Docosahexaenoic Acid (DHA) Reverses The Epithelial-mesenchymal Transition Of Human Hepatoma Cells Through Inactivation Of CD147

Hepatocellular carcinoma(HCC) is the most common primary malignant hepaticcarcinoma，it is characterized by rapid progression，invasiveness，metastasis，highlymalignant and poor prognosis．Surgical resection is a major therapeutic means of HCCtreatment，but the postoperative recurrence and metastasis greatly affected the long-termcure after the treatment of hepatocellular carcinoma．In recent years，studies have shownthat n-3polyunsaturated fatty acids can inhibit tumorigenesis，invasion and metastasis，and enhance the sensitivity of tumor cells to chemotherapy drugs，but the mechanismsremain unclear．Docosahexaenoic acid(DHA)，one of the n-3polyunsaturated fatty acids，presentsmainly in cold-water fish oil and marine microalgae．It is an important biocompound withstructural and functional roles in both normal and malignant cells．DHA has significanteffects on anti-inflammatory，treatment of cardiovascular disease，alleviation of metabolicdisease and anti-cancer effects．Researches indicate that DHA increases the cytotoxic effect of tumor cells through lipid peroxidation and oxidative stress．It can also induceautophagy features of tumor cells by stimulating mitochondrial ROS．Epithelial-mesenchymal transition(EMT) is one of the important mechanisms oftumor metastasis．It can be seen in cancer cells as they leave the primary tumor anddisseminate to other parts of the body to colonize distant organs and formmetastases．During this process，cell morphology changes from paving stone shape into along spindle, accompanied by the downregulation of epithelial protein markers(E-cadherin and alpha-catenin) and upregulation of mesenchymal protein markers(N-cadherin，Vimentin and Fibronectin)．Variety of autocrine factors, transcription factors，microRNAs，TGF–beta，Wnt/beta–catenin and Notch pathways as the important factorsto promote EMT．Recent studies have shown that CD147was also involving in theoccurrence of EMT in HCC．CD147is a membrane glycoprotein，belonging to the immunoglobulin superfamily.The molecular expressed highly in epithelial-derived tumor cells，participating in theprocess of invasion and metastasis of tumor cells．Over expression of CD147can degradeextracellular matrix through stimulating tumor cells，fibroblasts and endothelial cells tosecret MMPs．CD147have been reported closely associated with occurrence of EMT inHCC to promote tumor cell invasion．The aim of this study was to identify DHA reverses the EMT of human hepatomacells through inactivation of CD147．(1) Influence of DHA on cell viability andapoptosis．(2) Whether DHA can reverse EMT of SMMC-7721cells．(3) Effect of DHAon cell migration and invasion．(4) The role of CD147in DHA-reversed EMT．Part1．Influence of DHA on SMMC-7721cell viability and apoptosisSMMC–7721cells were exposed to different concentrations of DHA for24，48，72h.Cell viability and apoptosis were examed by MTT assay and flow cytometry．1．within the conditions（DHA con.≤100μM），the effect on cell viability was obvious bythe way of treated with100μM DHA for48h(P <0.05)．Other experimental groups had nosignificant influence compared with the negative control group；within another condition （DHA con.≥200μM），the cell viability of experimental groups were decreasedsignificantly(P <0.05) compared with negative control group after24，48，72h treatmentof DHA．2．Apoptotic rate of SMMC-7721cells treated with DHA (20，50，100，200μM) for48hwas increasing obviously after comparing with control group(P <0.05)．Conclusion: low concentrations of DHA had no effect on cell viability and high levels ofDHA after long time exposure inhibited cell viability and induced its apoptosis．Part2．Impact of DHA on SMMC-7721EMTThe changes of cell morphology was observed via optical microscope and theexpression of EMT markers(E-and N-cadherin) was detected by Western Blot．1．EMT reversion of cells was observed by optical microscope，and morphologicalchanged from paving stone shape into a long spindle2．Expression of E-cadherin in SMMC-7721cells increased after treated with DHA for8hand16h．3．Expression of N-cadherin enhanced after treated with DHA for8、16、24、48h．Conclusion: DHA reversed the EMT of SMMC-7721cells．Part3．Effect of DHA on SMMC-7721cell migration and invasionThe ability of cell migration and invasion was tested through wound healing andtranswell assay respectively．1． Cell migration decreased significantly (P <0.05) when different experimentalgroups(20，50，100，200μM) of the same time point respectively compared with controlgroup． Migration rate gradually decreased with the enhanced concentrations ofDHA．2．The ability of cell invasion was significantly reduced (P <0.05) when differentexperimental groups(20，50，100，200μM) of the same time point compared respectivelywith control group．Conclusion: DHA significantly inhibited cell migration and invasion through EMT． Part4．Effect of DHA on CD147protein and its mRNA expressionWestern Blot and Real-Time PCR detection of CD147protein and its mRNA levelrespectively．1．CD147protein expression was no obvious difference after DHA treatment for8h and16h．But the expression of CD147protein decreased with increasing dose of DHA after24h and48h treatment．2．Real-Time PCR detection found that CD147transcription level of experimental groupscompared with control group were significantly reduced(P <0.05) except one experimentalgroup(8h-20μM)Conclusion: DHA inhibited the expression of CD147protein and its mRNA，CD147wasa potential molecular to reverse EMT of SMMC-7721cells.