The Role Of MicroRNA-1266 In The Pathogenesis Of Cervical Cancer By Affecting Cell Biological Behaviour Of Cervical Cancer Cells Through Targeting DAB2IP

Background Cancer is a disease in which cells in the body grow out of control.Cancer is always named for the part of the body where it starts,even if it spreads to other body parts later.The cervix connects the vagina(birth canal)to the upper part of the uterus,when cancer starts in the cervix,it is called cervical cancer.All women are at risk for cervical cancer.Human papillomavirus(HPV)is the main cause of cervical cancer.Cervical cancer is due to the abnormal growth of epithelial cells which could invade or spread to other parts of the human body.It needs to go through low-grade squamous intraepithelial lesion(LSIL)to high-grade squamous intraepithelial lesion(HSIL),and then finally develop into invasive cervical cancer.These pathological changes indicate that the occurrence of cervical cancer is closely related to the abnormal proliferation,migration and invasion abilities of cervical epithelial cells.Therefore,it is of great significance for the prevention and treatment of cervical cancer to clarify the specific mechanism of cell proliferation,migration and invasion of cervical cancer.A micro RNA(miRNA)is a small non-coding RNA molecule(containing about 22 to 25 nucleotides)found in plants,animals and some viruses,that functions in RNA silencing and post-transcriptional regulation of gene expression.MiRNAs function via base-pairing with complementary sequences within m RNA molecules.As a result,these m RNA molecules are silenced,by one or more of the following processes: Cleavage of the m RNA strand into two pieces,Destabilization of the m RNA through shortening of its poly(A)tail,and less efficient translation of the m RNA into proteins by ribosomes.The first miRNA was discovered in the early 1990 s.However,miRNAs were not recognized as a distinct class of biological regulators until the early 2000 s.MiRNA research revealed different sets of miRNAs expressed in different cell types and tissues and multiple roles for miRNAs in plant and animal development and in many other biological processes.MiR-1266 is a newly discovered miRNA in recent years with abnormal expression levels in gastric cancer,prostate cancer and other cancers.These studies suggested that miR-1266 may be involved in the occurrence and development of cancer,but its expression in cervical cancer has not been reported yet.Disabled homolog 2-interacting protein(DAB2IP)is a protein in humans which is coded by the DAB2 IP gene,its total length are about 1036 amino acids.DAB2 IP is a newly discovered tumor suppressor gene,which is closely related to the occurrence of biological behavior processes such as the proliferation,migration and invasion of cancer cells.Therefore,we collected the tissues from cervical cancer(CC)patients and Control Group(CG),collected the serum from CC patients,LSIL patients,HSIL patients and normal control(NC),and collected the survival situation data of CC patients in order to clarify the role of miR-1266.We used Linked Omics to analyze the related miRNAs of DAB2 IP from TCGA cancer data,and found that miR-1266 has close relationship with DAB2 IP in cervical cancers,so we aimed to find the internal connection between miR-1266 and DAB2 IP in this study.Besides,we performed cervical cancer cell experiment in vitro in order to find out the functions of miR-1266 on cell survival,proliferation,migration and invasion.We performed animal model of cervical cancer xenograft in nude mice in vivo in order to find out the facilitation of miR-1266 on growth of cervical cancer.We hope to further reveal the pathogenesis of cervical cancer through these experiments,in order to provide theoretical supports for the prevention and treatment strategies.Section ? The expression level of miR-1266 in cervical cancer tissues and serum and the overall survival rates of cervical cancer patientsObjective 1.To clarify the expression level of miR-1266 in the cancer tissues and serum of cervical cancer patients.2.To evaluate the overall survival rates of cervical cancer patients with high expression miR-1266.Methods 1.Collection of cervical tissue specimens,a total of 50 cervical cancer(CC)tissues were collected from CC patients who underwent cervical surgical resection without preoperative systemic therapy,while 50 normal tissues were collected from patients with chronic cervicitis(Control Group,CG)at The Third Affiliated Hospital of Zhengzhou University between October 2013 and December 2017.2.Collection of Serum samples,a total of 50 patients with Low-grade Squamous Intraepithelial Lesion(LSIL),50 patients with High-grade Squamous Intraepithelial Lesion(HSIL),50 patients with cervical cancer(CC)and 50 healthy controls(HC)who received therapy or had a physical examination were recruited at The Third Affiliated Hospital of Zhengzhou University between October 2013 and December 2017.3.All CC patients were taken on 50 months follow-up.The interval from the date of surgery to the date of recurrence or death defined as the follow-up period.The last follow-up was recorded on December 1st,2017.4.MiR-1266 expression level in cervical cancer tissues and serum was evaluated by quantitative real-time polymerase chain reaction(q RT-PCR).5.Statistical analyses were performed by using Graph Pad Prism 5.0 and SPSS 21.0.All data were expressed as the means ± standard error of the mean,and data were tested for normality test.T-Test(Independent Samples)was used for comparison between two groups,and variance analysis was used for comparison between three or more groups after testing homogeneity of variance.The significance level was ?