Repair Founction Of ALR To Damaged Mitochondriaand The Regulatory Mechanism Of Nrf2 In Hypoxia

Objective: Hepatocellular carcinoma(HCC)is one of the most common malignancies and is the second leading cause of cancer-related mortalities worldwide.It seriously endangers the human public health.Among the risk factors of HCC,oxidative stress is the main factor of liver damage gradually evolved into hepatocellular carcinoma.HCC is characterized by excessive proliferation of cells.Rapid growth leads to hypoxia in tumor tissue,thus reducing the imbalance of REDOX state in HCC cells.Mitochondria,as an important target cell of oxidative stress,it's founction may be changed or even be damaged in this state.Studies have shown that the hypoxia cells relies on its own growing antioxidant active factors to counteract oxygen deprivation.ALR(augmenter of liver regeneration)can protect mitochondrial by regulating oxidative stress and apoptotic gene expression,due to it's characteristics of sulfhydryl oxidase.Nrf2(nuclear factor erythroid-2 related factor 2)is an important transcription factor regulating the oxidative stress response,which can significantly induce the endogenous antioxidant response of the organism.Both of them play an important role in resisting oxidative damage induced by extraneous chemicals and maintaining the normal oxidation and equilibrium in body cells.It is suggested that Nrf2 signaling pathway can up-regulate the expression of downstream antioxidant proteins and detoxification enzymes,ALR may be regulated by Nrf2.But there is no convincing evidence of a link between them.We intend to find out the regulatory mechanism between ALR and Nrf2 to further study the role of ALR in repairing damaged mitochondria.Methods: The Nrf2 overexpression system and interference system were constructed respectively by pAd Easy-1 and RNAi technology.ALR in PLC cells were detected in normal oxygen state after infected with Nrf2 overexpressed adenovirus and Nrf2-siRNA with qRT-PCR(Quantitative reverse transcription–polymerase chain reaction)and WB(Western blot).Cobalt chloride was used to establish the hypoxia model in PLC cells,and trigonelline(trigl)was used to block the transposition of Nrf2 in hypoxia.The localization and expression of Nrf2 and ALR in hypoxia were detected by qRT-PCR,WB and immunofluorescence technique(IF)to determine whether ALR is regulated by Nrf2.After PLC cells were added with cobalt chloride and trigonelline,the damage of mitochondria was observed by ATP detection,mitochondrial membrane potential and transmission electron microscope.After that,PBud CE4.1 vector was used to construct 23 KDa ALR overexpressionplasmid.The ALR overexpression plasmid and control plasmid were transferred into PLC cells separately which mitochondrial were already damaged.ATP detection,mitochondrial membrane potential detection,transmission electron microscope scanning and WB with mitochondrial related proteins: CytC(Cytochrome),AIF(apoptosis-inducing factor),Bcl-2(B-cell lymphoma-2),Bax(BCL2-Associated X Protein)and mtTFA(mitochondria transcription factor A)were performed.Qrt-pcr detection of mRNA expression of mt TFA gene was performed by qRT-PCR.The function of mitochondria was compared in the two groups,and whether ALR overexpression plasmid has the effect of repairing damaged mitochondria.Results: The recombinant adenovirus Ad-Nrf2 and Nrf2-sirna were successfully constructed.Nrf2 mRNA level and protein expression were enhanced/inhibited after verification by qRT-PCR and WB,whereas the mRNA level and protein expression of ALR did not change correspondingly.Cobalt chloride was used to induce hypoxia in PLC cells,and through the concentration screening and the effective expression of HIF-1?(Hypoxia inducible factor-1?),the hypoxia model of PLC cells was successfully constructed.On this basis,after detection with WB and IF,the expression of Nrf2 was enhanced and Nrf2 was transferred from cell cytoplasm into cell nucleus.After added trigonelline in hypoxia PLC cells,the expression of Nrf2 protein in the cytoplasm was higher than that in thenucleus,Nrf2 was still located in cytoplasm observed by IF.The results showed that transposition of Nrf2 was successfully blocked in hypoxia by trigonelline.Accordingly,the mRNA levels of ALR and protein expression were enhanced in hypoxia PLC cells.The enhancement was more obvious in the hypoxia/Ad-Nrf2 group.In the hypoxia/trigl group,ALR mRNA levels and protein expression were decreased,which indicated that the regulation of ALR was blocked accompanied by blocked Nrf2 translocation,so the expression of ALR gene was inhibited.Comparing the functional indexes of mitochondria in hypoxia group and hypoxia /trigl group of PLC cells,the ATP level and membrane potential in hypoxia/trigl group were lower than hypoxia group.Transmission electron microscopy also found that The damage of mitochondrial morphology in hypoxia/trigl group was more serious observed by transmission electron microscope.Inhibition the transposition of Nrf2 caused cells to lacked the protection of Nrf2 against oxidative stress,thus the damage of mitochondria was more serious.After the effectiveness of the ALR overexpression plasmid(pBudCE-ALR)was verified,pBudCE-ALR and pBudCE(con)were transferred into the hypoxia/trigl group separately.It was found that ATP level and membrane potential in hypoxia/trigl/ALR group were higher than hypoxia/trigl/con group,the difference was statistically significant(P<0.001).Compared with hypoxia/trigl/con group,in hypoxia/trigl/ALRgroup,the protein expression of CytC and AIF were increased in mitochondria,the expression of Bcl-2 and mtTFA protein were increased,and Bax protein expression was decreased,the differences were statistically significant(P<0.001).The mRNA level of mtTFA also was increased.Compared with hypoxia/trigl/con group,the mitochondrial morphology in hypoxia/trigl/ALR group showed that swelling of mitochondria and cristae fractured were improved.All the above experiments showed that ALR can repair the damaged mitochondria.Conclusions: In the stress state of hypoxia,the regulation of ALR gene was initiated after Nrf2 transposed into nucleus,both of them played the role of anti-oxidative stress.The inhibition of Nrf2 translocation in hypoxia state,caused the decrease of ALR protein expression,and the mitochondria were hited harder by oxidation.23 KDa ALR can not only protect the mitochondria under oxidative stress,but also can restore the damaged mitochondria,these results provide new evidence for ALR involved in the development of HCC cells.