Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells.

BACKGROUND: Expression of the microRNA miR-21 has been found to be altered in almost all types of cancers and it has been classified as an oncogenic microRNA or oncomir. Due to the critical functions of its target proteins in various signaling pathways, miR-21 is an attractive target for genetic and pharmacological modulation in various cancers. Cervical cancer is the second most common cause of death from cancer in women worldwide and persistent HPV infection is the main etiologic agent. This malignancy merits special attention for the development of new treatment strategies. In the present study we analyze the role of miR-21 in cervical cancer cells. METHODS: To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression in a cervical intraepithelial neoplasia-derived cell lines using siRNAs. The effect of miR-21 on gene expression was assessed in cervical cancer cells transfected with the siRNA expression plasmid pSIMIR21. We identified the tumor suppressor gene PTEN as a target of miR-21 and determined the mechanism of its regulation throughout reporter construct plasmids. Using this model, we analyzed the expression of miR-21 and PTEN as well as functional effects such as autophagy and apoptosis induction. RESULTS: In SiHa cells, there was an inverse correlation between miR-21 expression and PTEN mRNA level as well as PTEN protein expression in cervical cancer cells. Transfection with the pSIMIR21 plasmid increased luciferase reporter activity in construct plasmids containing the PTEN-3'-UTR microRNA response elements MRE21-1 and MRE21-2. The role of miR-21 in cell proliferation was also analyzed in SiHa and HeLa cells transfected with the pSIMIR21 plasmid, and tumor cells exhibited markedly reduced cell proliferation along with autophagy and apoptosis induction. CONCLUSIONS: We conclude that miR-21 post-transcriptionally down-regulates the expression of PTEN to promote cell proliferation and cervical cancer cell survival. Therefore, it may be a potential therapeutic target in gene therapy for cervical cancer.

Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells.