| Background Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Epidemiological studies have convincingly showed that chronic hepatitis B virus (HBV) infection is one of the main causes of HCC. Recent studies have revealed that hepatitis B virus X gene (HBx) plays a very important role in the development of HBV-associated HCC. HBx could influence hepatocyte apoptosis by many ways, which is one of the most important mechanisms in the pathogenesis of HBV-associated HCC. However, the role of HBx in the regulation of hepatocyte apoptosis remains controversial and the exact mechanisms remains unclear. HBx has been shown to promote apoptosis, conversely, HBx has also been shown to inhibit apoptosis. Interestingly, previous studies have demonstrated that HBx deletion, especially the COOH-terminal deletion of HBx, is a frequent event identified in HBV-associated HCC tissues. Deletions in the HBx-COOH-terminal alter a key role that the wild-type HBx plays in controlling cell proliferation, differentiation, and apoptosis, they may play a critical role in HBV-associated HCC. Our previous studies have also showed HBx deletion mutant could apparently promote the malignant transformation of hepatocytes, but its molecular mechanisms remains unclear.MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally and participate in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. They play a critical role in numerous diseases, including cancers. The overexpressed or underexpressed miRNAs in cancers might function as oncogene or tumor suppressors, respctively. Recent studies have already implicated that miRNAs are closely associated with the development of HCC. However, the exact mechanisms of miRNAs in HBV-associated HCC have not yet been well understood.Objective To study the biological characteristics, apoptosis and microRNA expression profiles of hepatocytes transfected by wild-type HBx (HBx) and HBx deletion mutant (HBx-d382), and its related pathogenesis in the malignant transformation of hepatocytes.Methods (1) E.coli DH5a containing plasmids pcDNA3.0, pcDNA3.0/HBx, pcDNA3.0/HBx-d382was recovered, respectively. Plasmids were extracted and verified by PCR and double-enzyme cleavage method. DNA sequences were detected and analyzed by bioinformatics software DNASIS MAX2.9and clusterW.(2) Gene transfection mediated by the lipofectamine was used to introduce the eukaryotic expression vector of pcDNA3.0, HBx and HBx-d382genes into L02cell lines. The selective medium containing G418was used to select the cell clones which express the HBx protein constantly.(3) L02cell lines in each group were exposed to actinomycin D (Act D) and tumor necrosis factor a (TNF-a) treatment to induce cell apoptosis. Hepatocyte apoptosis in L02, L02/pcDNA3.0, L02/HBx and L02/HBx-d382group was detected by Annexin V-fluorescein isothiocyanate/Propidium iodium (Annexin V-FITC/PI) double staining with flow cytometry and terminal deoxynuc1eotidy1transferase mediated dUTP nick end labeling (TUNEL). The mRNA and protein expression of Survivin and PTEN (phosphatase and tensin homology deleted on chromosome Ten, PTEN) were detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot respectively.(4) Microarray was used to analyze the miRNA expression profile in L02/HBx and L02/HBx-d382, L02/pcDNA3.0served as a mock control.Results (1) The correctness of wild-type HBx (HBx) and HBx deletion mutant (HBx-d382) eukaryotic expression vector was verified and human hepatic cell lines stably transfected HBx and HBx-d382genes were successfully estabilished.