The Relationship Between Expression Of MiR-218 And Malignancy Of Cervical Cancer And Pilot Studies Of Its Associated Mechanisms

Objects:The purpose of this study was to measure the expression level changes of microRNA-218 (miR-218) in normal cervical tissues, cervical intraepithelial neoplasm (CIN) and cervical cancer, and investigate the relationship between miR-218 expression levels with malignancy of cervical cancer. And miR-218 expression levels in serum were measured to study whether it could represent the changes of miR-218 in cervical cancer tissues. Then miR-218 concentrations were changed in cervical cell lines to study the effect of miR-218 on proliferation, apoptosis and cell cycle. Also the signal pathway of PI3K-Akt was determined to investigate whether this pathway was involved in the regulatory roles.Methods:1. We set up a method measuring the expression level of miR-218 using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and detected the expression levels of miR-218 in normal tissues, cervical cell line, cervical intraepithelial neoplasm (CIN) and cervical tissues.2. HPV types in tissues were measured using Hybrimax to study the effect of high risk HPV types on expression levels of miR-218.3. Small molecular RNA was extracted using extracting kit of Norgen company from serum and expression levels of miR-218 in serum were detected. 4. Using siPORT NeoFX, we transfected Pre-miR miRNA Precursor, Anti-miR miRNA Inhibitors with their negative control into cervical cell lines, and changes of mature miR-218 expression levels were measured in different time points,5. Proliferation, apoptosis and cell cycle of cervical cell lines were detected using MTT, flow cytometry, respectively.6. Changes of protein expression of PI3K regulatory subunit p85, p-Akt and Akt were determined using Western blotting and expression changes of p85a mRNA were measured by RT-PCR.Results:1. We developed a method successfully to determine the expression of miR-218 using universal probe library (UPL, Roche).2. We determined the expression of miR-218 in cervical cell lines and cervical tissues. In cell line, the expression levels from high to low were C-33A>Hela>SiHa. In cervical tissues, the expression levels were normal cervical tissues>CIN I>CINⅡ-Ⅲ>cervical cancer.3. Infection with high risk HPV types could reduce the expression levels of miR-218 in cervical tissues, and infection with low risk HPV types could not affect the expression levels in cervical tissues.4. The changes of miR-218 expression levels in serum had a close relationship with the changes of miR-218 in cervical tissues.5. After reverse transfection of miR-218 precursor/inhibitor into cervical cell, mature miR-218 expression levels were measured at three time points. The highest expression level of miR-218 was at 24 hour, and then gradually returned to normal.6. At 24 hour after reverse transfection, promoting the expression of miR-218 could enhance proliferation, decrease apoptosis of SiHa and HeLa cells. Otherwise, inhibiting the expression of miR-218 could reduce proliferation. And at 48 hour and 72 hour, proliferation and apoptosis were not affected by miR-218. Cell cycle of SiHa and HeLa cells were not affected by miR-218 at the three time points. Changes of miR-218 showed no effect on cell proliferation, apoptosis and cell cycle of C-33a cell.7. The up-regulation of miR-218 could increase the expression of p85a protein and phosphorylation of Akt, and down-regulation of miR-218 showed inverse effects. The mRNA of p85αwas not affected in this process.Conclusions:1. High risk HPV types could reduce the expression levels of miR-218, and low risk HPV types could not affect the expression of miR-218.2. The expression levels of miR-218 were reduced with the malignancy of cervical cancer, and this relationship could be represented in serum.3. miR-218 could regulate the proliferation and apoptosis of SiHa and HeLa, and these effects only existed when miR-218 was in the state of high concentrations.