1 The Effect Of ATF6 On Viability Of Hepatocellular Carcinoma Cell Lines 2 The Effect Of HBV On Thapsigargin Induced Apoptosis Via Inhibiting CHOP Expression In Hepatocellular Carcinoma Cells

Backgrounds:Endoplasmic reticulum is an important organelle, regulating the folding and modification of proteins. However, under the action of some physical and chemical factors, the environment within the endoplasmic reticulum changed a lot, leading to the endoplasmic reticulum stress and activating a signaling network called the unfolded proteins response (UPR). Activating transcription factor 6 (ATF6) is an endoplasmic reticulum membrane-anchored transcription factor with 90 kDa, which can promote the expression of chaperone and the ability of endoplasmic reticulum in protein folding and transshipment. Recent studies have showed that tumor cells often accompanied by unfolded proteins response (UPR) owing to its special internal and external environment. And ATF6, as an important transduction molecule in UPR, has a close relationship with various kinds of tumors. But the effect of ATF6 on liver cell cancer is rarely reported.Objective:To investigate the effects of ATF6 on viability, migration, invasion and colony formation in hepatocellular carcinoma HepG2 and SMMC7721 cells.Methods:p3X-Flag-CMV-14-ATF6 vectors were transfected into HepG2 and SMMC7721 cells which were set as transfection group, and then cells without treatment and cells transfected with p3X-Flag-CMV-14 vectors were set as blank group and negative group. MTT assay, wound assay, migration and invasion in Transwell and colony formation assay were taken to detecte the effect of ATF6 on HepG2 and SMMC7721 cells.Results:After transfected with ATF6 vector, the mRNA expression increased greatly in HepG2 and SMMC7721 cells. In MTT assay, the cell viability did not make difference in HepG2 and SMMC7721 cells after transfected with ATF6 vector. In wound assay, the migration rates of transfection group in HepG2 and SMMC7721 cells were higher than the negative group. In Transwell migration assay, the HepG2 and SMMC7721 cells migrated in the transfection group increase significantly when ATF6 mRNA expression increased. In Transwell invasion assay, the invasion capacity of transfection group increase significantly in HepG2 and SMMC7721 cells. The transfection group form more colonies than the negative groups in HepG2 and SMMC721 cells in colony formation assay.Conclusion:ATF6 promotes the capacity of migration, invasion and colony formation in HepG2 and SMMC7721 cells, however, does not work on cell viability.Background:Hepatocellular carcinoma (HCC) is responsible for many cancer-related deaths worldwide. Hepatitis B virus (HBV) infection is a major cause of HCC in China. Thapsigargin (TG) is a potential anti-tumor prodrug, eliciting endoplasmic reticulum (ER) stress via the inhibition of ER calcium pump, effectively inducing apoptosis.Objective:To investigate the role of HBV in TG induced apoptosis using two HCC cell lines, HBV positive HepG2.2.15 and HBV negative HepG2.Methods:0 h,24 h,48 h,72 h and 96 h after HepG2 and HepG2.2.15 cells treated with TG, images of cells were obtained using a microscope. MTT assay and Annexin V-FITC/propidium iodide staining were used to examined the apoptosis. Cell cycle test was used for analyzing proportion of cells in G2 phase. Real-time PCR was then used to detect the expression levels of genes in the ER stress pathway at different times after treatment with TG. Notably, the mRNA levels of the apoptosis factor CHOP increased significantly in HepG2 cells than in HepG2.2.15 cells.Western blotting was next to confirm the increased expression of CHOP. Additionally, HepG2.2.15 cells treated with interferon-α to examine the change of CHOP expression. The effect of overexpression or knockdown of CHOP mRNA in HepG2.2.15 or HepG2 cells on apoptosis status was examined by an MTT assay.Results:Images of cells show that HepG2 cells underwent significantly more apoptosis than HepG2.2.15 cells after treatment with TG. This phenomenon was confirmed by an MTT assay and Annexin V-FITC/propidium iodide staining. Cell cycle analysis show that TG reduced the proportion of cells in G2 phase. Real-time PCR and Western Blotting show that both the mRNA and protein levels of the apoptosis factor CHOP increased significantly in HepG2 cells than in HepG2.2.15 cells. Additionally, HepG2.2.15 cells treated with interferon-a had higher CHOP levels than untreated cells. Overexpression or knockdown of CHOP mRNA in HepG2.2.15 or HepG2 cells could reduce the difference in apoptosis status between the two cell lines.Conclusion:HBV may repress apoptosis induced by ER stress.