MiR-155 Negatively Regulates Weel Expression In A549 Cells

BACKGROUNDLung cancer remains the most frequent cause of cancer death worldwide. Non-small-cell lung cancer comprises 70%~80% of all cases of bronchogenic carcinoma. The relapse rate among patients with early-stage NSCLC is 25%~50% within 5 years after potentially curative treatment, and overall survival rate is very disappointing. The mechanisms of tumorigenesis,tumour development and metastasis are still not well understood.miRNAs are non-coding, single-stranded RNAs of ~22 nucleotides and constitute a novel class of gene regulators that are found in both plants and animals. They negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or nearly perfect complementarity to proteincoding mRNA sequences induce the RNA-mediated interference (RNAi) pathway. Briefly, mRNA transcripts are cleaved by ribonucleases in the miRNA-associated, multiprotein RNA induced-silencing complex (miRISC), which results in the degradation of target mRNAs. This mechanism of miRNA-mediated gene silencing is commonly found in plants, but miRNA-directed mRNA cleavage has also been shown to occur in mammals. However, most animal miRNAs are thought to use a second mechanism of gene regulation that does not involve the cleavage of their mRNA targets. These miRNAs exert their regulatory effects by binding to imperfect complementary sites within the 3′- untranslated regions (3′-UTRs) of their mRNA targets, and they repress target-gene expression post-transcriptionally, apparently at the level of translation, through a RISC complex that is similar to, or possibly identical with the one that is used for the RNAi pathway. Consistent with translational control, miRNAs that use this mechanism reduce the protein levels of their target genes, but the mRNA levels of these genes are barely affected. They regulate diverse biological processes, and bioinformatic data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway.Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer. Nozomu Yanaihara, et al. reported that pre-miR-155 was frequently overexpressed in NSCLC. Patients with high expression of pre-miR-155 were found to have a significantly worse prognosis. The mechanisms of miR-155 worsening the prognosis of NSCLC patients have not been reported. Yoshida, et al. reported that absence of Wee1 expression was observed immunohistochemically in about two-thirds of the NSCLC patients. The reduction in Wee1 expression was associated with a poorer prognosis and a higher recurrence rate. The absence of Wee1 expression enhanced proliferation of NSCLC cells. Furthermore, Wee1 expression at the protein level may be regulated by other mechanisms, and not by gene expression at the DNA level. The effect of miR-155 on Wee1 expression in NSCLC cells is not well understood. After reviewing relative articles, using bioinformatics computational algorithms, we predicted the interaction between miR-155 and Wee1 mRNA 3'-UTR. We speculate that Wee1 mRNA may be the target mRNA of miR-155. miR-155 might regulate Wee1 expression and hence worsen the prognosis of NSCLC patients.OBJECTIVESTo determine the expression levels of mature miR-155 in NSCLC tissues and cell lines, observe the effects of miR-155 on expression levels of Wee1 mRNA and protein, and .investigate the biological significance of miR-155 regulating expression of Wee1.METHODS1. Expression of mature miR-155 in lung adenoma tissues, lung squamous cell carcinoma tissues, A549 cell line and H157 cell line: (1) Mature miRNAs were extracted using mirVanaTM miRNA Isolation Kit, then identification of mature miRNAs was performed using denaturing polyacrylamide gel electrophoresis and radioautography. (2) Using mirVanaTM qRT-PCR miRNA Detection Kit, mirVanaTM qRT-PCR Primer Set and SuperTaqTM Thermostable DNA Polymerase, absolute copy numbers of mature miR-155 in lung adenoma tissues, lung squamous cell carcinoma tissues, corresponding normal tissues, A549 cell line and H157 cell line were determined by constructing a standard curve of mature miR-155. Mature miR-155 was synthesized by Integrated DNA Technologies. Real-time quantitative reverse transcription PCR was performed using standard protocols on an Applied Biosystems 7500 Sequence Detection System.2. Prediction of the interaction between miR-155 and the 3'-UTR of Wee1 mRNA. To begin to investigate Wee1 gene as a target of miR-155 regulation, 3'- UTR of Wee1 mRNA sequence was analyzed by various computational algorithms which utilize distinct parameters to predict the probability of a functional miRNA binding site within a given mRNA target. Due to performance values (i.e. sensitivity and specificity in target prediction), we utilized the following three bioinformatics algorithms to predict the target sites. These three bioinformatics algorithms were miRanda (http://microrna.sanger.ac.uk), TargetScan (http://www.targetscan.org) and PicTar (http://pictar.bio.nyu.edu). The free energy (△G) of the 70 nucleotides flanking the 3'and 5'sides of the predicted miR-155 binding sides was determined using mFold Software.3. Effect of miR-155 on expression of Wee1 mRNA: 100nmol/L AMOs were used to inhibit the activity of mature miR-155 in A549 cells. Using Gapdh mRNA as internal control, relative real-time quantitative reverse transcription PCR was performed to detect the expression levels of Wee1 mRNA in A549 cells.4. Effects of miR-155 on expression of Wee1 protein and Phospho-cdc2 (Tyr15) protein: 100nmol/L AMOs was used to inhibit the activity of mature miR-155 in A549 cells. Using Histone H3 as internal control, western blot was performed to detect the expression levels of Wee1 protein and Phospho-cdc2 (Tyr15) protein.5. Effects of miR-155 on proliferation of A549 cell line and H157 cell line: A549 and H157 cells were divided into two groups: control group and AMOs group. AMOs administration group was subdivided into three groups by the concentration of AMOs: 10nmol/L, 50nmol/L and 100nmol/L. AMOs were used to inhibit the activity of miR-155, [3H]-TdR incorporation was measured by liquid scintillation counting. MTT assay was used to observe the inhibitory percentage of proliferation of A549 and H157 cells. Cell cycle was measured by flow cytometry.RESULTS1. Real-time PCR is the gold standard for gene expression quantification. Mature miRNAs were successfully extracted and identified using using denaturing polyacrylamide gel electrophoresis and radioautography. Compared with corresponding normal tissues, the expressiong level of mature miR-155 in lung adenoma tissues showed an increase by 35.38 folds. The expressiong level of mature miR-155 in A549 cells showed an increase by 37.95 folds versus normal tissues. Compared with corresponding normal tissues, the expression level of mature miR-155 in lung squamous cell carcinoma tissues showed an increase by 18.24 folds. The expression level of mature miR-155 in H157 cells showed an increase by 19.42 folds versus normal tissues.2. Predictionof the interaction between miR-155 and the 3'-UTR of Wee1 mRNA: All of three bioinformatics algorithms predicted the same miR-155 binding site. The△G was calculated using nucleotide sequence surrounding the predicted Wee1 miRNA binding site. The site had a lower△G than randomly expected (△G=-14.01kcal/mol).3. Effect of miR-155 on expression of Wee1 mRNA: After the Ct values were measured, Livak Method, also known as the 2△△Ct Method was used to determine the expression level of Wee1 mRNA in the test sample relative to the control sample. A549 cells treated with 100nmol/L AMOs expressed Wee1 mRNA at a 1.07-fold higher than control cells.We set the 2-fold as the threshold. If the change of expression level is beyond 2-fold, the result will be accepted as significance. According to this standard, compared with control cells, 100nmol/L AMOs did not exert significant effect on expression level of Wee1 mRNA in A549 cells.4. Effects of miR-155 on expression of Wee1 protein and Phospho-cdc2 (Tyr15) protein: In contrast to control cells, 100nmol/L AMOs significantly increased expression level of Wee1 protein in A549 cells (29.30±0.18 versus 9.51±0.29, P<0.05). In addition, 100nmol/L AMOs significantly increased expression level of Phospho-cdc2 (Tyr15) protein in A549 cells (37.02±0.11 versus 15.15±0.20, P<0.05).5. Effects of miR-155 on proliferation of A549 cell line and H157 cell line: Compared with control, the cell morphology of AMOs group was not significantly changed under microscope. 10nmol/L AMOs significantly reduced [3H]-TdR incorporation into A549 cells and H157 cells compared with control (P<0.01). Increasing concentration of AMOs had significant effect on reduction in [3H]-TdR incorporation in a dose-dependent manner (P<0.01). Compared with control, 10nmol/L AMOs significantly reduced OD of A549 cells and H157 cells (P<0.01). Increasing concentration of AMOs had significant effect on reduction in OD, simultaneously, inhibitory percentage was increased in a dose-dependent manner (P<0.01). Compared with control, 100nmol/L AMOs significantly decreased the cell ratios of G2/M phase and S phase, simultaneously increased the cell ratio of G0/G1 phase (P<0.01).CONCLUSIONS1. Mature miR-155 was over-expresssed in lung adenoma tissues, lung squamous cell carcinoma tissues, A549 cells and H157 cells.2. The miR-155 bingding site harbored in 3'-UTR of Wee1 mRNA was predicted by bioinformatics analysis. The miR-155 binding site was predicted to be accessible to miR-155.3. AMOs had no significant effect on expression level of Wee1 mRNA.4. AMOs significantly increased expression level of Wee1 protein and Phospho-cdc2 (Tyr15) protein.5. AMOs significantly inhibited proliferation of A549 cells and H157 cells in a dose-dependent manner.