Experimental Study Of HAb18G/CD147 In Regulation Of Autophagy In Human Hepatoma Cell Lines

Cancer is common diseases and has a serious hazard to human health, which not only endangers the lives of patients, but brings enormous social impact of the medical world. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and ranks sixth among cancers as cause of death worldwide and third in China. It has an annual incidence rate of 564000 cases, and 55% of those are in China.High invasion and metastasis abilities of HCC are the important reasons to the high mortality. In the process of tumor metastasis, matrix metalloproteinases (MMPs) play an important role in the degradation extracelluar matrix. HAb18G/CD147 is inducer of MMPs, which plays key functions in cancer metastasis. HAb18G/CD147, a plasmolemma glucoprotein, takes part in many physiological and pathological processes and plays a majar role in many functions, including tumor invasion and metastasis, inflammation, cell adhesion and embryonic development. Our lab prepared monoclonal antibody HAb18 in 1987, which is specific to HCC. We obtained a HCC associated antigen, HAb18G using HAb18 as the probe to screen the HCC cDNA library. The ORF (open reading frame) sequence of HAb18G is homologous to that of CD147 molecule. HAb18G has generally been acknowledged as a new member of CD147 superfamily. Our previous studies showed that HAb18G was highly expressed in HCC tissues and lowly expressed in nomal tissues. We have discovered that the function of HAb18G is similar to CD147/EMMPRIN (Extracellar matrix metalloproteinase inducer) and this molecule could induce HCC cells and fibroblast cells to secret MMPs and promote tumor invasion.One of most important functions of HAb18G/CD147 is to induce the production of MMPs. However, whether there are other mechanisms explaining the role of HAb18G/CD147 in tumor progression remains to be elucidated. In this study, we investigated the functional effects of HAb18G/CD147 expression on autophagy in hepatoma cell line SMMC 7721 using immunostainning, Western Blot and transmission electronmicroscopy. We found that HAb18G/CD147 could inhibit starvation-induced autophagy in SMMC 7721 cells. The result of RNA interference experiment for HAb18G/CD147 indicated that autophagy significantly increased in SMMC 7721 cells, compared with scramble control. Meanwhile, cell death ratio increased in SMMC7721 cells transfected with HAb18G siRNA. In addition, we found that HAb18G/CD147 inhibited starvation-induced autophagy via down-regulating Beclin1 expression, which led to decrease the formation of autophagosome. Furthermore, HAb18G/CD147 inhibiting ATG6/Beclin 1 expression via the Class I PI3K/AKT pathway, which is confirmed by class I PI3K inhibitor LY294002. Our findings indicate that HAb18G/CD147 can protect cancer cells by inhibiting excessive autophagy to avoid autophagic death pathway in starvation condition. These results may lead to a better understanding of regulation mechanism of human SMMC7721 liver cancer cells in starvation condition.Part 1: HAb18G/CD147 expression in hepatoma cells under starvation conditionTo detect HAb18G/CD147 expression in hepatoma cells under starvation condiction, we selected three hepatoma cell lines (SMMC7721, HepG2 and HCC9204), which were synchronized by starvation in amino acids free EBSS buffer for 0, 1, 3, 6, 12, 24 hours at 37°C. HAb18G/CD147 expression was detected by using Western Blot. These results showed that HAb18G/CD147 expression was significantly increased when the hepatoma cells were starvated for 1, 3, 6, 12 h.Part 2: HAb18G/CD147 inhibits starvation-induced autophagy in human hepatoma cell SMMC7721.In this part, we investigated the functional effects of HAb18G/CD147 on autophagy in hepatoma cell line SMMC7721 using immunofluoscence staining, Western Blot and transmission electronmicroscopy. Our data showed that HAb18G small interference RNA considerably down-regulated the expression of HAb18G/CD147 in SMMC7721 cells at both mRNA and protein levels. The down-regulation of HAb18G/CD147 significantly promoted the starvation-induced autophagy in a dose-dependent manner. Using trypan blue exclusion assay, we found that HAb18G/CD147 notably enhanced the survival of SMMC7721 cells through inhibiting starvation-induced autophagy.Part 3: Signaling pathway of starvation-induced autophagy inhibition by HAb18G/CD147 In this part, we first assayed the epression of autophagy-relative genes including ATG5, ATG6/Beclin 1, and ATG8/LC3 using RT-PCR. We demonstrated that HAb18G/CD147 down-regulated the expression of autophagy-regulating gene ATG6/Beclin 1 in SMMC7721 cells. Then, our result showed that HAb18G/CD147 down-regulated the expression of ATG6/Beclin 1 protein in SMMC7721 cells. Furthermore, our data indicated that HAb18G siRNA-transfected SMMC7721 cells had a significantly decreased level of phosphorylated serine-threonine kinase Akt (pAkt) and the expression of Beclin 1 was inversely associated with the level of pAkt, suggesting that the Class I phosphatidylinositol 3-kinase-Akt pathway might be involved in the down-regulation of ATG6/Beclin 1.