Mechanistic study of microRNA-135a on transformation of HPV-infected cervical epithelial cells in vitro

Abstract

Cervical cancer is the third leading cause of morbidity in female cancers. Persistent infection of high-risk type human papillomavirus (HPV) is a prerequisite of cervical cancer development. Nevertheless, chronic HPV infection is inadequate to induce cervical cancer formation while additional endogenous or exogenous cues are needed with chronic HPV-infection to cause cervical cancer. Stimulation of the Wnt/β-catenin signaling pathway has been demonstrated to enhance the tumorigenecity of the cervical cancer cell lines, and is also able to transform HPV-infected keratinocytes into cancer cells.

Aberrant microRNA (miRNA) expression is one the commonest factors leading to cervical cancer formation. MiR-135a is an oncogenic miRNA that transforms HPV-immortalized cervical epithelial cells to cancer cells. It was demonstrated that miR-135a triggers transformation by activation of the Wnt/β-catenin signaling pathway through suppressing the Wnt/β-catenin negative regulator SIAH1. A single miRNA can suppress many genes and affects multiple pathways; however, the summation effect of miR-135a in cervical carcinogenesis was unknown. Besides, the molecular events occurred during miR-135a induced transformation was unclear.

In this study, in order to investigate the molecular events occurred during miR-135a mediated transformation, an HPV immortalized non-carcinogenic cervical epithelial cell line NC104-E6E7 has been adopted as a model of the starting point of the transformation process. MiR-135a was force-expressed in the NC104-E6E7 cells by serial transient force-expression of miR-135a. The tumorigenic properties (invasion, migration, cell cycle progression and the percentage of CD〖133〗^+ subpopulation) of the miR-135a force-expressed cells were examined during the serial force-expression process. It was obvious that the tumorigenicity of the cells increased with increasing number of miR-135a force-expression. Such transformation process was accompanied by the initiation of epithelial-mesenchymal transition (EMT), when the markers E-cadherin decreased and N-cadherin increased their expression within this process. Moreover, the cells experienced 3 and 5 times of miR-135a force-expression could generate tumors in severe combined immunodeficiency (SCID) mice while the tumors formed from 5 time of miR-135 force-expressed cells are larger.

The expression profile of the serially transfected cell lines was determined by microarray analysis. Compared with the parent cell line NC104-E6E7, the miR-135a serially force-expressed cell lines 1#135a, 3#135a and 5#135a experienced the activation of the carcinogenic signaling pathways, such as ECM-receptor interaction, pathways in cancer and Toll-like receptor signaling pathway. Meanwhile, several genes relates to tumor formation have been differentially expressed. We validated the expression of CXCL14, ITGB6, FGF2 and IL-8 through quantitative polymerase chain reactions (qPCRs). Results of qPCRs were similar to that observed in microarray analysis. In the serially transfected cell lines, CXCL14 and ITGB6 were down-regulated, while FGF2 and IL-8 were up-regulated after the cells experienced serial miR-135a expression. To conclude, to the best of my knowledge, this is the first study to mimic the multistep-transforming process of cervical cancer, and to explore the molecular events during transformation. Serial miR-135a force-expression successfully transformed HPV-immortalized cervical epithelial cells into cancer cells. Expression profiles determined in this process provided clues for the early diagnosis and possible treatment of cervical cancer.