The Regulatory Effects On PUMA In Lung Cancer Cells XWLC-05by MiR221/222

Objectives①To identify the predition targets of miR221/222by luciferase reporter assays.②Methylthiazolyl tetrazolium (MTT) assay and caspase activity assay was applied to investigate the effect of miR221/222on proliferation and apoptosis in XWLC-05cells.③To study the roles of miR221/222in regulating targeted gene in XWLC-05cells, the expressions of PUMA gene in lung cancer cells were detected by RT-PCR technology.Methods①Gene-cloning method was used to construct a luciferase report gene vector.②Then, we transfected it into XWLC-05cell line.③Lciferase activity was detected by luciferase reporter assays to certified whether PUMA is the target gene of miR221/222.④Cell proliferation was measured by MTT assay.⑤Caspase-3,7,9activities were detected by colorimetric methods to evaluate the apoptosis of cells respectively.⑥PUMA mRNA differential expression was observed by RT-PCR.Results①Each vector was identified by sequencing analysis.②Luciferase reporter assays revealed that miR221/222could directly act on the3"UTR distal end sequence of PUMA.③MTT assay indicated that In-expression groups could availably inhibit the growth of cancer cells, on the contrary, Over-expression groups could promote cell proliferation.④Colorimetric methods suggested that the activity of Caspase-3,7,9was down-regulated in Over-expression and the activity of Caspase-3,7,,-9was up-regulated in in-expression.⑤After transfection of various vector, The PUMA mRNA of Over-expression groups and In-expression groups had no statistical difference.Conclusion①We successfully constructed a luciferase report gene vector.②PUMA proved to be the target gene of miR221/222.③miR221/222have the ability to affect the cell proliferation and apoptosis.④According to the level of mRNA, miR221/222showed no effects on the degradation rate of mRNA.