| Background and PurposesCervical cancer is the second most common gynecological malignant tumor in the world.In 2018,there were approximately 570,000 new cases and 311,000 new deaths worldwide owing to cervical cancer,accounting for 11.1%of the global female cancer-related deaths.At the time of diagnosis,80%of patients have developed invasive cancer,and the age at the time of diagnosis is slowly declining.In addition,the morbidity and mortality of cervical cancer remain high as a result of metastasis and recurrence.Thus,it is a necessity to identify novel and reliable biomarkers and therapeutic targets for cervical cancer.Long non-codingRNAs（lncRNAs）are widely defined as a transcript composed of more than 200 nucleotides and do not encode proteins owing to the lack of an open reading frame of the necessary length.More and more studies reported that lncRNAs is related to the molecular mechanism of cancer,and emphasize that it can be used as an early molecular marker for cancer diagnosis and therapeutic target.DNA methylation is a common epigenetic modification,which can enhance tumor progression and invasiveness by promoting cell proliferation or migration,disrupting cell communication and down-regulating apoptosis.Moreover,DNA methylation is an early molecular event in the process of tumor malignant transformation.Therefore,it is great significance to identify the aberrant methylation of lncRNAs for the early diagnosis and treatment of cervical cancer.LncRNA SOX21-AS1 is a newly discovered cancer-related lncRNA,located on human chromosome 13q32.1 with a length of 3230bp and its transcriptional direction is opposite to that of SOX21.It has been reported that SOX21-AS1 plays a key role in the progression of colorectal cancer,nephroblastoma,oral cancer and hepatocellular carcinoma.However,the role and mechanism of SOX21-AS1 in cervical cancer is not clear.Our provious study found that SOX21-AS1 has a certain relationship with cervical cancer by methylation chip and bioinformatics analysis.Therefore,this study takes this as a starting point to explore the role of SOX21-AS1in the pathogenesis of cervical cancer by analyzing the expression of SOX21-AS1 in cervical cancer cells.Materials and Methods1.Participants:patients who received routine outpatient TCT examination in the third affiliated Hospital of Zhengzhou University from January 2016 to January 2018were selected as subjects.In this study,the patients included in the study group displayed cytological and pathological indications of squamous cervical carcinoma（SCC）and HPV infection.Conversely,the patients included in the normal group displayed cytological features of mild inflammation without HPV infection.All patients were sexually active,not pregnant,without total hysterectomy,no history of miscarriage,medical treatment or surgery.Finally,a total of 43 clinical samples,including 23 normal cervical specimens（median age 48 years,range 26-65 years）and20 cervical cancer specimens（median age 50 years,range 28-68 years）were randomly selected for this research.Ethical consent was obtained from the Ethics Committee of the Third Affiliated Hospital,Zhengzhou University;2.The methylation levels of SOX21-AS1 in the two groups was detected by pyrophosphate sequencing;3.The expression levels of SOX21-AS1 in the two groups was detected by RT-PCR,and the data were analyzed by 2-△△Ct relative quantitative;4.Multiple experimental matrices（MEM）and annotation,visualization and comprehensive discovery database（DAVID）)were used to analyze the target genes and related pathways of SOX21-AS1;5.Small interferenceRNA and plasmids of SOX21-AS1 were used to transfect Si Ha cells.RT-PCR was used to detect the efficiency of silencing or overexpression of SOX21-AS1;6.Small interferenceRNA of FZD3 was used to transfect Si Ha cells,and RT-PCR and western blot methods were used to detect the efficiency of FZD3 silencing;7.The effects of SOX21-AS1 on FZD3 and Wnt pathyway were detected by western blot;8.The invasive ability of Si Ha cells was detected by transwell assays,the migration rate of Si Ha cells was detected by wound healing assays,the