=0.05.Results 1.Expression level of miR-1266 in the cervical cancer tissues of cervical cancer patients and cervical tissues of the control group.The expression level of miR-1266 in cervical cancer tissues determined by q RT-PCR was significantly increased compared with the Control Group,the difference was statistically significant(P < 0.05).2.Expression level of miR-1266 in the serum samples of the cervical cancer group,HSIL group,LSIL group and HC group.(1)The expression level of miR-1266 in the CC group was increased compared with HC group,the difference was statistically significant(P < 0.05).(2)The expression level of miR-1266 in the HSIL group was increased compared with HC group,the difference was statistically significant(P < 0.05).(3)The expression level of miR-1266 in the LSIL group was increased compared with HC group,the difference was statistically significant(P < 0.05).3.Survival analysis results measured by kaplan-meier analysis showed that a total of 50 CC patients with low miR-1266 had higher overall survival rates than patients with miR-1266 high expression.Brief summary MiR-1266 has a significant correlation with the survival and prognosis of patients with cervical cancer.Section ? The role of miR-1266 in regulating survival,proliferation,migration and invasion in cervical cancer cells through targeting DAB2 IP in vitro1.To evaluate the functions of miR-1266 in survival,proliferation,migration and invasion in cervical cancer cells.2.To clarify the internal relationship between miR-1266 and DAB2 IP.3.To definite whether miR-1266 could regulate the survival,proliferation,migration and invasion of cervical cancer cells by targeting DAB2 IP.Methods 1.Bioinformatics analysis websites(MiRanda,Target Scan and miRDB)were used to predict the potential downstream targets of miR-1266,and then Venn analysis was used to analyze the data.2.Western blot was used to analyze the expression level of DAB2 IP protein in cervical tissues of the control group and cervical cancer tissues of cervical cancer patients.3.Pearson correlation coefficient was used to analyze the correlation between miR-1266 and DAB2 IP in cervical cancer tissue.4.Using Dual-Luciferase Reporter Gene assay,luciferase reporter vectors containing the wild 3?-Untranslated Regions sequences of the DAB2 IP gene(WT-DAB2 IP 3?-UTR)or the mutant 3?-Untranslated Regions sequences of the DAB2 IP gene(MUT-DAB2 IP 3?-UTR)were co-transfected into cervical cancer cells with miR-1266 mimics or miR-negative control,respectively.The experiment was divided into four groups:(mimics + WT)group,(mimics + MUT)group,(NC + WT)group and(NC + MUT)group.Then the luciferase activities of each group were detected in order to verify the predicted results.5.Human cervical cancer cells He La and Si Ha cells were assigned to the miR-1266 mimics group(mimics),the miR-1266 inhibitor group(inhibitor)and the negative control group(NC),respectively.Cell counting kit-8 proliferation assay,Colony formation assay,Wound-healing assay and Transwell invasion assay were used to detect the survival,proliferation,migration and invasion abilities of cervical cells in each group,respectively.6.Cervical cancer cell lines He La and Si Ha were cultured.Lipofectamine 2000 was used to transfect DAB2 IP overexpressed plasmid.The experiment was divided into three groups: He La and Si Ha cells were co-transfected the miR-1266 mimics with DAB2 IP overexpressed plasmid(mimics + plasmid),were co-transfected the miR-1266 mimics with blank plasmid(mimics + control plasmid),and were co-transfected the miR-1266 negative control with DAB2 IP overexpressed plasmid(NC + plasmid),respectively.Western blot was used to analyze the expression level of DAB2 IP protein in cervical cancer cells.Cell counting kit-8 proliferation assay,Colony formation assay,Wound-healing assay and Transwell invasion assay were used to detect the survival,proliferation,migration and invasion abilities of cervical cells in each group,respectively.7.Graph Pad Prism 5.0 and SPSS 21.0 were used for statistical analyses.Data were expressed as the means ± standard error of the mean,and data were tested for normality test.T-Test(Independent Samples)was used for comparison between two groups,and variance analysis was used for comparison between three or more groups after testing homogeneity of variance.Correlations between DAB2 IP protein levels and miR-1266 levels were measured by using Pearson correlation coefficient analysis.The significance level was ?