Expression Of MIR-574-5p With Squamous Cervical Cancer And Its Its Effect On Siha Cells

1 Objectives and significanceCervical cancer is the most common in female reproductive malignant carcinoma and squamous cell carcinoma is its main pathological types,which causing more than three hundred thousand deaths each year.Although the cervical cytology screening benifites a lot in early detection and early treatment in recent decades,cervical cancer morbifity rates remain high,and there were more and more young cases recently.Infiltration and metastasis are two important factors for the rapid development of malignant tumor,and the clinical research shows that the prognosis of the patients with metastatic cervical cancer is poor.Therefore,it is important to explore the mechanism of cervical cancer development for clinical effective treatment of cervical cancer.MicroRNA(miRNAs)are small nod-coding RNAs of approximately 19-22 nucleotides(nt)and act as post-trsnscriptional regulators genes involved in diverse the malignancy biological processes.Our early experiments by hybridization arrays compared the miRNA expression profile between cervical cancer and healthy controls,which showed that 89 miRNAs were examed,and miR-574-5p was up-regulated significantly.In this study,we aim to evaluate the expression of tissue/serum miR-574-5p by Real-Time PCR,and analysis the miR-574-5p expression level’s correlativity with clinicopathologic features in squamous cervical cancer.Transfecting artificially synthesized of miR-574-5p mimics and inhibitor to up-regulate and down-regulate its expression in SiHa cells,we can observe the effect of miR-574-5p on proliferation/invasion and migration/cell cycle and apoptosis of SiHa cells.Using bioinformatics technology such as miRBase,TargetScan databases to forecast miR-574-5p’s target gene and validating its target gene by qRT-PCR and Western-Blot in mRNA and protein respectively.Through the above methods to up-regulate and down-regulate expression of miR-574-5p,we can illuminate the molecular mechanism of processes in cervical cancer which provide a new idea and method to more effective treat in clinical.2 Methods(1)All the samples were collected in Guangxi Medical University Affiliated Tumor Hospital from surgery of cervical biopsy or total hysterectomy from March 2011 to April 2013.All the tissue and serum samples were collected before treated with any therapies.The expression level of tissue and serum miR-574-5p in 80 patients with squamous cervical cancer and 80 women with normal cervical tissue as controls were detected by Real-Time PCR,and the relationship between the level of tissue and serum miR-574-5p and its clinicopathologic features were analyzed by Spearman correlation analysis.(2)Cultured the cervical cancer SiHa cell,and transfected the miR-574-5p mimics and inhibitor in the cells through lipo2000,and divided into four groups(Blank,NC,Mimics and Inhibitor)according to the objective.The transfection rate was detected by the qRT-PCR.Cellular proliferation capacity was assayedby the CCK-8 and flat panel clone experiment.The abilities of migration and invasion were detected by Transwell experiment,Cell cycle and cell apoptosis were tested by FCM.(3)Using bioinformatics technology such as miRBase,TargetScan databases to forecast miR-574-5p’s target gene and validate its target gene by qRT-PCR and Western-Blot.3 Results(1)We found that the expression levels of tissue and serum miR-574-5p were significantly higher in patients with squamous cervical cancer than that in normal controls(P<0.001);up-regulation expression of miR-574-5p had a significantly difference and a positive correlation with the International Federation of Gynecology and Obstetrics(FIGO)stage,lymph node metastasis and pathological grade of squamous cervical cancer(P<0.05),while it had no expression difference with the patients,age and menopausal status(P>0.05).(2)The expression of miR-574-5p inhibitor and miR-574-5p mimics were lower or higher than another two guoups(P<0.001),but there was no diference between NC and Blank(P>0.05)according to the qRT-PCR.(3)Test of SiHa cell function:The result of CCK-8 showed that the cell proliferation ability of the miR-574-5p mimics was ascended significantly from the time of 24th hour to 120th hour;and there was opposite result in the inhibitor compared to another two groups.The colony formation rate of the four groups had the same result in CCK-8 test.The result of Transwell invasion and migration tests showed that the number of cells in the miR-574-5p mimics group which crossed the wound region was significantly much more when compared with the control group(P<0.01);and the number was much less in miR-574-5p inhibitor group.The result of cell apoptosis shouwed that the apoptotic cells in the miR-574-5p inhibitor group was significantly more than the other two guoups;and mimics group had opposite result.With the cell cycle detection showed that there was no significantly in mimics and inhibitor compared to Blank and NC(P>0.05).(4)The prediction and validation of miR-57-4p’s target gene revealed that FOXO3 was selected as the target gene after searching the database.The relative expression level of FOXO3 mENA and FOXO3 protein with miR-574-5p mimicis were significantly more than the other there guoups detected by qRT-PCR and Western-Blot(P<0.05),and the result was opposite with miR-574-5p inhibitor.4 ConclusionsThe relative expression of miR-574-5p in tissue and serum were higher than normal group,and it increased gradually with tumor differentiation,clinical stage,tumor diameter and lymph node metastasis.We can inferred that miR-574-5p should promoted the progress of cervical cancer which provided the basis of subsequent experiments.MiR-574-5p can promote the SiHa cell ability in proliferation,migration and invasion;and it can also inhibit the cells’ apotosis to promote the cervical cancer.Inhibiting the expression of FOXO3 which was one of miR-574-5p’s targets should be the molecular mechanism in promoting progress of cervical cancer.