| Hepatocellular carcinoma(HCC)is one of the common malignant tumors with the highest morbidity and mortality world wild.About 60 million new cases of HCC patients occur in the world annually and about half of them happened in China which is a serious threaten to the health,social and psychological problems of Chinese citizens.The HCC patients are often diagnosed in the late stages because the onset of the disease is often concealed and losed the effective treatment.At present,the treatments for advacend HCC patients are limited,and the multiple protein kinase inhibitor Sorafenib is one of the most effective treatment method.But both from clinical research and experience,HCC patients are commonly prone to get drug resistance during the treatment of sorafenib,which leads to treatment failure and disease progression.Sorafenib resistance has become a hot issue in the field of liver cancer treatment.Although many researches about sorafenib resistance have been conducted,but the resistance status of HCC patients have not been improved based on the results of the current study.So,more researches showed be conducted in discovering sorafenib resistance of HCC.And find the key molecules and the regulation mechanism will be beneficial for the patients with hepatocellular carcinoma.Methods: We set up hypoxia induced hepatocellular carcinoma cells by hypoxia incubator and Co Cl2 treatment,and the hypoxia status was identified by Western-blot.Subsequently,the sensitivity of hepatocellular carcinoma cells to sorafenib treatment was further detected by flow cytometry and CCK8 assays.Immunofluorescence,immunohistochemistry,Western-blot,q RT-PCR and other techniques were used in finding the key molecules and the mechanism.Subsequently,we constructed stably gain-and loss-of mitochondrial membrane protein ATAD3 A HCC cells(Huh7 and LM3)using lentivirus stable transfection,and further explored the role of ATAD3 A in sorafenib treatment by flow cytometry and CCK8 assay.At the same time,the role of ATAD3 A in the malignant biological behavior of hepatoma cells was explored through cell cycle,clone formation,CCK8 assay,and in vivo tumorigenesis.In terms of specific regulatory mechanisms,the correlation between mi R-210-5P and ATAD3 A was examined by luciferase reporter gene detection.The regulatory effect of mi R-210-5P and mitochondrial membrane protein ATAD3 A on the phosphorylation level of m TOR signaling pathway was detected by Western-blot.Results:(1)In Huh7 and LM3 cells,flow cytometry showed that Sorafenib treatment combining with hypoxia induced significantly lower proportion of apoptosis than that in normal oxygen condition.The average apoptosis rate for 96 h was 16.87%(LM3)or 18.88%(Huh7)in hypoxia microenviroment,and the average apoptosis rate under normoxic condition was 47.8%(LM3)or 48%(Huh7),the difference was significant(p<0.05).CCK8 cell proliferation assay showed that the proliferation inhibition of Huh7 and LM3 was significantly lower than that in the normal oxygen condition,and the difference was statistically significant(p<0.05).(2)Under hypoxia treatment,the expression level of hypoxia inducible factor HIF1α in liver cancer cells was obviously up-regulated,while the expression level of ATAD3 A was obviously down-regulated,and the difference was statistically significant(p<0.05).IHC staing of 85 HCC patients,the expression of mitochondrial membrane protein ATAD3 A and hypoxia inducible factor HIF1α was negatively correlated(p<0.05).We further confirmed the TCGA database that the expression of ATAD3 A was negatively correlated with HIF1α expression(r=-0.1733,P=0.0003).(3)Western-blot analysis showed that ATAD3 A has an obviously lower expression in the liver cancer tissues compared with the para-cancerous tissues;Study also showed that the expression level of ATAD3 A in normal liver cell(L02)was significantly higher than that of hepatocellular carcinoma cells(7701,Huh7,LM3,7721,Hep G2 and PLC).q RTPCR showed that the expression level of mitochondrial membrane protein ATAD3 A in normal liver cell(L02)was significantly higher than that of hepatocellular carcinoma cells(Huh7,LM3,7721,Hep G2 and PLC),and the difference was statistically significant(p<0.05).However,the expression of ATAD3 A in 7701 cells was not significantly lower than that in L02.The expression level of ATAD3 A is negatively correlated with the tumor size and histopathological classification of the HCC patients.ATAD3 A is predicted to be an important prognostic factor of liver cancer patients.The patients with higher expression of ATAD3 A has a better prognosis than the lower ones.(4)Stable gainand loss-of ATAD3 A expression Huh7 and LM3 cells were constructed.CCK8 and flow cytometry assay showed that stable gain-of ATAD3 A expression significantly increased the sensitivity of the hepatocellular carcinoma cells to sorafenib treatment,and the proportion of apoptotic cells up-regulated significantly(p<0.05);while,stable loss-of ATAD3 A expression decreased the sensitivity of the hepatocellular carcinoma cells to sorafenib treatment and the proportion of apoptotic cells significantly down-regulated in HCC cells(p<0.05).