| Objective:1. To investigate the isolation, purification, phenotypic analysis and biological features of pancreatic cancer stem-like cells.2. To investigate the effect on the proliferation of pancreatic cancer stem cell-like cells regulated by microRNA.3. To investigate the mechanisms on the the regulation of proliferation in pancreatic cancer through the Hedgehog signaling pathway.Methods:1. Pancreatic cancer tissue samples were collected after surgery, primary pancreatic cancer cells were isolated and prepared for being cultured in vitro. Pancreatic stem-like cells were isolated by tumor stem cell spheroid assay, cultured, stained by HE, and identified by immunofluorescence. In order to enrich tumor cells, primary pancreatic cancer cells were used to establish pancreatic cancer xenograft models in nude mice. The expressing of epidermal stem cell key markers in pancreatic cancer cells were detected byRealtimeRT-PCR. The primary pancreatic cancer cells were transfected by the plasmid vector constructed with the key factor promoter combined with fluorescent protein and the xenograft models were established again. We used flow cytometry(FCM) for analyzing the content and percentage of positive cells and sorting cells. Cell viability was measured by MTT assay. The proliferation and colony forming efficiency of positive cells and negative cells were measured by clonogenic assay. The tumor spheroids forming efficiency of key factors positive cells and negative cells were measured by tumor stem cell spheroid assay. Tumor formation rates of contrast in both cells were measured in xenograft models.2. In order to detect the changes of miRNA expressions with the stem-cell key markers in positive and negative cells respectively, used miRNA microarray and comparative analysis. It showed the potential miRNA which was associated with the stem cell factor.RealtimeRT-PCR were used to detect the expression of specific mi-RNA in pancreatic cancer tissues and cell lines. The target gene fragment was amplified, and the type of point mutations were predicted, and then they were constructed to form plasmid vectors which were contained with the p GL4 luciferase, and were cotransfected with miRNA to the normal pancreatic cells. Double reporters of luciferase activities for gene assay explicitly showed the relationship between the selected miRNA and stem cell factors, it also indicated the potential gene targeted by selected miRNA which was associated with the interference in the growth of pancreatic cancer. After finished the construction of the recombinant lentiviral vectors and the selected miRNA, they were transfected to the pancreatic cancer cells of which target gene expressions were positive. The selected miRNA was upregulated and the changes of the target gene were detected byRealtimeRT-PCR in positive cells which were transfected with the selected and recombinant miRNA. The changes of protein in positive cells were detected by western blot. To measure colony forming efficiency by colony formation assay, cell apotosis rates were analized by flow cytometry, nude mice were observed to determine the changes of the interference rates for pancreatic cancer cell growth after the gene targeted by the selected miRNA. The changes of migration and invasion in the positive cells and control group were observed by transwell invasion assay after they were recombinant transfected. The changes of metastasis and EMT(epithelial-Mesenchymal transition) markers were detected by cells immune-fluorescence and western blot in the transfected positive cells and the control group cells.3. After the cells were treated with recombinant SHH-N or cyclopamine which is an inhibitor of SHH signal pathway, the impact of them on the growth rates of pancreatic cancer cells was detected by MTT method. The expressions of mRNA for the key factors related to SHH signal pathway or stem cells,and the miRNA related to the stem cells in pancreatic cancer cells were detected byRealtimeRT-PCR. The impact of recombinant SHH-N and cyclopamine on MMPs and PI3 K / Akt in the pancreatic cancer cells was detected by western blot assay.Results:1. Primary cells came out from the small tissure blocks which were obtained from digested pancreatic cancer tissue samples. By using inverted microscope and phase contrast microscope, cells could be seen had been confluent into a single layer of adherent cells after 5-6 days. The confluence was more than 85%. Cells were clear and connected to each other tightly. Besides, it seemed they had contiguous capacities. The morphology of H & E stained pancreatic cancer primary cells was observed by microscope. More than 90% of primary pancreatic cancer cells were confluent and adherent. The cells were polygonal with wide cytoplasms in the enlarged vision of microscope. The results of morphological examination showed that pancreatic cancer stem-like-cell has a capacity of self proliferation and differentiation. Compared with other key factors of stem cells, the results of immunofluorescence showed that expression level of Oct-4 was higest.RealtimeRT-PCR analysis showed that the expressions of Oct-4, C-myc, CD133 which were commonly used as the key stem cells or tumor stem cells markers were positive both in primary cells and tumor tissue specimens of pancreatic cancer cell xenograft. Compared with primary cells, the expressions of Oct-4 in tumor xenograft specimens were higher. Therefore, Oct-4 was selected as a specific marker of pancreatic cancer stem-like