| Part I Bmi-1maintains the properties of GCSCs and participates in chemotherapy resistance.Background:Cancer stem cells(CSCs) theory well explains the properties of cancer, like incurable property, recurrence and metastasis of cancer. More and more researches support the existence of CSCs. CSCs is a small part of cells with properties of stem cells, was considered as the orgin of cancer. Though CSCs accounts for only a very small proportion of cancer cells, its chemoradiation resistance due to its still state become the main reason for tumor recurrence and treatment failure. Several kinds of CSCs have been isolated from different solid tumors, like breast cancer, glioma, liver cancer and colorectal cancer, while little is known about gastric cancer stem cells (GCSCs). Till now, CD44+or CD133+gastric cancer cells was considered to have the properties of CSCs, exhibiting the higher tumorigenicity and chemoradiation resistance, Side population cells(SPs) isolated from gastric cancer has higher abdominal adhesion and metastatic ability. The isolation and identification methods for GCSCs is still far from clear, especially lack of treatment strategy for CSCs. Bmi-1plays an important role in proliferation and self-renewal of stem cells and cancer cells. Bmi-1was also found to play roles in maintaining the properties of normal mammary stem cells and breast cancer stem cells. So Bmi-1might also maintain the properties of GCSCs, which might be potential target of treatment for CSCs.Methods:Two classical methods for CSCs were used in our research:Serum free culture, Fluorescence Activated Cell Sorting (FACS) by cell surface markers (CD44, CD133). Western blotting and Immunoflurence(IF) were used for indentification of CSCs marker, CCK8assay was used to evaluate the chemotherapeutic sensitivity, the in-vivo tumorigenity was used to detect the tumorgenetic capability of isolated cells. The change of ectopic Bmi-1expression was used to verify the correlation of Bmi-1with GCSCs and chemotherapeutic sensitivity.Results:CSC-like cells isolated from MKN28, MKN45and primary gastric cancer cells using serum free culture method exhibits the properties of CSCs. CD133or CD44postive cells exhibits the properties of CSCs; Bmi-1plays role in chemotherapeutic resistance, higher proportion of GCSCs was found in chemotherapy resistant cells; Bmi-1maintains the properties of GCSCs. Conclusions:Bmi-1maintians the properties of GCSCs, and plays roles in chemotherapeutic resistance, Bmi-1interference by Ad-Bmi-1i transfection can partially reverse its stem cells properties, which offers a new stategy for treatment targeting CSCs.Part Ⅱ Effect and mechanimsm of Bmi-1interfering adenovirus driven by Bmi-1promoter targeting for gastric cancer in vivo and in vitroBackground:The exploration of tumor-specific targets and development of specific RNA interference (RNAi) vectors are two important aspects of the current RNAi gene therapeutic research. Cancer stem cells (CSCs) is one of the main reasons for the failure of cancer treatment, which has become a new therapeutic target. Bmi-1plays roles in maintaining the self-renew of stem cells. Our previous research found that Bmi-1is highly expressed in gastric cancer cells and gastric cancer tissues specifically, and correlates with depth of invasion, lymph node metastasis and prognosis, suggesting its potential role as a good therapeutic target. Thus, we aim to construct Bmi-1promoter mediated Bmi-1interfering adenovirus, and check its effect on gastric cancer cells or gastric cancer stem cells(GCSC) with different Bmi-1expression level in vivo and in vitro.Methods:Luciferase reporter assay was used to detect the promoter activity of Bmi-1, then construct Bmi-1interference adenovirus driven by Bmi-1promoter (Ad-Bmi-1i);1) In vitro study:check the effect of Ad-Bmi-1i on cellular proliferation, apoptosis, senescence, cellular mobility and migration, Western blot was used to detect the poteintial downstream target proteins, including p53, p16, Erk, Oct4, Akt pathways, etc.2) In-vivo study:Firstly, build up stablely Bmi-1silencing gastric cancer cells in Bmi-1lowly expressed and Bmi-1highly expressed gastric cell lines, then check the tumorigenity of Bmi-1silencing in vivo; Check the killing effect of Ad-Bmi-1i on gastric cancer cells by intra-tumor injection; In-situ tissue β-galactosidase staining to detect the induction of senescence by Bmi-1interference in vivo; IHC was applied to detect the potential mechanism of Bmi-1.3) Gene therapy:To clarify the in-vivo mechanism of Bmi-1interference and the effect of Ad-Bmi-1i on CSCs. HE staining and IHC were use to clarify the killing effect of Ad-Bmi-1i adenovirus on GCs and GCSCs in vivo. Results:Bmi-1interference by Ad-Bmi-li transfection can induce cellular senescence and reduce maglinant transformation phenotype in gastric cancer cells obviously in vitro, especially for cancer cells with higher Bmi-1expression, suggesting its specificity for targeting Bmi-1in vitro. Ad-Bmi-li adenovirus also inhibits tumorigenesis in vivo, and its inhibition efficiency on tumorigenesis depends on its Bmi-1level, which reveals Ad-Bmi-li adenovirus also has specific targeting for Bmi-1in vivo. Besides these, Our finding also showed that Ad-Bmi-li has a perfect killing effect on gastric cancer in vivo by direct in-tumor injection. More importantly, we also found that Ad-Bmi-li can partially kill CSC-like cells. Bmi-1interference by Ad-Bmi-1i transfection can induce cellular senescence via upregulating p53or p16, and(or) downregulation of Akt activity both in vitro and in vivo.Conclusions:Both in-vivo and in-vitro study showed that Ad-Bmi-li adenovirus has a good killing effect on gastric cancer, or potential killing effect on cancer stem cells, this project may provide highly effective targeted therapy for gastric cancer and provide a new idea and method for targeting CSCs. |
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