Abstract
Cervical cancer is the second most deadly gynecological tumor worldwide. MicroRNAs (miRNAs) play very important roles in tumor oncogenesis and progression. The mechanism of post-transcription regulation of WTX gene is still unknown. A series of differential miRNAs were discovered by microarray analysis comparing three pairs of primary cervical cancer specimens and their relapsed tumors from three patients. Quantitative reverse transcriptase PCR (qRT-PCR), Western Blot (WB) and Immunohistochemistry (IHC) was used to detect the expression of miR-4524b-5p and WTX in cervical cell lines and tissues. The biological function of miR-4524b-5p and WTX was investigated through knockdown and overexpression with inhibitor/siRNA and mimic/plasmid in vitro and in vivo. In this study, we found that miR-4524b-5p is highly expressed in relapsed cervical cancer specimens. Combined in vitro and in vivo experiments, showed that miR-4524b-5p could regulate the migration and invasion ability of cervical cancer. Furthermore, we also found that miR-4524b-5p could regulate the migration and invasion of cervical cancer by targeting WTX and that WTX could regulate the expression of β-catenin. Taken together, our data identified a miR-4524b-5p/WTX/β-catenin regulatory axis for cervical cancer, and miR-4524b-5p may be a potential target for cervical cancer therapy.
Highlights
Methodshuman embryonic kidney (HEK)-293T and H8 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, USA) with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA)
To identify miRNAs associated with relapse and the metastasis of cervical cancer patients, we performed microarrays to evaluate the differential expression of miRNAs in three pairs of tumor samples obtained from three patients who were diagnosed with early-stage cervical cancer and recurrence in 2~3 years (Fig 1A)
Using Quantitative reverse transcriptase PCR (qRT-PCR), we found that miR-4524b-5p was significantly increased in the relapsed cervical cancer specimens (n = 39) relative to the primary specimens (n = 50) (Fig 1B)
Summary
HEK-293T and H8 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, USA) with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA). The cervical cell lines SiHa, HeLa and me-180 were cultured routinely in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% FBS, and incubated at 37 oC with 5% CO2. The miRNA mimic, miRNA inhibitor and siRNA were designed and synthesized by RiboBio (Guangzhou, China). The pCDH plasmids encoded the full-length cDNA sequence of WTX. The miR-4524b-5p mimic, inhibitor, siRNA or control was transfected into cells with Lipofectamine 2000 Reagent (Life Technologies) according to the manufacturer’s protocol.
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