Hsa?circ?0000839 Inhibits Papillary Thyroid Carcinoma Proliferation By Targeting CDC27

Objective: Thyroid cancer is one of the most common endocrine tumors.Papillary thyroid carcinoma(PTC)is the most common pathological subtype of thyroid cancer.In recent years,the incidence of PTC has increased dramatically,and a large number of papillary thyroid microcarcinoma(PTMC)with a diameter of less than 1 centimeter has been detected.Some scholars pointed out that the incidence of thyroid cancer in recent years was caused by overdiagnosis.Therefore,it is necessary to explore the biomarkers of PTC and the related molecular mechanisms of their effects to predict the prognosis of thyroid cancer before surgery.Circular RNA(circRNA)is a kind of recently discovered endogenous non-coding RNA,which is characterized by the circular structure.Previous studies have revealed the evolutionary conservation,high abundance and structural stability of circRNA in various tissues and cell lines.The stable structure of circRNA makes it an ideal diagnostic and prognostic biomarker for many diseases.In addition,circRNA has many important molecular biological functions,including micro RNA(miRNA)sponges,combining with RNA binding proteins to regulate the expression of target genes.In recent years,more and more reports have shown that circRNA can competitively bind to miRNA and affect the expression level of the target genes,thereby further regulating tumor development.In this study,we aim to study the circRNA associated with the PTC progression,and further study the molecular mechanism of circRNA in the PTC cell lines.Our research found that hsa?circ?0000839 was significantly underexpressed in the tissue and plasma samples of PTC patients.Moreover,hsa?circ?0000839 could inhibit the cell proliferation of PTC by regulating CDC27,and further explain the specific mechanism of hsa?circ?0000839 regulating the expression level of CDC27.Methods: 1.We collected 118 pairs of PTC and adjacent non-cancerous tissues and the related clinicopathological data at the First Affiliated Hospital of China Medical University,from May 2018 to May 2019.Meanwhile,preoperative serum samples from50 PTC patients and matched healthy volunteers were collected at the First Affiliated Hospital of China Medical University,from May 2018 to August 2020.2.The expression levels of hsa?circ?0000839 in tissues and serum samples were detected by q RT-PCR.Chi-square test was used to analyze the relationship between the expression of hsa?circ?0000839 in PTC tissues and clinicopathological data.3.We downregulated or upregulated the expression of hsa?circ?0000839 in PTC cell lines by transfecting si RNA or lentivirus,and used q RT-PCR to detect the interference efficiency.CCK8 and Edu were used to detect the effect of hsa?circ?0000839 on PTC cell proliferation.Flow cytometry was used to detect the effect of hsa?circ?0000839 on PTC cell cycle.4.After silencing hsa?circ?0000839,the expression of m RNA was detected by RNA sequencing.The expression of CDC27 was detected by q RT-PCR and western blotting,after downregulating or upregulating the expression of hsa?circ?0000839.5.We used q RT-PCR to detect the expression of CDC27 in 118 pairs of PTC and adjacent tissues.Spearman correlation was used to test the relationship between hsa?circ?0000839 and CDC27 expression in PTC tissues.The TCGA database was used to analyze the relationship between the expression of CDC27 in PTC tissues and clinicopathological data.6.We downregulated the expression of CDC27 in PTC cell lines by transfecting si RNA,and used q RT-PCR to detect the interference efficiency.CCK8 and Edu were used to detect the effect of CDC27 on PTC cell proliferation.7.We used the biological information database to predict miRNAs that could bind to both hsa?circ?0000839 and CDC27.After changing the expression of hsa?circ?0000839,q RT-PCR was used to detect the expression of miR-149-5p.The expression of CDC27 was detected by q RT-PCR and western blotting in PTC cells transfected with miR-149-5p mimics.8.We used RIP and luciferase reporter gene assays to prove that miR-149-5p can directly bind to hsa?circ?0000839 and CDC27.9.The expression of CDC27 was detected in PTC cells co-transfected with hsa?circ?0000839 lentivirus and miR-149-5p mimics.CCK8 and Edu were used to detect in co-transfected PTC cells to confirm that hsa?circ?0000839inhibited the cell proliferation by competitively binding miR-149-5p.Results: 1.Hsa?circ?0000839 was significantly underexpressed in the tissues and plasma samples of PTC patients,and its expression was negatively correlated with tumor diameter.2.Hsa?circ?0000839 could inhibit the proliferation of PTC cells in vivo and in vitro.Changing the expression of hsa?circ?0000839 in PTC cells could affect the cell cycle progression.3.Silencing the expression of hsa?circ?0000839 in PTC cells could significantly downregulate the m RNA and protein levels of CDC27,while overexpression of hsa?circ?0000839 had the opposite effect.4.CDC27 was significantly underexpressed in PTC tissues,and is significantly related to the expression of hsa?circ?0000839.5.Silencing CDC27 in PTC cells could inhibit the cell proliferation of PTC.6.RIP and luciferase assays proved that miR-149-5p could directly bind to hsa?circ?0000839 and CDC27,respectively.7.Silencing or overexpression of hsa?circ?0000839 in PTC cells could affect the expression of miR-149-5p,and overexpression of miR-149-5p in PTC cells reduced the expression of CDC27.8.Hsa?circ?0000839 regulated the expression of CDC27 by competitively binding miR-149-5p.9.Hsa?circ?0000839 inhibited cell proliferation by competitively binding miR-149-5p.Conclusions: Hsa?circ?0000839 is significantly underexpressed in the PTC tissues and plasma samples of PTC patients,and its expression is negatively correlated with tumor diameter.Hsa?circ?0000839 regulates the expression of CDC27 by competitively binding miR-149-5p in PTC and inhibits the cell proliferation.Hsa?circ?0000839 can serve as a potential biomarker and therapeutic target of PTC.