Unknown,Transcriptomics,Genomics,Proteomics

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Next Generation Sequencing Facilitates Quantitative Analysis of Argonaute 2 (Ago2)-immunoprecipitation (IP) after miR-195 or miR-497 overexpression in HepG2


ABSTRACT: To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines. Further expression analyses using a large panel of HCC cell lines and primary tumors demonstrated four miRNAs, miR-101, -195, -378 and -497, as candidate TS-miRNAs frequently silenced in HCCs. Among them, two clustered miRNAs miR-195 and miR-497 showed significant growth-suppressive activity with induction of G1 arrest. Comprehensive exploration of their targets using Argonute2-immunoprecipitation-deep-sequencing (Ago2-IP-seq) and genome-wide expression profiling after their overexpression, successfully identified a set of cell-cycle regulators, including CCNE1, CDC25A, CCND3, CDK4, and BTRC. Our results suggest the molecular pathway regulating cell cycle progression to be integrally altered by downregulation of miR-195 and miR-497 expression, leading to aberrant cell proliferation in hepatocarcinogenesis. Identification of miR-195 and miR-497 target genes by sequencing Ago2-binding mRNAs and total mRNAs of miR-195 or miR-497 overexpressed, or non-treated Hep G2 cell. Deep sequencing of RNAs in Ago2-IP fraction and mRNAs extracted from miR-195 or miR-497 overexpressed, or non-treated Hep G2 cell.

ORGANISM(S): Homo sapiens

SUBMITTER: Johji Inazawa 

PROVIDER: E-GEOD-41074 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The tumor-suppressive miR-497-195 cluster targets multiple cell-cycle regulators in hepatocellular carcinoma.

Furuta Mayuko M   Kozaki Ken-ichi K   Tanimoto Kousuke K   Tanaka Shinji S   Arii Shigeki S   Shimamura Teppei T   Niida Atsushi A   Miyano Satoru S   Inazawa Johji J  

PloS one 20130327 3


MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and commonly deregulated in carcinogenesis. To explore functionally crucial tumor-suppressive (TS)-miRNAs in hepatocellular carcinoma (HCC), we performed integrative function- and expression-based screenings of TS-miRNAs in six HCC cell lines. The screenings identified seven miRNAs, which showed growth-suppressive activities through the overexpression of each miRNA and were endogenously downregulated in HCC cell lines.  ...[more]

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