 could efficiently to transmit the siRNA into HepG2 cells,the transfection efficiency was about 85%.3.The 4 specific siRNAs targeting DcR3 all decreased the expression of DcR3 mRNA,DcR3 siRNA4 could decrease the mRNA expression by 62.9%,siRNA1,siRNA2,siRNA3 decreased the mRNA expression by 60.7%,32.1%,23.0%respectively,which was significant lower than that untransfeced group(P＜0.05).So,we used transfected with DcR3 siRNA4 as spesific interfrence group. Immunocytochemistry result revealled that the expression of DcR3 protein in spesific interfrence group was highly weaken than that in the untransfected group,the group transfected with liposome and the group transfected with nonspecific siRNA(P＜0.01).4.After transfection 24h,48h and 96h,MTT assay revealled that the value of the absorbance in spesific interfrence group was much lower than the untransfeced group,the group transfected with liposome and the group transfected with nonspecific siRNA(P＜0.001), the inhibition rate of proliferation in 24h,48h and 96h was 20.71%,35.00%and 34.04%respectively.5.Compared with the untransfeced group,the group transfected with liposome and the group transfected with nonspecific siRNA,the rate of the cell in G1-phase in the spesific interference group was increasing while the rate in the G2/M-phase was decreasing(P＜0.01).6.FCM showed the apoptosis rate in the spesific interfrence group was higher than other groups(P＜0.001);the spesific interfrence group's positive rate in DNA ladder experiment was 83.3%,higher than other groups(P＜0.01)7.The relative mobility of the spesific interfrence group after transfection 24h,48h and 96h was all smaller than other groups (P＜0.001).ConclusionsDcR3 siRNA could efficiently supress DcR3 gene expression, arrest the cell cycle in G1/S-phase,inhibite the cell proliferation, induce the tumor cells apoptosis and repress the transferring ability in human hepatocellular carcinoma cell lines HepG2.The results indicate that.DcR3 gene plays an important role in progression and metastasis of HCC.