The Expression Of Activating Transcription Factor3and Its Role In Human Hepatocellular Carcinoma

Objective: To detect the expression of Activating Transcription Factor3(ATF3) in humanhepatocellular carcinoma (HCC), and investigate the relationship between ATF3expression and theclinicopathological features of HCC patients. To study the role of ATF3in HCC, ATF3proteinover-expression was induced by LV-ATF3-EGFP in HepG2cell. To select and verify the proteinswhich interact ATF3and collaborate with ATF3to promote hepatocarcinogenesis.Methods:(1) RT-PCR, IHC and WB were used to detect ATF3expression in HCC, peritumoral livertissues and normal liver tissues, and to explore the correlation between ATF3expression and theclinicopathological features of HCC patients.(2) MTT, Transwell, FCM (which was used for detecting the cell cycle and apoptosis) was used toobserve the biological behavior changes of HepG2, which was infected by LV-ATF3-EGFP; thencombined with the variation of cell proliferation, migration ability, cell cycle and apoptosis, toexplore the role of ATF3in HCC and the correlative cytological mechanism.(3) IP, co-IP and protein spectrum analysis were used to select and verify the protein which wasinteract with ATF3in HepG2. IHC and WB were used to detect the expression of ATF3and itscandidate protein in HCC tissue sections, in order to investigate the correlation betweenprotein-protein interaction of ATF3and its interactive protein and the biological behavior of HCC.Results:(1) ATF3gene level was examined in53cases HCC and the corresponding peritumoral liver tissues,and10cases normal liver tissues by using Real time PCR. The result showed that ATF3gene wasexpressed in all of the groups; the lowest level was recorded in HCC than the other groups (P＜0.0001). There was no significant difference between peritumoral liver tissues and normal livertissues (P=0.6240). ATF3gene level was lower in capsule invasion tissues than the without group (P＜0.05), while not significantly associated with other main clinic-pathological features, such aspatient genders, tumor size, vessel metastasis, pathology classification, histomorphologic type,HBs-Ag status, AFP level and BCLC stage(P＞0.05). Additionally, there was no correlation between ATF3gene level and patients’ prognosis analyzing by Cox Regression (P＝0.0960).(2) ATF3protein was examined in20cases HCC and the corresponding peritumoral liver tissues，and10cases normal liver tissues by using IHC. The result showed that ATF3was located in cell nuclei,and the weakest dye was detected in HCC.(3) ATF3protein level was examined in53cases HCC and the corresponding peritumoral livertissues, and10cases normal liver tissues by using WB. The result showed that the lowest level wasalso recorded in HCC, and it was significantly lower than the other two groups (P＜0.05), whilethere was no significant difference between peritumoral liver tissues and normal liver tissues (P=0.3260). ATF3protein level was lower in capsule invasion tissues than the without group (P＜0.05),while not significantly associated with other main clinic-pathological features, such as patientgenders, tumor size, vessel metastasis, pathology classification, histomorphologic type, HBs-Agstatus, AFP level and BCLC stage(P＞0.05). Additionally, there was no correlation between ATF3protein level and patients’ prognosis analyzing by Cox Regression (P＝0.8390). The key results wereconsistent with the Real-time PCR analysis of ATF3gene level.(4) The biological behavior changes of HepG2, which was infected by lentiviral vector with ATF3over-expression was detected by using MTT, Transwell, FCM (which was used for detecting the cellcycle and apoptosis); The result showed that cell proliferation was slower, cell apoptosis wasaccelerated, cell cycle was retardant (P＜0.05), while the change of cell migration was not obvious(P＝0.262).(5) The protein expression differences were detected by using IP in HepG2groups. One group wasinfected by lentiviral vector with ATF3over-expression, the other group was infected lentiviralvector without ATF3over-expression. Then several protein bands with expression diversity wereanalyzed by protein spectrum.(6) Protein spectrum result showed that there were eight candidate proteins which may be relatedwith ATF3. Peptide sequences were analysised by Mascot software and NCBI database. Combinedwith the reported literatures and study results one by one, Gelsolin (GSN), which function had beenconfirmed clearly, was selected as one of the combined proteins with ATF3closely.(7) The combination of ATF3and GSN was verified by using co-IP technology. The correlationbetween ATF3and GSN was detected by using IHC and WB. Then related molecular mechanism ofATF3for HCC was speculated according to the definite function of GSN and the interaction between ATF3and GSN proteins.Conclusion:(1) The low expression of ATF3mRNA and protein were observed in HCC tissue. And the level waslower in patients with capsule invasion than the ones without capsule invasion. In addition, there wasno significant association between ATF3expression and the other clinical pathological features inpatients. These results indicated that ATF3may serve as a tumor suppressor during humanhepatocellular oncogenesis.(2) Slow cell proliferation, accelerated cell apoptosis, and retardant cell cycle were shown in HepG2group with ATF3over-expression. These results indicated that ATF3played its suppressor role inHCC by inhibiting tumor cell growth, accelerating cell apoptosis, blocking cell cycle process.(3) The correlation between ATF3and its interactive protein GSN was verified. And the expressionpattern of GSN was consistent with ATF3in HCC. GSN may be help for the nuclear localization,functional activation and transcriptional regulation of ATF3. These results indicated that ATF3played its suppressor role in HCC via protein–protein interactions with Gelsolin.