proliferation ability of Si Ha cells was detected by Cell Counting Kit-8（CCK-8）,and the apoptosis rate of Si Ha cells was detected by flow cytometry;9.SPSS 25.0 software was used for statistical analysis.Measurement data were expressed as mean±standard deviation（SD）,the differences between two groups were analyzed by means of T-test,and correlation analysis among the two data was conducted using Pearson correlation.ROC curve was used to evaluate the sensitivity and specificity of methylation in the diagnosis of cervical cancer.P<0.05 was considered to be statistically significant.Results1.The methylation level of SOX21-AS1 promoter in the study group was significantly lower than that in the control group（t=3.457,P<0.01）.According to the diagnostic criteria of Federation International of Gynecology and Obstetrics（FIGO）,cervical cancer samples were divided into two groups:Ib-IIa group and IIb-IIIc group.The results showed that the methylation level of SOX21-AS1 in IIb-IIIc group was significantly lower than that in the Ib-IIa group（t=3.46,P<0.01）;2.The expression levels of SOX21-AS1 and SOX21 was significantly increased in cervical cancer compared with the normal cervix（SOX21-AS1:t=4.636,P<0.001;SOX21:t=5.186,P<0.001）.In addition,the expression of SOX21-AS1 was positively correlated with SOX21（r=0.715,P<0.001）;3.Methylation statues of the SOX21-AS1 promoter region was negatively correlated with the expression levels of SOX21-AS1 and SOX21（SOX21-AS1,r=-0.531;SOX21,r=-0.540;both P<0.001）;4.Hypomethylation of SOX21-AS1 has high diagnostic value for cervical cancer,and the area under ROC curve is 0.775.When the Youden index was at its maximum value,SOX21-AS1 methylation showed 95.0%sensitivity and 55.2%specificity in the ROC analysis;5.Bioinformatics analysis showed that SOX21-AS1 might regulate Wnt signal pathway through FZD3;6.CCK-8 analysis manifested that knocking out SOX21-AS1 inhibited the proliferation of Si Ha cells（P<0.01）,The transwell experiment results manifested that knocking out SOX21-AS1 can restrain the invasion of Si Ha cells（P<0.01）,The results of the scratch experiment manifested that knocking out SOX21-AS1 could suppress the migration of Si Ha cells（P<0.01）,Meanwhile the results of apoptosis experiments indicated that the silence of SOX21-AS1 can promote Si Ha cell apoptosis（P<0.01）;7.CCK-8 analysis indicated that the up-regulation of SOX21-AS1 increased the proliferation of Si Ha cells（P<0.01）,The results of transwell experiments indicated that SOX21-AS1 boosted the invasion of Si Ha cells（P<0.01）,The scratch test manifested that SOX21-AS1 promotes the migration of Si Ha cells（P<0.01）,The apoptosis experiments showed that SOX21-AS1 inhibited Si Ha cell apoptosis in the meantime（P<0.01）;8.Western blot assays manifested that the down-regulation of SOX21-AS1 can down-regulate the protein expression level of FZD3 in cervical cancer cells,while the up-regulation of SOX21-AS1 can up-regulate the protein expression level of FZD3（P<0.01）,Apoptosis experiments indicated that silencing FZD3 can promote the apoptosis of Si Ha cells,and the stable expression of SOX21-AS1 can reverse the increase in apoptosis caused by FZD3 knockdown（P<0.01）.In addition,the results of transwell experiments manifested that silencing FZD3 can inhibit the invasion ability of Si Ha cells,and the stable expression of SOX21-AS1 can compensate for the reduced invasion ability caused by FZD3 knockdown（P<0.01）;9.Western blot results showed that the down-regulation of SOX21-AS1 could down-regulate the protein expression levels ofβ-catenin and c-myc in cervical cancer cells,while the up-regulation of SOX21-AS1 could up-regulate the protein expression levels ofβ-catenin and c-myc（P<0.01）.Conclusions1.SOX21-AS1 is an oncogene of cervical cancer,it can participate in the physiological and pathological process of cervical cancer by up-regulate the expression of FZD3 and activating Wnt pathway;2.SOX21-AS1 has potential value in the diagnosis of cervical cancer and is expected to play a certain role in clinical diagnosis and treatment in the future. |
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