(5)Our study also showed that stable loss-of ATAD3 A expression in the LM3 and Huh7 cells increased the clone formation efficiency,the cell proliferation,cell cycle progression,the expression of cell cycle related protein Cyclin A2,Cyclin D1,CDK2,and the tumorigenesis ability.The difference was statistically significant(p<0.05).Conversely,stable-gian of ATAD3 A expression get the opposite results.(6)We have previously found that two hypoxia induced methods could obviously increase the expression of hypoxia induced mi R-210-5P.Bioinformatics analysis showed that ATAD3 A was the target gene of hypoxia induced mi R-210-5P.And we further confirmed that hypoxia induced mi R-210-5P has a direct binding target in the 3’UTR region of ATAD3 A by luciferase reporter gene assay.We then transfected hepatocellular carcinoma cells with mi R-210-5P mimic,and find that mi R-210-5P mimc could down regulate the protein and gene expression level of ATAD3 A.On the contrary,mi R-210-5P inhibitor could up-regulate the protein and gene expression level of mitochondrial membrane protein ATAD3A(p<0.05).Results also showed that the expression of ATAD3 A was significantly reduced after Co Cl2 treatment,and the effect of Co Cl2 treatment on ATAD3 A expression could be reversed by mi RNA-210-5P inhibitor(p<0.05).(7)hypoxia treatment partially reversed the effect of gain-of ATAD3 A expression on the malignant biological behavior of hepatoma cells.Flow cytometry showed that liver cancer cells with higher expression of ATAD3 A were more sensitive to sorafenib treatment.After hypoxic treatment,the sensitivity of the cells to sorafenib treatment was reduced.Except this,the proliferation,clone formation and cell cycle progression were more efficiency after hypoxia treatment compared to the higher ATAD3 A expression ones,and the difference was statistically significant(p<0.05);the expression of cell cycle related protein Cyclin A2,Cyclin D1,CDK2 was significantly decreased in the gain-of ATAD3 A expression hepatoma cells which were significantly increased after hypoxia treatment.(8)Flow cytometry showed that mi R-210-5P mimic could reduce the sensitivity of sorafenib treatment,and could partly restore the effects of gain-of ATAD3 A expression in hepatoma cells.The difference was statistically significant(p<0.05).On the contrary,the mi R-210-5P inhibitor could enhance the sensitivity of sorafenib treatment obviously.While,mi R-210-5P inhibitor has no obvious response to ATAD3 A lower expression hepatoma cells,which further indicates that mitochondrial membrane protein ATAD3 A is a direct functional target gene of hypoxia induced mi R-210-5P.(9)Western-blot showed that mi R-210-5P mimic and inhibitor were involved in regulating the phosphorylation level of m TOR.The p-m TORser2448 expression level of the hepatoma cells increased significantly after the mi R-210-5P mimic treatment,and the expression level of p-m TORser2448 in the hepatocellular carcinoma cells decreased significantly after mi R-210-5P inhibitor transfected.The expression level of p-m TORser2448 was obviously increased in the loss-of ATAD3 A expression hepatoma cell.And the expression level of pm TORser2448 was significantly decreased in the gain-of ATAD3 A expression hepatoma cells.Furthermore,our study also showed that mi R-210-5P mimc could restore the effect of ATAD3 A overexpression on the level of phosphorylation of m TOR.Conclusion:(1)Hypoxic microenvironment could induce the insensitivity of sorafenib treatment in hepatoma cells.(2)Hypoxic microenvironment can reduce the expression level of mitochondrial membrane protein ATAD3 A in hepatoma cells.(3)The expression of mitochondrial membrane protein ATAD3 A is lower in the cancer tissues and cells compared to the adjacent normal tissues and cells.The expression is correlated with the prognosis of HCC patients,the tumor size and the other clinicopathological features.(4)the lower expression of mitochondrial membrane protein ATAD3 A is closely related to the hypoxia state of hepatoma cells which is negatively correlated with the expression of HIF1α.Furthermore,hypoxia induced mi R-210-5P can directly target the 3’UTR region of the mitochondrial membrane protein ATAD3A;(5)hypoxia can partially restore the malignant biological behavior of gain-of ATAD3 A expression in hepatocellular carcinoma cells.(6)mi R-210-5P mimic can partially restore the effect of gain-of ATAD3 A expression on the malignant biological behavior of hepatoma cells,while the inhibitor of mi R-210-5P has no effect.It is further confirmed that ATAD3 A is an important functional target gene of mi R-210-5P.(7)ATAD3A is involved in regulating the activation of m TOR signaling pathway which could be regulated by hypoxia induced mi R-210-5P. |
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