Tumor-suppressing Effects Of ATF4and MiR-4458on Human Hepatocellular Carcinoma And Its’ Preliminary Underlying Molecular Mechanism

Background&objectiveHepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is the sixth most common cancer worldwide and the third leading cause of cancer related death. The difficulty to diagnose early cancer stages, the aggressive behaviors of HCC, and the poor effectiveness of therapeutic treatments, represent the reasons for the quite similar deaths per year and incidence number. Considering the fact that the diagnosis of HCC typically occurs in the advanced stages of the disease when the therapeutic options have only modest efficacy, the possibility to identify early diagnostic markers could be of significant benefit. So far, a large number of biomarkers have been associated to HCC progression and aggressiveness, but many of them turned out not to be of practical utility. This is the reason why active investigations are ongoing in this field. This is the reason why the scientific community is actively investigating this field as demonstrated by the number of papers published (740items in PubMed using the key words:"hepatocellular carcinoma" and "marker" in2012and2766items this year).Transcription is one of the most important life processes, transcription factors play an important role in this process.The activating transcription factor (ATF) belongs to the family of basic zipper-containing proteins. A variety of micro-environments stress can cause ATF4upregulated, including amino acids depletion, oxidative stress and endoplasmic reticulum stress. Expression of ATF4and induction of ISR colocalizes with hypoxic areas in human solid tumors and in tumors derived from transformed MEFs. In some ER stress-sensitive cells UPR rapidly induces apoptotic cell death involving caspase-dependent apoptosis, caspase-independent necrosis and/or autophagy. In contrast, the prosurvival measures activated by PERK and ATF4may be important for maintenance of solid tumors. But the role of ATF4in tumors, particularly with the survival of HCC patients, prognosis of HCC and related molecular mechanism is not clear. MicroRNAs are small (approx18-25nt) non-coding double-stranded RNAs with the capacity to regulate the expression of target genes mainly by impairing mRNA translation, but also by inducing mRNA degradation. In the last several years the role of miRNAs in HCC development has become evident. Due to their capacities to control gene expression behaving like tumor suppressors or oncogenes, miRNAs are considered to play a crucial role in carcinogenesis. The key role of many miRNAs in the control of cell proliferation,apoptosis and invasion underlies the relevance of their de-regulation in promoting cancer.Our research studied the relationship of ATF4and miRNA4458with the survival of HCC patients, we also studied the correlation of prognosis of HCC patients and ATF4and miRNA4458. We wish our study may afford new potential predictors of HCC prognosis and prospective therapeutic approach for HCC.Methods:The expression of ATF4was assayed by RT-PCR. Experimental determination of ATF4protein in tissue microarray was assayed by immunohistochemistry. The expression of miR-4458was up-regulated by miR-4458mimics transfection, or down-regulated by miR-4458ASO transfection. Cell proliferation was assayed by MTT assay. MiRNAs and mRNA expression were assayed by qRT-PCR. These potential targeted genes of SMiR-4458were predicated by bioinformatic algorithm. Dual Luciferase reporter assay system was used to analyze the interaction between miR-4458and IKBKE. IKBKE protein level was assayed by Western blot. The role of miR-4458or IKBKE in the survival of HCC patients was revealed by Kaplan-Meier plot of overall survival.Results:Lower ATF4expression level in HCC tissues correlated with worse prognosis of HCC patients.Lower miR-4458expression level or higher IKBKE level in HCC tissues correlated with worse prognosis of HCC patients. MiR-4458mimics inhibited the HCC cells growth. MiR-4458ASO promoted HCC cell proliferation. MiR-4458played its role via targeting3’UTR of IKBKE.Statistical analysisData is presented as the mean±SD from at least three independent experiments. The difference between groups were analyzed using two-tailed Student’s t test when only two groups were compared. The difference between groups were analyzed using ANOVA when three or more than three groups were compared. Correlation analysis was performed by two-tailed Person’s correlation coefficient analysis. Overall survival of patients was estimated by the Kaplan-Meier method. Statistical analyses were performed using SPSS software (version16.0). P<0.05was considered significantly different.ConclusionsATF4may be a potential predictor of HCC prognosis.MiR-4458or IKBKE may be potential predictors of HCC prognosis. Restoration of miR-4458or inhibition of IKBKE could be a prospective therapeutic approach for HCC.