(2) By Annexin V-FITC/PI double staining with flow cytometry, the percentages of early apoptosis in L02, L02/pcDNA3.0, L02/HBx-d382and L02/HBx group before ActD and TNF-a treated were (3.38±0.53)%,(3.95±0.29)%,(3.23±0.69)%,(10.34±0.74)%, respectively. After being treated for12hours, the percentages of early apoptosis in each group were (4.07±0.27)%,(4.69±1.04)%,(4.09±0.16)%,(9.83±1.02)%, respectively. After being treated for24hours, the percentages of early apoptosis in each group were (19.30±3.00)%,(20.82±2.43)%,(18.18±2.48)%,(28.37±3.50)%, respectively. After being treated for48hours, the percentages of early apoptosis in each group were (35.25±5.17)%,(35.73±4.21)%,(31.13±4.50)%,(45.65±3.37)%, respectively. The percentages of early apoptosis in L02/HBx group were always higher than in L02, L02/pcDNA3.0and L02/HBx-d382group before and after ActD and TNF-a treated (p<0.05). However, there was no significant difference in the percentages of early apoptosis between L02/HBx-d382, L02/pcDNA3.0and L02group before and after ActD and TNF-a treated (p>0.05). There was no significant difference in the percentages of early apoptosis between L02/HBx-d382, L02/pcDNA3.0and L02group before and after ActD and TNF-a treated for12hours (p>0.05). Compared with L02cell lines in each group before Act D and TNF-a treated, the percentages of early apoptosis in L02, L02/pcDNA3.0, L02/HBx and L02/HBx-d382group increased after Act D and TNF-a treated for24hours and48hours (p<0.05), and they were higer after being treated for48hours than being treated for24hours (p<0.05); By TUNEL assay, the apoptotic index in L02, L02/pcDNA3.0, L02/HBx-d382and L02/HBx group after ActD and TNF-a treated for24hours was (21.97±4.91)%, (23.49±3.26)%,(22.55±4.86)%,(33.68±4.43)%, respectively. The apoptotic index in L02/HBx group was also higher than in L02, L02/pcDNA3.0and L02/HBx-d382group after ActD and TNF-a treated for24hours (p<0.05). However, there was no significant difference in the apoptotic index between L02/HBx-d382, L02/pcDNA3.0and L02group after ActD and TNF-a treated for24hours (p>0.05). The relative expression intensity of Survivin mRNA and protein products in L02, L02/pcDNA3.0, L02/HBx-d382and L02/HBx group was0.66±0.09and0.69±0.12,0.66±0.07and0.76±0.09,0.63±0.13and0.77±0.14,0.26±0.09and0.13±0.12, respectively. The relative expression intensity of PTEN mRNA and protein products in each group was0.21±0.09and0.19±0.13,0.24±0.12and0.21±0.07,0.22±0.11and0.23±0.06,0.52±0.09and0.71±0.04, respectively. Survivin mRNA and protein expression were significantly lower, and PTEN mRNA and protein expression were significantly higher in L02/HBx group than in L02, L02/pcDNA3.0and L02/HBx-d382group (p<0.01). However, there was no significant difference in the mRNA and protein expression of Survivin, PTEN between L02/HBx-d382, L02/pcDNA3.0and L02group(p>0.05).(3) HBx transfection induced4miRNA up-regulated (hsa-miR-7, hsa-miR-1274a, hsa-miR-137, hsa-miR-663) and11miRNA down-regulated (hsa-miR-338, hsa-miR-24, hsa-miR-29, hsa-miR-744, hsa-miR-455, hsa-miR-324, hsa-miR-551b, hsa-miR-340, hsa-miR-590, hsa-miR-660, hsa-miR-113). HBx-d382transfection induced7miRNA up-regulated (hsa-miR-501, hsa-miR-595, hsa-miR-1307, hsa-miR-1180, hsa-miR-497,hsa-miR-1246, hsa-miR-623) and5miRNA down-regulated (hsa-miR-338, hsa-miR-551b, hsa-miR-1, hsa-miR-455, hsa-miR-200c). Although hsa-miR-338, hsa-miR-455and hsa-miR-551b down-regulated both in L02/HBx hepatocytes and in L02/HBx-d382hepatocytes, the other changes of miRNA expression profiles in L02/HBx hepatocytes and in L02/HBx-d382hepatocytes were different.Conclusion (1) The correctness of wild-type HBx (HBx) and HBx deletion mutant (HBx-d382) eukaryotic expression vector was verified and human hepatic cell lines stably transfected HBx and HBx-d382genes were successfully estabilished, which might establish the foundation for studying HBx and HBx-d382genes and proteins in the development of HBV-associated HCC in vitro.(2) It was different that wild-type HBx (HBx) and HBx deletion mutant (HBx-d382) influenced on hepatocyte apoptosis. Wild-type HBx (HBx) could promote hepatocyte apoptosis by down-regulating the expression of Survivin and up-regulating the expression of PTEN, but HBx deletion mutant (HBx-d382) could alter the role of HBx in promoting apoptosis. HBx deletion mutant might play a role in the development of HCC through modifying the biological functions of HBx.(3) HBx and HBx-d382transfection could induce the changes of miRNA expression profiles in hepatocytes. However, the changes of miRNA expression profiles in L02/HBx hepatocytes and in L02/HBx-d382hepatocytes were different, which might induce the different biological impact of HBx and HBx-d382on hepatocyte apoptosis. |
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