| PurposeCervical cancer is one of the most common malignant tumors among women,accounting for the fourth cancer death among women.Most of the onset of cervical cancer associated with the type of high-risk human papilloma virus,the existing research evidence shows that the type of high-risk human papillomavirus and cervical cancer there is a cause-and-effect relationship,therefore,can be more perfect before cervical cancer screening to prevent the occurrence and development of cervical cancer,and primary prevention of cervical cancer is the best way to reduce cervical cancer mortality.However,screening for cervical precancerous lesions has not been fully promoted and applied in developing countries,which leads to the increasing incidence and mortality of cervical cancer in many underdeveloped areas.At the same time,high-risk HPV screening means can not completely prevent the occurrence of cervical cancer,the collection of specimens in the process of location,whether the specimen is sufficient and the cost of screening,all have adverse effects on the patient’s body and mind.Therefore,it is imperative to understand the molecular mechanism of the occurrence and development of cervical cancer and to find reliable and effective therapeutic targets.As one of the endogenous non-coding RNAs,mature circRNAs do not have a 5 ’cap and a 3’ polyadenylate tail,but form a covalent circular closed structure through the form of anti-splicing.This circular structure makes it highly stable and difficult to be hydrolyzed by RNA enzymes,and plays a key role in the occurrence of cancer.circSPECC1(hsa＿circ＿000745)has been proved to be related to the occurrence and development of malignant tumors,but its role and mechanism in cervical cancer are seldom reported.This study is to investigate the relationship between the two.Methods1.This study first retrieve the GEO,cervical cancer circRNA expression spectrum data in the database and get the GSE102686 cervical cancer data set,we applied GEO2 R online software analysis found that differentially expressed circRNA(P < 0.05,| log FC | > 2);2.The expression of circSPECC1 in 60 cases of cervical squamous cell carcinoma tissues and corresponding paracancerous tissue specimens as well as in cervical cancer cell lines Siha cells and Caski cells was detected by q RT-PCR method,and the correlation between circSPECC1 and some clinicopathological parameters of these 60 cases was analyzed.3.We designed two kinds of small interfering RNAs to change the expression levels of circSPECC1 in cervical cancer cell lines Si Ha and Caski,and detected the effects of circSPECC1 on the proliferation of cervical cancer cell lines Si Ha and Caski by clone formation assay and CCK-8 assay.The effect of circSPECC1 on the migration ability of cervical cancer SIHA and Caski cells was detected by Transwell assay.4.According to our previous experiments,firstly,we detected whether the expression of SHP-2 protein was changed when the expression of circSPECC1 was changed by Western-blot test;Then we transfected SHP-099(a SHP-2 inhibitor)to down-regulate the expression of SHP-2 to observe whether it had any effect on the promotion of circSPECC1 on the proliferation and migration of cervical cancer SIHA and Caski cells.5.Finally,the miRNAs with common binding sites of circSPECC1 and SHP-2 were predicted by TCGA and miRWalk database.6.We verified that hsa-miRNA-409-3p had direct binding sites with circSPECC1 and SHP-2m RNA through in situ immunofluorescence assay,q RT-PCR,RIP assay and double luciferase reporter gene assay.7.The dual luciferase reporter gene assay,q RT-PCR assay and Western-blot assay were used to verify the regulatory effect of hsa-miRNA-409-3pmimics on its target gene SHP-2protein.8.Finally,CCK-8,clone formation assay and Transwell assay were used to test the effect of up-regulation of hsa-miRNA-409-3p expression on the proliferation and migration of circSPECC1 cells.Results1.The expression of circSPECC1 was significantly increased in cervical cancer tissues and cells,and was significantly correlated with FIGO stage and tumor size of patients.2.Circ SPECC1 promotes the proliferation and migration of cervical cancer SIHA and Caski cells;3.The overexpression of circSPECC1 and SHP-2 increased in clinical tissues of cervical cancer;4.Overexpression of circSPECC1 can promote the proliferation and migration of cervical cancer Siha and Caski cells,while inhibition of SHP-2 can weaken this promotion.5.Dual luciferase reporter gene assay and RIP assay proved that hsa-miR-409-3p had a direct binding site with circSPECC1;Dual luciferase reporter gene assay proved that hsa-miR-409-3p had direct binding sites with SHP-2m RNA.6.When the expression of miR-409 was changed,the expression of SHP-2protein was significantly changed.7.The overexpression of hsa-miRNA-409-3p can reverse the promoting effect of circSPECC1 on the proliferation and migration of cervical cancer cells.Conclusion1.The expression of circSPECC1 was up-regulated in cervical cancer tissues,and its expression level was statistically correlated with FIGO stage,tumor size,T stage,etc.2.Downregulation of circSPECC1 expression can inhibit the invasion and migration of cervical cancer cells;3.Circ SPECC1 may affect the proliferation and migration of cervical cancer cells through SHP-2.4.Circ SPECC1 can affect the proliferation and migration of cervical cancer cells by regulating SHP-2 protein by adsorption of hsa-miR-409. |
