Effects Of RNF2 On The Maliganant Behavior HCC And Its Interaction With MANF

Ring2/RNF2(ring finger protein 2), a member of PRC1(polycomb repressor complex 1) belongs to Pc G(polycomb group) proteins. It has a ring finger domain. RNF2 is also known as an E3 ubiquitin ligase. It is recruited through the recognition of the tri-methylation of H3K27 and mediates histone H2 A ubiquitination at lysine 119, and then it can maintain the transcription repression. Pc G proteins have closely related in tumor development. The expressions of many polycomb group proteins(RING1, RNF2, BMI1, MEL18, HPH1, and RYBP) have significant change in the different tumor and normal tissues. RNF2 is highly expressed in many tumors, however the research about the expression of RNF2 in hepatocelluler carcinoma(HCC) has not yet reported. RNF2 was screened as an interacting protein with MANF(mesencephalic astrocyte-derived neurotrophic factor) by using of yeast two-hybrid assay. RNF2 has a strong interaction with MANF in HCC tissue. MANF is an ER stress inducible protein. As we know, it plays a remarkable protective role against various injuries to neurons. Our experiments had proved that MANF also has a suppressive effect on HCC. We found that RNF2 dependently stabilizes MANFprotein level. Therefore, we speculate that RNF2 may inhibit HCC by interacting with MANF.ObjectiveTo observe the effects of RNF2 on HCC and explore the underlying mechanisms involved in HCC.MethodsImmunohistochemical method was used to detect the expression of RNF2 in paracancer and cancer tissues of HCC patients. SMMC-7721 cells were transfected with RNF2 or RNF2-si RNA. Then the cells were in treated with oxygen glucose deprivation(OGD) for 2.5 h. The number of dead cells were observed using PI staining. The expressions of Caspase-3, RNF2, and MANF were detected using western blot and immunofluorescence assay. MTT assay, colony formation assay, transwell experiment, and wound healing assay were used to detect the cell viability, the ability of clone formation, invasion, and migration, respectively. To investigate the interaction between RNF2 and MANF, Co-immunoprecipitation assay and GST-pull down were used. The m RNA and protein level of MANF were determined by using RT-PCR and western blot in RNF2 knockdown and over-expression cells, respectively.Results1. The expression of RNF2 in HCC9 136 non-tumor controls were detected by immunohistochemical assay. We found that RNF2 was expressed in all the tissues. However, compared to the non-tumor tissue, the cases of HCC with high expression of RNF2 was significantly less(p <0.01).2. RNF2 increased the number of dead cells of hepatoma cellsSMMC-7721 cells were transiently transfected with RNF2-myc or RNF-si RNA, and then treated with oxygen glucose deprivation(OGD) for 2.5 h. PI staining showed that after transfected with RNF2-myc, the number of dead cells was increased by counting the PI-positive cells. On the contrary, transfection with RNF2-si RNA decreased the number of dead cells, suggesting that RNF2 may promote OGD-induced cell death of hepatoma cells.3. RNF2 promote apoptosis of SMMC-7721 cellsUsing flow cytometry to detect cell apoptosis, we found that RNF2-si RNA reduced the number of apoptotic cells treated with OGD. We used western blot to detect the expression of caspase-3. We found that RNF2 inhibited the expression of caspase-3. These results suggest that RNF2 may promote hepatoma cells apoptosis.4. Effects of RNF2 on the malignant behavior of hepatoma cellsIn SMMC-7721 cells and TEL-7402 cells, we transfected with RNF2-myc or RNF2-si RNA and detected the proliferation of cells by using MTT assay and colony formation assay. We found that RNF2 inhibited the proliferation of hepatoma cells. This result was supported by the wound healing experiment. The Transwell assay showed that RNF2 inhibited the invasion and migration of hepatoma cells.5. RNF2 interacts with MANFImmunofluorescent staining assay showed that RNF2 colocalized with MANF in the liver tissue of HCC. Immunoprecipitation assay confirmed the interaction of RNF2 and MANF in HCC. OGD or tunicamycin induced RNF2 co-localized with MANF in nucleus.6. RNF2 stabilizes the protein level of MANFTo detect the effects of RNF2 on the protein level of MANF, we transfected SMMC-7721 cells with RNF2-si RNA or RNF2-myc plasmid. Western blotting and immunofluorescent staining were used to examine the expression of MANF. The results suggested that RNF2 knockdown reduced the expression of MANF. RNF2 over-expression dose-dependently increased the expression of MANF after treatment with OGD or tunicamycin. These results indicated RNF2 stabilizes MANF protein level. However, RT-PCR assay showed that RNF2 did not affected the transcriptional level of MANF.Conclusions:1. RNF2 promoted apoptosis of hepatoma cells. 2. RNF2 inhibited the malignant behavior of hepatoma cells. 3. RNF2 interacted with MANF. 4. RNF2 stabilizes MANF protein level and MANF dose dependence on RNF2.