cells. Then the primary pancreatic cancer cells were transfected by the plasmid vector constructed with the Oct-4 promoter combined with yellow fluorescent protein. In order to enrich the tumor cells, they were transplanted to the nude mice for establishing the xenograft models. As the results of FACS showed, the percentage of Oct-4 positive cells in primary pancreatic cancer cells was accounted for 2.79%, and was accounted for 7.84% of positive cells in nude mice xenograft model which was significantly increased, P<0.05. The viability, tumor formation rate, colony-forming ability and tumor-spheroid-forming ability of Oct-4 positive cells were higher than negative cells, P <0.05.These data indicated that the tumor-forming abilities of Oct-4 positive cells were higher than the negative cells.2. miRNA microarray assay showed the expression of miR-335 in Oct-4(+) cells was significantly lower than Oct-4(-) cells. The expressions of mi-335 in 18 Groups of pancreatic cancer specimens and cell lines were significantly lower than those of normal tissue or cells. Target Scan showed there may be some interference targets for mi-335 in gene of Oct-4. The luciferase activity of p GL4-OCT4-WT in pancreatic cells group which were co-transfected with mi-335 mimics was much lower than the control group. Compared with the control group, expression levels of endogenous mRNA of Oct-4 and protein in the Oct-4(+) cell group which was transfected with recombinant miR-335 were decreased. Compared with the control group, colony formation of Oct-4(+) cell group transfected with recombinant miR-335 was decreased significantly. Apoptosis rate of Oct-4(+) cell group transfected with recombinant miR-335 increased, tumor formation rate dropped significantly, migration and invasion of tumor cells decreased significantly. Compared with the control group, in the Oct-4(+) cells group which were transfected with of recombinant miR-335, the expressions of mesenchymal markers such as Fibronectin, Vimentin, α-SMA, and SNAIL1 were downregulated, epithelial marker E-cadherin was upregulated.Recombinant SHH-N could increase growth rate of pancreatic cancer cells. Cyclopamine which was the inhibitor of Shh signaling pathway could suppresses growth rate of pancreatic cancer cells. Compared with control group and Oct-4(-)group, after being treated with recombinant SHH-N, the expression of factors in signal pathway of SHH and Oct-4 were increased significantly in Oct-4(+) pancreatic cancer cells, while the expression of miR-335 was decreased significantly. After being treated with Cyclopamine, we could only see the expression of Smo and Oct-4 were decreased significantly in Oct-4(+) pancreatic cancer cells, while the expression of miR-335 was increased significantly. After incubated withRecombinant SHH-N and Cyclopamine respectively, the protein expressions of MMP-2 and MMP-9 in pancreatic cancer cells increased in group ofRecombinant SHH-N while decreased in group of Cyclopamine.Recombinant SHH-N could increase the expression of Akt phosphorylation in pancreatic cancer cells after incubated for 24 h, while cyclopamine could reduce the expression of Akt phosphorylation in pancreatic cancer cells which was in a dose-dependent manner. So SHH signaling pathway plays a regulatory role in the PI3 K / Akt signaling pathway. The activation of SHH signaling pathway may increase the expression of MMP-2, MMP-9 protein and phosphorylation of Akt in pancreatic cancer cells, while down-regulate the expression of miR-335, and then increase the expression of Oct-4, thus affecting the stem characterizations of pancreatic cancer cells dry, and promote the growth and proliferation of tumor.Conclusion:1. We isolated pancreatic cancer stem-like cells, and established subcutaneous tumor xenograft model to enrich tumor cells especially the tumor stem cells, usedRealtimeRT-PCR and immunefluorescence chemical to detect and identify cell markers, selected Embryonic stem cell transcription factor Oct-4 as a specific marker of pancreatic cancer stem-like cells, used FCM to analyse content and proportion of positive expression cells, purify and sort cells. By measuring cell viability, colony-forming efficiency and xenograft tumor formation rate,we confirmed there were some biological characteristics of stem-like cells in Oct-4-positive cells.2. The expressions of miR-335 in Oct-4(+) cells was significantly lower than the Oct-4(-) cells. The expressions of mi-335 in pancreatic cancer specimens and cell lines were significantly lower than those of normal tissue or cells. By recombining and transfecting cells with overexpressed miR-335, it may induce significantly decreasing of Oct-4 expression in tumor cells, while suppressing the abilities of proliferation and metastasis invasion. So it was clarified that miR-335 could inhibit tumor development and the changes of stem-like cell characteristics by targetting gene of Oct-4 in the pancreatic cancer, which could be a potential new treatment of pancreatic cancer.3. SHH signaling pathway plays a regulatory role in the PI3 K / Akt signaling pathway. The activation of SHH signaling pathway may increase the expression of MMP-2, MMP-9 protein and phosphorylation of Akt in pancreatic cancer cells, while down-regulate the expression of miR-335, and then increase the expression of Oct-4, this process plays a vitally role in regulating the growth and proliferation of pancreatic cancer stem-like cells. |
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