Expression Of Peroxiredoxin Ⅱ In Hepatocellular Carcinoma And Its Biological Function

Objective To evaluate mRNA and protein expression of peroxiredoxinâ…¡(Prxâ…¡)in hepatocellular carcinomas(HCC)of tree shrew(Tupaia belangeri chinensis)induced by aflatoxin B₁(AFB₁)and in human HCC tissues,and to explore the function of Prxâ…¡in hepatocarcinogenesis and the possible underlying mechanism.Methods The expression levels of Prxâ…¡mRNA and protein were detected by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot on 6 cases of tree shrew HCC induced by AFB₁,their surrounding liver tissues (para-HCC)and the biopsies taken before HCC had developed(pre-HCC)from the same animals.The biopsies from 6 animals in control group were used as contemporaneous controls.The results were verified on 18 cases of human HCC sample,the corresponding para-HCC and 17 cases of human normal liver tissue,as well as on three liver cancer cell lines Hep3B,SMC7721,MHCC97L and normal liver cell line LO2.Afterward,RNA interfering(RNAi)technique was applied on cell lines Hep3B by transfecting two pairs of chemically synthesized small interfering RNA(siRNA)targeted on Prxâ…¡,Prxâ…¡-1 and Prxâ…¡-2,through Lipofectamine^TM2000.Following confirming the interfering effects of Prxâ…¡-1 and Prxâ…¡-2 by Real-Time PCR and Western blot,the biological characters of Prxâ…¡-silenced Hep3B cell were observed by MTT,colony formation and flow cytometry assays.Finally,the products of oxidative reaction such as the reactive oxygen species(ROS)and malondialdehyde(MDA)in the Prxâ…¡-silenced Hep3B cells were measured by Dichlorodihydrofluorescein diacetate(DCFH-DA)and Thibabituric acid(TBA)assays.Results Both the expression levels of Prxâ…¡mRNA and protein in tree shrew HCC tissues were higher than that in para-HCC,pre-HCC and the control tissues (P＜0.05).The expression levels of Prxâ…¡mRNA and protein were also higher in human HCC tissues than that in para-HCC and normal liver tissues(P＜0.05),as well as were higher in the three liver cancer cell lines than that in normal liver cell line.Results from Real-Time PCR showed the expression level of Prxâ…¡mRNA was reduced more than 80%in the two Prxâ…¡-silenced groups(Hep3B cells transfected with siRNA Prxâ…¡-1 or Prxâ…¡-2)than that in blank control group(Hep3B cell without transfection)or mock control group(Hep3B cell transfected with non-specific small dsRNA)after transfecting for 48 hours.The MTT results showed the cell growth inhibition rates in the two Prxâ…¡-silent groups were more than 40%after transfecting 96 hours comparing to the two control groups(P＜0.05).Result from colony formation assay showed the numbers of colony in the two Prxâ…¡-silent groups were 42.0±2.83 and 40.5±0.71 respectively,while they were 121.5±2.12 and 130.0±1.41 in the two control groups respectively(P＜0.05). Results from flow cytometry assay showed the cell apoptosis ration in the Prxâ…¡-1, Prxâ…¡-2 and mock group was 8.40%,7.59%and 5.14%respectively when transfected with the final concentration 50 nM/L,while it was 19.40%,33.25%and 5.96%respectively when with the final concentration 100 nM/L,and was 4.91% in the blank control group.The results from DCFH-DA and TBA assays showed the levels of endogenous ROS and MDA were obvious higher in the two Prxâ…¡-silent groups than that in the two control groups(P＜0.05).Conclusion The up-regulated expression of Prxâ…¡in HCC tissues may play an important role in hepatocarcinogenesis,possibly through the mechanism of antioxidation by which providing a favorable microenvironment for cancer cell surviving and progressing.Prxâ…¡might be used as a molecular target for HCC prevention and treatment.