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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
MiR-135b-5p is an oncogene in pancreatic cancer to regulate GPRC5A expression by targeting transcription factor KLF4
Daren Liu1, Yun Jin2, Jinhong Wu2
1Department of General Surgery, Second Affiliated Hospital, Zhejiang University, Hangzhou, China. darenliu@zju.edu.cn.
Abstract:
KLF4 is implicated in tumor progression of pancreatic cancer, but the molecular regulatory mechanism of KLF4 needs to be further specified. We aimed to probe molecular regulatory mechanism of KLF4 in malignant progression of pancreatic cancer. qRT-PCR or western blot was completed to test levels of predicted genes. Dual-luciferase and chromatin immunoprecipitation (ChIP) assays were designed to validate binding between genes. Cell viability and oncogenicity detection were used for in vitro and vivo functional assessment. KLF4 was a downstream target of miR-135b-5p. KLF4 could regulate GPRC5A level. MiR-135b-5p was notably increased in cancer cells, and overexpressing KLF4 functioned a tumor repressive role, which could be restored by miR-135b-5p. Besides, cell malignant phenotypes could be inhibited through reducing miR-135b-5p level, but they were restored by GPRC5A. Our results stressed that KLF4, as a vital target of miR-135b-5p, could influence promoter region of GPRC5A, thus affecting the malignant progression of pancreatic cancer.
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