Expression And Functional Study Of BRMS1 In Hepatocellular Carcinoma And The Regulation Of Sodium Butyrate On The Growth Of Hepatocellular Carcinoma Cells

BRMSl (Breast cancer metastasis suppressor1) was one of the tumor metastasis suppressor genes originally cloned in breast cancer. In metastatic breast cancer and melanomas, BRMS1could inhibit tumor metastasis to lymph nodes and lung tissues. BRMS1is widely expressed in normal human tissues. However, BRMS1is significantly down-regulated in malignant breast cancer and melanoma cells and the expression levels of BRMS1correlated with the prognosis of breast cancer patients.However, the role of BRMS1in hepatocellular carcinoma (HCC) remains poorly understood. Our previous work found that BRMS1was significantly down-regulated in cancer tissues and it exhibited higher expression levels in non-metastatic HCC cell lines than those in metastatic HCC cells.In this section, we first investigated the expression levels of BRMS1in paired HCC specimens (cancer tissues and neighboring noncancerous tissues) and various HCC cell lines through quantitative real-time PCR and western blotting analysis. This finding further indicated an involvement of BRMS1in hepatocarcinogenesis and HCC development.To further explore the biological function of BRMSl in HCC, a metastatic HCC cell line SK-Hepl was selected to establish cell lines stably expressing BRMS1and the effects of BRMS1expression on SK-Hepl cells were then studied. The results show that:expression of BRMS1did not affect cell cycle distribution and in vitro cell growth rate under normal culture condition; however, the growth ratio of BRMS1-expressing stable cell lines was obviously decreased in the absence of serum, and the apoptotic levels of BRMS1-expressing stable cell lines were significantly increased when kept in suspension. Meanwhile, in vivo study also showed that exogenous expression of BRMS1in SK-Hepl suppressed spontaneous metastasis to lymph nodes and lungs in nude mice whereas it had no effect on primary tumor growth. These results together indicated that BRMS1was likely to suppress tumor metastasis in HCC through regulating cell apoptosis. Lentivirus-based RNA interference was utilized to establish cell lines with knocked down endogenous BRMS1in two non-metastatic cell lines, Hep3B and Huh7. It is further found that by contrast to control cells, cells with knocked down BRMS1grew faster in the absence of serum, and the apoptotic levels was lower when cultured in suspension. These results further demonstrated the potential role of BRMS1in regulating cell apoptosis and tumor metastasis. During the exploration of the mechanisms accounting for the function of BRMS1in cell apoptosis and tumor metastasis, we found that BRMS1could regulate the expression of osteopontin (OPN), which was identified as one critical regulator protein in HCC metastasis. Overexpression of BRMSl inhibited OPN expression at both mRNA and protein levels while knocking down of BRMS1exerted an opposite effect on OPN expression. Further experiments analyzing cell lines with double genes (both BRMSl and OPN) knocked down revealed that knocking down of OPN could reverse the elevated anti-anoikis effect induced by knocking down of BRMS1. These results strongly suggest that the regulatory role of BRMS1on OPN expression is probably responsible for its activity on tumor metastasis suppression.In conclusion, our study investigated the expression alternation, cellular function and molecular mechanism of BRMS1in regulating HCC development. We reported for the first time that BRMS1is down-regulated in HCC tissues and it exerts a suppressive role in HCC metastasis. Via regulating OPN expression, BRMS1could play a role on cell apoptosis and tumor metastasis. Our finding not only brings more information into current knowledge of the molecular biology of HCC, but also provides solid experimental evidence for developing a more effective tumor maker or a novel protective/therapeutic approach in treating HCC. Sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor, has been shown to inhibit cell growth, induce cell differentiation and apoptosis in multiple cancer cells. However, little is known about NaBu in hepatocellular carcinoma (HCC).In present study, we investigated the in vitro effect of NaBu on two different HCC cells and revealed the bilateral effects of NaBu in HCC. Treating SK-Hepl and SMMC-7721cells with low concentration of NaBu induced a significant increase in the percentage of cells in S phase and cell growth ratio, accompanied by a reduced p21Cipl expression level at both mRNA and protein levels. By contrast, high concentration of NaBu significantly induced G1arrest through up-regulation of p21Cipl and p27Kipl protein expression. A decrease of p45expression was also noted in HCC cells treated with high concentration of NaBu, indicating that the ubiquitin-mediated protein degradation plays a role in NaBu-induced up-regulation of p21Cip1and p27Kip1. Furthermore, high concentration of NaBu triggered cell apoptosis and PARP cleavage in a longer culture period.Taken together, these results indicate that while high concentration of NaBu plays a suppressive role in regulating cell growth in a p21Cipl and p27Kipl-dependent manner, low concentration of NaBu exhibits a completely opposite effect mainly through p21factor. This finding might be of great help in developing more effective tumor therapeutic protocols of NaBu.