Study On MicroRNA Expression Profiles And The Relationship With Its Target Gene In Cervical Cancer Of Uygur Women

Objective:To investigate the expression profiles of microRNAs(miRNAs)in cervical cancer of Uygur women. To detect the expression of most obvious differential microRNA in cervical cancer of Uygur women. To predict target genes of the miRNAs and detect the expression levels of target gene in cervical cancer tissues.To investigate the relationship between miRNA and its target gene.To provide new idea and approach for the molecular mechanism and play a foundation for exploring related biomolecular markers of cervical cancer.Methods:(1) Total RNA was isolated from 3 fresh samples of cevical cancer and 3 chronic cervicitis tissues by Trizol method. Then the total RNA samples were labelized with Cyanine 3-pCp by Agilent miRNA Complete Labeling and hyridized with miRNAs microarray V4.0 with 576 human miRNA probe. The hybridization microarrays were washed and scanned with Agilent microarray scanner. The data in the microarrays was extracted with Feature Extraction software. The significant differential expression miRNAs between cevical cancer and chronic cervicitis tissues were determined by t-test (P<0.05 and FoldChange?2 or?0.5) using Gene Spring software. To select two miRNAs were screening for Real-time PCR to validate; (2) To detect the expression of miR-101 in cervical cancer and chronic cervicitis tissues by real-time PCR.3. To detect the expression of most obvious difference miRNA of cervical cancer and chronic cervicitis tissues using Locked Nucleotide Acid In Situ Hybridization (LNA-ISH). (4) Three bioinformatics softwares for predicting miRNAs:Targetscan,PicTar and MiRanda were utilized to predict the target genes of miR-101, and selected the most closely related target gene of miR-101 refering the reported literature; (5) To detect the expression of EZH2 protein of cervical cancer and chronic cervicitis tissues using Western blot method; (6) Immunohistochemistry was used to detect the EZH2 protein expression in the cervial cancer and chronic cervicitis tissues, and then investigated the correlation between EZH2 protein expression and various clinicopathological parameters in cervical cancer.Results:(1) Aberrant microRNA expression in cervical cancer of Uygur women: From the microarray results we found that a set of aberrant miRNAs could clearly differentiate cervical cancer from chronic cervicitis tissues. A significant decrease of 12 miRNAs were detected in cervical cancer in comparison to control group, including hsa-miR-101, hsa-miR-572, hsa-miR-424, hsa-miR-409-3p, hsa-miR-365, hsa-miR-675, hsa-miR-187, hsa-miR-1247, hsa-miR-634, hsa-miR-1224-3p, hsa-miR-1238, hsa-miR-125a-5p. Then the result of quantitative RT-PCR analysis showed that has-miR-101 and has-miR-125a-5p were significantly downregulated in cancer tissues in comparison to control group (P<0.05), which inaccordance to the microarray results. The relative abundance of has-miR-101 in the experimental group and control group were 0.91?0.22 and 2.96±1.24 (P<0.05). The relative abundance of has-miR-125a-5p were 11.52±3.33 and 39.95±11.08 (P<0.05); (2) The expression of miR-101 in cervical cancer of Uygur women:RT-PCR results:The relative abundance of miR-101 in the experimental group and control group were 1.41±0.84 and 19±0.94 (P< 0.05). LNA-ISH results:Positive rate of miR-101 in the control group and experimental group were 72% (18/25) and 8%(2/25) (P<0.05); (3) The expression of target genes of miR-101 and its clinical significance in cervical carcinoma of Uygur women,and the relationship of miR-101 and its target gene:The all target genes of miR-101 by three bioinformatics softwares predicted were 103 genes. Refering the reported literature, EZH2 was a target of miR-101. Western blot results:Protein expression levels of EZH2 in cervical cancer and control group were 1.17±0.06 and 0.99±0.06 (P<0.05). Immunohistochemical results:Positive signal of EZH2 protein was yellow or brown located in the nucleus. The positive expression rate of cervical cancer and the control group were 61%(36/59) and 28. 9%(13/45)(p<0.05). The expression rate of EZH2 protein in cervical cancer was not correlated with all the parameters:age, the degree of differentiation and clinical stage (P >0.05). The correlation between miR-101 and EZH2 in quantitation results:R=-0.394, (P>0.05). But the correlation miR-101 and EZH2 in qualitation results of miR-101 and EZH2: R=-0.394, (P<0.05).Conclusion:(1) The study found 12 differential miRNAs in cervical cancer of Uygur women which were all downregulated in cervical cancer. The set of miRNAs might be involving in cervical tumorgenesis; (2) The quantity of miR-101 transcript in was much less than that of in chronic cervicitis tissue, and the quantity of EZH2 protein in cervical cancer was much more than that of in chronic cervicitis tissues. The expression of EZH2 protein in cervical cancer was not correlated with the age, the degree of differentiation and clinical stage of cervial cancer. There was a possible negative correlation between miR-101 and EZH2. The down-regulation of miR-101 and up-regulation of EZH2 protein may be one of the mechanisms in cervical cancer of Uygur women; (3) Microarray is a high-throughput detection technology of miRNAs expression profiles and Real-time PCR was a valid verification method. LNA-ISH was a convenience and reliable method of miRNA in situ expression.