=0.05.Results 1.Expression level of DAB2 IP protein in cervical cancer tissues of cervical cancer patients and cervical tissue of control group.The expression level of DAB2 IP in cervical cancer tissues determined by Western blot was significantly increased compared with the Control Group,the difference was statistically significant(P < 0.05).2.The correlation between miR-1266 and DAB2 IP in cervical cancer.(1)The results measured by Pearson correlation coefficient analysis showed that there was a negative correlation between miR-1266 and DAB2 IP in cervical tissues of patients with cervical cancer,and the correlation coefficient r=-0.4255,the difference was statistically significant(P < 0.05).(2)The results analyzed by bioinformatics analysis softwares showed that miR-1266 may target DAB2 IP expression through binding its 3?-UTR,the sequences are located in the 3?-UTR region of DAB2 IP m RNA 996 to 1003 nucleotides.(3)The results analyzed by Dual-Luciferase Reporter Gene assay showed that the luciferase activities of cervical cancer cells in(mimics + MUT)group did not change obviously compared with(NC + MUT)group(P?0.05),the luciferase activities of cervical cancer cells in(mimics + WT)group were decreased obviously compared with(NC + WT)group,the difference was statistically significant(P < 0.05).3.Changes in the survival,proliferation,migration and invasion abilities of He La and Si Ha cells after transfection with miR-1266 mimics or inhibitors.(1)The survival,proliferation,migration and invasion abilities of He La and Si Ha cells when transfected with miR-1266 mimics were increased compared with the negative control group,the difference was statistically significant(P < 0.05).(2)The survival,proliferation,migration and invasion abilities of He La and Si Ha cells when transfected with miR-1266 inhibitor were decreased compared with the negative control group,the difference was statistically significant(P < 0.05).4.The expression level of DAB2 IP protein and changes in the survival,proliferation,migration and invasion abilities of He La and Si Ha cells after co-transfection of miR-1266 mimics and DAB2 IP overexpression plasmids in DAB2 IP gain or loss experiment.(1)The expression level of DAB2 IP protein in(mimics + plasmid)group was higher compared with(mimics + control plasmid)group,and was lower compared with(NC + plasmid)group,the difference was statistically significant(P < 0.05).(2)The survival,proliferation,migration and invasion abilities of He La and Si Ha cells in(mimics + plasmid)group were decreased compared with(mimics + control plasmid)group,and were increased compared with(NC + plasmid)group,the difference was statistically significant(P < 0.05).Brief summary MiR-1266 could promote the proliferation,migration and invasion of He La and Si Ha cells through targeting the expression of DAB2 IP.Section ? MiR-1266 promotes cervical cancer growth in vivoObjective Based on the results of Section? that miR-1266 works as a tumor promoter in cervical cancer in vitro,we continued to clarify the tumor positive role of miR-1266 in vivo though establishing cervical cancer xenograft model in nude mice.Methods 1.The cervical cancer cell line He La was cultured and transfected with miR-1266 mimics or negative control,respectively.2.Establish cervical cancer xenograft model in nude mice,we randomly divide 10 female nude mice into two groups including treatment group and control group.Treatment group,He La cells transfected with miR-1266 mimics were inoculated into subcutaneous of the back near the right hind limb in nude mice.Control group,He La cells transfected with miR-1266 negative control were inoculated into subcutaneous of the back near the right hind limb in nude mice. 3.Dietetic state and weight change of the He La tumor-burdened nude mice were observed and the tumor volumes and changes in body weight were monitored every 4 days for 28 days.Atotal of 7 times were recorded.4.After 28 days,we killed the nude mice,removed the tumor masses completely,and recorded the weight and measured the volume.The curves of tumor growth were drawn according to the tumor volumes.5.MiR-1266 expression level in cervical cancer tumor in nude mice was evaluated by q RT-PCR.6.The statistical analysis was similar to the methods used in Section?.Results 1.The cervical cancer tumor volume in the experimental group and control group.The tumor volumes in the treatment group were larger compared with the control group,the difference was statistically significant(P < 0.05).2.The cervical cancer tumor weight in the experimental group and control group.The tumor weight in the treatment group was higher compared with the control group,the difference was statistically significant(P < 0.05).3.The expression of miR-1266 in the experimental group and control group.The expression of miR-1266 determined by q RT-PCR in the treatment group was increased compared with the control group,the difference was statistically significant(P < 0.05).Brief summary MiR-1266 could promote the growth of cervical cancer in vivo,which could use as a new therapeutic marker.