| Part1Systemic analysis of miRNAs targeting OCT4Objective: To systematically characterize the microRNAs targeting OCT4by bindingthe3’UTR from all the known miRNA of human.Method: First we used the5bioinformation prediction software to match the miRNAwith entire sequence of OCT43’UTR, then we construct the dual-luciferase reporter systemand cotransfected with miRNAs one by one in293T cells. We check the relative luciferaseactivity, and after Western blot was applied to test the OCT4protein level. we confirmedmiRNAs with the site mutation of seed sequence.Result: Targetscan has predicted54miRNAs can binding the OCT43’ UTR (50miRNAs with a score to "poorly conserved" and4miRNAs with "conserved"),7miRNAspredicted by miRDB with default parameter,11miRNAs predicted by miRanda with defaultparameter. We got61miRNA by predicted bioinformation software.20miRNAs were testedby dual-luciferase reporter system for it can change the relative luciferase activity. Westernblot assay was used to analyzed and8miRNAs can depress the OCT4protein. After checkedby mutation of seed sequence,we obtained3miRNAs: miR335, miR-4657, miR-3654.Conclusion: The precess of systemic analysis contains four key steps: l.predicted bybioinformation software online.2.check the relative luciferase activity in vitro. Idetectionthe expression of OCT4protein after transfected miRNA into cells.4.re-check the relativeluciferase activity with mutantion of seed sequences. It is indicated that this program is usefuland efficiency for lab application. Part2The3miRNAs depress the proliferation of bladder cancer celllines by targeting OCT4Objective: To study the expression of OCT4in bladder cancer tissues and its relationto clinicopathology. And to investigate the mechanism of suppression of miR-335, miR-4654and miR-3657in bladder cancer cell lines5637and HT1376.Method: The expression of OCT4was detected in the pathological specimens from31cases by using immunohistochemistry and16pairs cases of bladder cancer and nearbynormal tissue. Transfected bladder cance cell line5637and HT1376with the3miRNAindependently. Then MTS assay and colony formation have testedResult: The expression rate of OCT4in bladder cancer was83.9%, and the postiverate and intensity of OCT4expression had obvious relationship with the grade(p<0.05). Theproliferation assay by MTS and colony formation potential of5637and HT1376transfectedwith3miRNAs independently were significantly lower than the control group, and theexpression of OCT4has been down-regulated by3miRNAs.Conclusion: It is indicated that the3miRNAs (miR-335, miR-4654, miR-3657)suppresse cell growth of bladder cancer cell5637and HT1376directly by targeting OCT4.Part3miR-335regulate the biological behavior of cancer stem cell inrenal cancer ACHN cell line by targeting OCT4Objective: To investigate the mechanism of suppression of miR-335in cancer stem cellof renal cancer cell line ACHNMethod: Sorted and indentified cancer stem cells with cell surface antigen label CD105in renal cancer cell line ACHN. Transfected the CD105+indentified cancer stem cell of ACHN with miRNA-335by lentivirus, Western blot assay was used to analyze the expressionof OCT4in transfectec cells, cell cycle and sphere formation were assayed.Result: The sorted cells were indentified as cancer stem cells for its cell surface antigenlabel of Sox-2/c-Myc/Nanog/OCT4/CD44/nestin/Pax2/DLK/CXCR4/Musashi.After dealed with cisplatin by24h or48h, the cell viability of ACHN CD105+is higher thanthat of ACHN CD105-. Phase G0in cell cycle of ACHN CD105+is longer than ACHNCD105-. The expression of protein OCT4has been down-regulated by miR-335transfection,and the characteristic of CSC was decreased with the flCT4.Conclusion: It is indicated that miR-335supressed CSC migration and induce CSCtowards differentiation by targeting OCT4. The CSCs ability of sphere formation wasdisappeared, phase GO of cell cycle was shorten, and cell migration decreased. |
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