WAPAL Gene And Its Target MicroRNA In Cervical Cancer

ObjectivemicroRNAs (miRNAs), newly found RNAs of 19-25nt single stranded noncoding, are distributed widely in Eukaryotes from elegans to human beings. miRNAs have the characteristic of highly evolutionarily conservation and exhibit tissue-specific or developmental-stage-specific expression. miRNAs act as gene regulation molecules and function through the regulation of target genes by imperfect or perfect base-pairing with their target mRNAs, then either lead to the degradation of the target transcript or the inhibition of the protein translation. Therefore, miRNAs play an important role in gene regulation network, including the cellular differentiation, proliferation, apoptosis and oncogenesis.Human wings apart-like (WAPAL) is a new sister chromatid islocated regulation factor. Recent research has found that the WAPAL plays an important role in normal cellular growth and tumorigenesis. The expression of WAPAL is closely related with cervical cancer. miR-15a and miR-26b have been known as the function of tumor suppressor gene in many tumors. However, the expression of miR-200b is significant difference in the different tissues of tumor. The expression of these three miRNAs has a notable correlation with therapeutic effect and prognosis of patients. We found that miR-15a, miR-26b and miR-200b might be the target micoRNAs of the WAPAL gene by using bioinformatic methods.In reviewing the literature, very little was found on the association between miRNAs and the expression of WAPAL gene. To explore the effect of predicted miRNAs on the expression of WAPAL gene, this project did a series of research. First, we examined the WAPAL gene expression in cervical cancer and investigated its relation with HPV infection. Then, we detected the expression of miR-15a, miR-26b and miR-200b in human cervical cancer. The results showed that the expression of miR-15a and miR-26b decreased in cervical cancer. In contrast, the expression of miR-200b significantly increased in cervical cancer. A significant negative correlation was observed between the expression of miR-26b and WAPAL mRNA. The expression of miR-200b was positively correlated with the expression of WAPAL. Based on our results, miR-26b is reasonably predicted to be the most potential target miRNA of the WAPAL gene. Furthermore, miR-26b eukaryotic vector was constructed and transfected into SiHa cells of cervical cancer cell line. mRNA expression and protein expression of WAPAL gene in transfected SiHa cells was tested by RT-PCR, Western and immunohistochemical method. This study may provide new ideas and methods for understanding the regulation mechanism of WAPAL gene expression and for the gene diagnosis and therapy of cervical cancer in future. Methods一,Specimen source:For the immunohistochemistry analysis of WAPAL protein,176 examples of paraffin-embeded tissues were selected from January 2006 to December 2008 in pathology department of the second affiliated hospital of Zhengzhou University, including 29 cases of cervical intraepithelial neoplasia (CINI),35 cases of CINⅡ,39 cases of CINⅢ,48 cases of cervical cancer, and 25 cases of normal cervical tissues. The patient's age is 29～57 years old, average age 43 years old.36 fresh tissues examples of operative cervical cancer were collected from September 2007 to December 2008 in gynecology department of the second affiliated hospital of Zhengzhou University, including 9 cases of G1 grade (high-differentiated cancers),19 cases of G2 grade (middle-differentiated),8 cases of G3 grade (low differentiated carcinoma). At same time,36 examples of normal cervix were collected from the excised uterus of benign gynecologic disease. All specimens were confirmed pathologically with HE dyeing. Tumor nuclei of cervical cancer specimens were more than 80%. The specimen divided into cervical cancer group and the normal cervical group. All patients did not receive any pre-radiotherapy or pre-chemotherapy.二,Experimental Method1. SP immunohistochemistry was used to examine the expression of WAPAL in cervical cancer samples, cervical intraepithelial neoplasia(CIN) and normal cervical samples. The expression mRNA level of the WAPAL gene in cervical cancer tissues and normal cervix was detected by real time PCR. Microarray tech was used to detect the HPV infection of different cervical samples.2. Bioinformatics strategy predicted the target miRNAs of WAPAL gene. The expression level of miRNAs (miR-15a, miR-26b and miR-200b) in human cervical cancer and normal cervix was measured by TaqMan real time PCR. 3. miR-26b eukaryotic vector was constructed. After miR-26b eukaryotic vector was transfected into SiHa cells by Lipofectamine 2000, fluoremicroscope was applied to observe the transfectional effect. The expression of WAPAL gene in transfected SiHa cells was tested with Western and immunohistochemistry methods.三,Statistical MethodsThe experimental data were analyzed by SPSS 11.0 software. One-way ANOVA was used for the statistics of immuno-staining score. Chi- square test was used for the statistics of positive rate. For real-time PCR results, the independent sample t-test was used to examine the differences between the group of normal cervix and group of cervical cancer. a=0.05 was considered significant.Results1. The expression of WAPAL gene significantly increased in cervical cancer. The protein expression of WAPAL gene increased gradually with increasing of cervical intraepithelial neoplasia. The expression mRNA level of the WAPAL gene was significantly higher in tumor tissues than in normal tissues. The WAPAL protein expressions in cervical lesions of HPV 16/18 infection were significantly increased, compared with the lesions of negative HPV and HPV 6/11 infection.2. The mRNA expression of miR-15a was lower in tumor tissues than in normal tissues(P>0.05), and expression fold change was 2.985. The mRNA expression of miR-26b was lower in tumor tissues than that in normal tissues (P<0.01), and expression fold change was 5.618. The mRNA expression of miR-200b was obviously higher in tumor tissues than normal tissues (,P<0.05), and expression fold change was 45.71.The mRNA expression of WAPAL gene was negatively correlated with expression of miR-26b (P<0.05), and positively correlated with expression of miR-200b (P<0.05).3. Restriction enzyme digestion and sequencing analysis showed the vector sequence was coincidence with miR-26b.We successfully obtained recombination vectors. Green fluorescence could be observed in post-transfectional SiHa cells with fluoremicroscope, thus demonstrating that the transfection also was successful. The protein and mRNA expression of the WAPAL gene was dramatically decreased after the miR-26b was transfected in to SiHa cells (P<0.05)Conclusions1. WAPAL gene is special over-expressed gene in cervical cancer, and the expression level of WAPAL is significantly associated with the HPV infection.2. miR-26b and miR-200b are abnormally expressed in cervical carcinoma tissues, and their expression is obviously associated with the WAPAL expression. All these indicate that miR-26b might play a tumorgenesis role, miR-200b as a role of tumor suppressor gene in the development of cervical carcinoma. This study may provide a new method for the gene diagnosis and therapy of cervical cancer in future.3. miR-26b might involve the regulation mechanism of WAPAL gene.