| Objective Transcription factor Twist1, a member of basic helixloop-helix transcription factors family, is a hallmarker of tumor epithelial-mesenchymal transition. Twist1 promote tumor EMT by binding to promoter of E-cadherin and repress the expression of it, which lead to disassembly of adherens junctions and increased migratory potential. A clear link between Twist1 expression and metastasis is well established. Bcl-2 is an oncogene, which paly a crucial role in intrinsic apoptosis pathway, also known as the mitochondrial apoptosis pathway. In most types of cancer, Bcl-2 is overexpressd. Some researches indicated that Bcl-2 may regulated tumor EMT, however, the role of Bcl-2 in mediating EMT has not been well clear. This study was to clarify expression of Bcl-2 and Twist1 in oral squamous cell carcinoma(OSCC) cell lines and to explore the interaction between Bcl-2 and Twist1 and synergetic mechanism of Bcl-2 and Twsit1 in OSCC invasion and metastasis.Method 1. Oral squamous cell carcinoma cell lines(Tca8113and Tb3.1) were selected as the experimental object. The expression of Bcl-2 and Twist1 were induced by Co CL2 to analysis their mechanism. The expression trend of Bcl-2 and Twist1 over hypoxia time were detected by using real-time PCR. The interaction mechanism of Bcl-2 and Twist1 was determined by using protein immunoprecipitation. 2. Tca8113 and Tb3.1 cell lines were transfected by si RNA(Lipofectamine2000 mediated Bcl-2-si RNA and Twist1-si RNA) or infected by lentiviral system(LV-RFP-sh-Bcl-2 and LV-GFP-sh-Twist1). To analysis their function, Bacl-2 and Twist1 were knocked down respectively or together. Wound scratch assay and Transwell assay were used to evaluate cell the migration and invasion ability. EMT associated proteins and invasion-related markers were detected by using Western blot. The expression of E-cadherin and N-cadherin were determined by immunofluorescent assay. 3.The oral squamous cell carcinoma cell lines with stable expression of Bcl-2 and/or Twist1 were injected into the mouth floor of nude mice to establish in-situ model. The mice were into 4 groups(sh-BCL-2 group, sh-Twist1 group, sh-Bcl-2/Twist1 group and control group). Tumor size was measured every 3 days. After 30 days, the mice were sacrificed, then the tumor and peripheral lymph nodes were dissected. HE staining was used to observe the morphology of tumor cells and to identify positive lymph nodes. The expression of E-cadherin, N-cadherin, Vimentin and invasion-related markers(MMP2, MMP9) were determined through immunohistochemistry.Result 1. After hypoxia treatment in Co CL2 induced fashion, the expression of Bcl-2 and Twist1 increased over time, then the expression of both gene were gradually decreased after the peak. The expression trend of Bcl-2 and Twist1 were similar. The peak expression of Bcl-2 and Twist1 of Tca8113 and Tb3.1 were observed at 12 h and 24 h, respectively. The immunoprecipitation assay showed that Bcl-2 and Twist1 could form a complex. Western blot showed that the expression of Bcl-2 or Twist1 in cytoplasm and nucleus were decreased after knock-down. The expression level were even lower when both genes were knock-down. 2. Using lentivirus-mediated system, oral squamous cell carcinoma cell lines with stable expression of Bcl-2 or/and Twist1 were established. Compared with control, knock-down of Bcl-2 or/and Twist1 led to the up-regulation of E-cadherin and the down-regulation of N-cadherin and Vimentin. Moreover, down-regulation of MMP2 and MMP9 were also observed. The down-regulation of these genes were more significant in dual knock-down group. The scratch closure rate of three treatment groups were lower than the control group. The transwell assay showed that knockdown of Bcal-2/Twist1 led to reduction of invasion ability. The immunofluorescent assay showed the similar results as Western blot. 3. Compared with control, down-regulation of Bcl-2 and Twist1 could exert in vivo antitumor effect. The inhibition ability of sh-Twist1 group and sh-Bcl-2/Twist1 group were more significant than sh-Bcl-2 group. HE staining showed that 2 of 24 lymph nodes were positive in control group, while no lymph node metastasis were observed in 3 treatment groups. Immunohistochemistry results further confirmed that knockdown of Bcl-2 and Twist1 led to the up the up-regulation of E-cadherin and the down-regulation of N-cadherin,Vimentin, MMP2 and MMP9,and inhibited epithelial-mesenchymal transition(EMT) of tumor.Conclusion 1. Bcl-2 and Twist1 were co-overexpressed in oral squamous cell carcinoma. Bcl-2 and Twist1 exert their function by forming complex. Interference of the expression of Bcl-2 or Twist1 could inhibit the formation of complex. 2. Bcl-2 and Twist1 involved in EMT in oral squamous cell carcinoma. Hypoxia condition could induce the formation of Bcl-2/Twist1 complex and enhanced the induction of EMT in oral squamous cell carcinoma and thus influence the invasion and metastasis of oral squamous cell carcinoma. This may related to the increasing of Twist1 nuclear import which enhanced by the up-regulation of Bcl-2 under hypoxia condition. Dys-regulation of Bcl-2/Twist1 could impact tumor cell formation, the expression of EMT-related proteins, migration and invasion of tumor cells. 3. Interfering the expression of Bcl-2/Twist1 could significantly inhibit growth in mice model, down-regulate EMT-related proteins and recover the expression of E-cadherin. These results revealed a new regulation mechanism of EMT in oral squamous cell carcinoma. The Bcl-2/Twist1 may serve as a new therapeutic target of oral squamous cell carcinoma metastasis. |
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