Suppressive Effects Of MANF Or The Malignant Biological Behavior Of Hepatoma Cells

Hepatocelluler carcinoma,(HCC),the most common type of liver cancer, is one of the most common malignant tumor in China. Its etiology is complex. Although the clinical diagnosis and management of early-stage HCC has improved, survival of HCC patients remains dismal due to the lack of adequate therapies. The neoplastic lesion usually originates on a bed of chronic inflammation that sequentially progresses from fibrosis to cirrhosis and finally culminates in HCC. Endoplasmic reticulum stress is a kind of eukaryotic cells’ protective stress reaction, through activating the unfolded protein response(UPR) pathway to reduce the abnormal accumulation of the unfolded proteins in ER lumen to protect cells. There are some studies which have show that the reaction and development of ERS are closely related to the occurrence of diseases caused by many factors. Mesencephalic astrocyte-derived neurotrophic Factor(MANF) gene is the most sensitive gene to ER stress screened out from 30000 genes in the early stage of the study. We had found that MANF expressed more in the liver tissue of paracancer than that in carcinoma in both m RNA and protein levels. Therefore, MANF may play an important role in the process from hepatitis to HCC.ObjectiveTo observe the inhibitory effect of MANF on the proliferation, cloning formation, migration, and invasion of HCC.MethodsThe liver tissues were collected from the patients with HCC. We made use of immunohistochemical method to investigate the differential expression of MANF protein in the tissues of carcinoma and its adjacent part. After establishing the cell lines of hepatocellular carcinoma which stably express lower MANF, we observe the ability of cell proliferation, migration, invasion, the single cell proliferation and the effects on tumor growth in nude mice through the methods of MTT, wound healing assay, transwell, and clone formation assay.Results1. Expression of MANF in HCC Using immunohistochemical method, we found that the expression level of MANF was decreased in HCC, compared with that in the paracancer. The m RNA level of MANF was similar to the protein level that was reduced in HCC by using of in situ hybridization method. These results suggest that the transcriptional and translational levels of MANF were suppressed in HCC. 2. Effects of MANF on the proliferation of hepatoma cells We have established the cell line of hepatocellular carcinoma which stably expresses lower MANF and its control. The proliferation of cells was detected by MTT. We found that the proliferation rate of the cells stably expressing lower level of MANF was higher than that of the control group. As shown above, MANF may inhibit the proliferation of hepatoma cells. 3. Effects of MANF on the migration ability of hepatoma cells The migration ability of the hepatoma cells stably expressing lower MANF wasdetermined using the method of scratch experiment. We found that the migration ability of the cells stably expressing lower MANF is higher than that of the control group. This result suggests that MANF may inhibit the migration of hepatoma cells. 4. Effects of MANF on the invasion of hepatoma cells To investigate the effects of MANF on the invasion of hepatoma cells, we detected the invasion ability of hepatoma cells stably expressing lower MANF using the method of Transwell. We found that the invasion rate of the hepatoma cells stably lower expressing MANF is higher than that of the control group. The results suggest that MANF may inhibit the invasion ability of hepatoma cells. 5. Influence of MANF on malignant liver cancer cell proliferation Using the hepatoma cells stably expressing lower MANF, we detected the ability of clone formation of the cell line using soft agar. We found that the malignant proliferation capacity of the cell line stably expressing lower MANF is higher than that of the control group. This result suggests that MANF may inhibit the malignant proliferation of hepatoma cells. 6. Effect of MANF on the tumor growth in nude mice The female nude mice, 4 to 6 weeks after birth, were subcutaneously injected with the cells stably expressing lower MANF. We found that the size of tumor from the mice subcutaneously injected the cells stably expressing lower MANF was greater than that in the control group. After the formation of tumor, the mice subcutaneously injected the cells stably expressing lower MANF were also treated with the recombinant MANF protein or PBS solution through tail intravenous. We found that the size of tumor from the mice injected recombinant MANF protein through the tail intravenous was smaller than that of the group injected with PBS. These results suggest that MANF may inhibitthe tumor growth of nude mice.Conclusions:1. The expression level of MANF was decreased in HCC. 2. MANF suppressed the ability of cell proliferation, migration, invasion and the single cell proliferation of hepatoma cells. 3. MANF inhibited tumor growth of the tumor in nude mice.