MicroRNA-372 Is Down-regulated and Targets Cyclin-dependent Kinase 2 (CDK2) and Cyclin A1 in Human Cervical Cancer, Which May Contribute to Tumorigenesis

Rui-Qing Tian,Xing-Hua Wang,Li-Juan Hou,Wei-Hua Jia,Qian Yang,Yi-Xuan Li,Min Liu,Xin Li,Hua Tang

doi:10.1074/jbc.m111.221564

Rui-Qing Tian, Xing-Hua Wang + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m111.221564

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Abstract

MicroRNAs are a class of noncoding RNAs that are ~22 nucleotides in length. MicroRNAs have been shown to play important roles in cell differentiation and in cancer. Recently, studies have shown that miR-372 is tumorigenic in human reproductive system cancers. However, we provide evidence that miR-372 acts as a tumor suppressor gene in cervical carcinoma. miR-372 was found down-regulated in cervical carcinoma tissues as compared with adjacent normal cervical tissues. Growth curve and FACS assays indicated that ectopic expression of miR-372 suppressed cell growth and induced arrest in the S/G₂ phases of cell cycle in HeLa cells. We used bioinformatic predictions to determine that CDK2 and cyclin A1 were possible targets of miR-372 and confirmed this prediction using a fluorescent reporter assay. Taken together, these findings indicate that an anti-oncogenic role of miR-372 may be through control of cell growth and cell cycle progression by down-regulating the cell cycle genes CDK2 and cyclin A1.

Highlights

Deregulation of the cell cycle machinery is considered to be a factor in tumor generation
We found that overexpression of CDK2 or cyclin A1 with miR-372 reversed the inhibition of cell growth and the arrested cell cycle at S/G2 phase caused by miR372 (Fig. 7, A and B)
It has been reported that miR-372 and miR373 are overexpressed in some cancers [14, 24, 25] and may play an oncogenic role by targeting the tumor suppressor LATS2 [14, 15], our studies showed that miR-372 was down-regulated in human cervical cancer tissues

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The human cervical cancer-derived cell lines, HeLa and C33A, were obtained from the American Type Culture Collection and cultured in RPMI 1640 (Invitrogen). The culture medium was supplemented with 10% fetal bovine serum (FBS), 100 ␮g/ml streptomycin, and 100 IU/ml penicillin and maintained at 37 °C in a humidified atmosphere with 5% CO2. Transfection and Stable Cell Line Selection—The HeLa cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection efficiency was monitored by fluorescence microscopy 48 h after transfection with pcDNA3/EGFP and Cy5-oligonucleotides. Transfected cells were selected by adding 0.4 mg/ml G418 to the culture medium.

Oligonucleotides used in this work

Findings

DISCUSSION