The Research Of Temozolomide Inhibits The Proliferation Of Glioma Cells By Regulating MiR-223/PAX6

Objective:Temozolomide (TMZ) is a novel alkylating chemotherapeutic drug characterized as its effect and safety. TMZ has been verified to ameliorate glioma prognosis based on recent clinical researches, and it has been listed on first-line choices. However, the mechanism of its therapeutic effect on patients with malignant glioma remains to be discussed. Accumulating evidence showed that interactions between drugs and gene especially miRNAs help us understand the underlying mechanism and provide us novel molecular targets to improve chemotherapeutic effects of these drugs.miRNAs is the largest family of noncoding RNAs involved in gene silencing. The5’UTR nucleotide completely or incompletely binds to mRNA3’UTR of target gene, thus suppress its expression through direct translation inhibition, mRNA dissociation or deadenylation. Paired Box Gene6(PAX6) is a highly conserved transcription factor which has been associated with tumor suppressor. Besides, it has also been reported that PI3K/AKT signaling pathway is pivotal to regulate glioma growth and proliferation.This project aims to identify the role of PAX6on TMZ therapeutic mechanism through U251cell line proliferation monitoring, and find potential PAX6miRNAs against TMZ therapeutic effects. In addition, we will investigate the impact of interactions between those miRNAs and PAX6on TMZ inhibited U251cell proliferation, and test whether this effect was carried out via PI3K/Akt pathway. Methods:(1) Four groups of U251cells were treated with0,100,200,400μmol/L TMZ respectively, then the expressions of PAX6gene and protein in each group were quantified using real-time quantitative reverse transcription-PCR (qPCR) and Western Blot.(2) The expressions of PAX6and its protein in PAX6-shRNA transfected U251cells was quantified by qPCR and Western Blot.(3) The expressions of PAX6and its protein in PAX6ORF clone transfected U251cells was quantified by qPCR and Western Blot.(4) Proliferation rate was respectively detected using MTT and BrdU in the grouped U251cells pretreated by the following conditions: pcDNA3.1, PAX6ORF clone, TMZ+pcDNA3.1or TMZ+PAX6ORF clone.(5) Proliferation rate was respectively detected in grouped U251cells pretreated by Con-shRNA. PAX6-shRNA、TMZ+Con-shRNA or TMZ+PAX6-shRNA using MTT and BrdU.(6) PAX6targeted miRNAs were screened and predicted using miRanda and targetscan software.(7) The levels of miR-223, miR-590-3p, miR-190, miR-190b and miR-7were quantified in U251cells respectively treated with0,100,200or400μmol/L TMZ using qPCR.(8) The levels of miR-223in U251cells transfected by miR-223mimics or miR-223Sponge were tested using qPCR.(9) Proliferation rate was respectively detected using MTT and BrdU in the grouped U251cells pretreated by Vector, miR-223Sponge, TMZ or TMZ+miR-223Sponge.(10) Proliferation rate was respectively detected using MTT and BrdU in the grouped U251cells pretreated miR-SCR, miR-223, TMZ+miR-SCR or TMZ+miR-223.(11) The effect of miR-233overexpression on PAX6protein production was tested using dual-luciferase reporter system.(12) Proliferation rate was respectively detected in the grouped U251cells pretreated by:Vector+Con-shRNA, Vector+PAX6-shRNA, miR-223Sponge+Con-shRNA or miR-223Sponge+PAX6-shRNA using MTT and BrdU.(13) The expressions of PAX6, PI3K, p-PI3K, Akt, p-Akt protein were respectively detected in the grouped U251cells pretreated by miR-SCR, TMZ+miR-SCR or TMZ+miR-223using Western blot.(14) The expressions of PAX6, PI3K, p-PI3K, Akt, p-Akt protein were respectively detected in the grouped U251cells pretreated by miR-SCR+PCDNA3.1, miR-223+PAX6ORF clone or miR-223+PCDNA3.1using Western blot.Results:(1) The mRNA and protein expression levels of PAX6were significantly increased in those U251cell groups TMZ were given compared to the controlled group, especially in the group of400μmol/L concentration.(2) The mRNA and protein expression levels of PAX6in the PAX6-shRNA group were significantly decreased compared to the Con-shRNA group.(3) The mRNA and protein expression levels of PAX6in the pcDNA3.1group was significantly increased compared to the PAX6ORF clone group.(4) The U251cell proliferation was significantly inhibited in the PAX6ORF clone group. Compared to TMZ+PAX6ORF clone group with TMZ+pcDNA3.1group, U251cell proliferation was inhibited more apparent in TMZ+PAX6ORF clone group.(5) The U251cell proliferation was most significantly promoted in the PAX6-shRNA group while it was most significantly inhibited in the TMZ+Con-shRNA group. The difference between the Con-shRNA group and the TMZ+PAX6-shRNA group was not statistically significant.(6) The possible target miRNA of PAX6was screened as below: miR-223, miR-590-3p、miR-190、miR-190h、miR-7.(7)Compared with the control group, the expression of miR-223and miR-590-3p was significantly decreased while the expression of miR-190, miR-190b and miR-7was significantly increased in the TMZ treated cells of different concentration, especially in the group of400μmol/L(8) The expression of miR-223was significantly increased in the miR-223mimics transfected U251cells compared to the miR-SCR mimics group, while it was significantly decreased in the miR-223Sponge group than the Vector group.(9) The cell proliferation of U251cell lines was inhibited in the miR-223Spong group and TMZ group, and this inhibition impact is even more obvious when combination of these two drugs were applied.(10) The cell proliferation of U251cell lines was promoted in the miR-223group while it was inhibited in the TMZ+miR-SCR group. And this promotion is relieved in TMZ+mir-223group.(11) The dual luciferase reporter system demonstrated that miR-223can bind to the3’UTR region of PAX6mRNA directly.(12) The cell proliferation of U251cell lines was promoted in the Vector+PAX6-shRNA group while it was inhibited in the miR-223Sponge+Con-shRNA group. Besides, this promotion and inhibition was relieved in the miR-223Sponge+PAX6-shRNA group.(13) PI3K/Akt pathway was activated by miR-223, and miR-223can reverse the inhibition of PI3K/Akt pathway caused by TMZ.(14) PAX6ORF clone can reverse the activation of PI3K/Akt caused by miR-223. Conclusions:(1) PX6-ORF clone inhibits U251cells proliferation, which has synergistic effect with TMZ on anticancer effects.(2) miR-223silencing can inhibit U251proliferation, which has synergistic effect with TMZ on anticancer effects.(3) TMZ inhibits the proliferation of glioma cell by inhibiting the activity of PI3K/Akt pathway via regulating miR-223/PAX6.