| Purposes.1.To find highly expressed circRNA in papillary thyroid carcinoma(PTC)by High-throughput sequencing.2.To verify the expression level and circular structure of hsa?circ?0076710 in PTC cells.3.To investigate the biological function and molecular mechanism of has?circ?0076710 in PTC cells.4.To explore the clinical value of hsa?circ?0076710 as a PTC tumor marker.Methods.1.The Differential expression profile of circRNA in PTC was construct by 5 pairs of PTC tumor and tumor-distant normal tissues with high-throughput sequencing.According to this,we screened the differential circRNA and selected the target circRNA.2.The expression level was verified in thyroid cancer cell lines by quantitative real time polymerase chain reaction(q RT-PCR).Structural verification included:(1)Sequence verification: divergent primers was designed to perform Sanger sequencing with the PCR product of hsa?circ?0076710;(2)Primer verification: Design convergent primers and divergent primers respectively,and perform polymerase chain reaction(PCR)amplification on c DNA and g DNA.(3)RNase R tolerance verification: After adding RNase R,hsa?circ?0076710 and its parental m RNA were amplified by q RTPCR.(4)Reverse transcription verification: The reverse transcription of hsa?circ?0076710 and its parental m RNA was performed with Oligo(d T)and random hexamer primers,respectively.3.After interfering with hsa?circ?0076710,the effect of hsa?circ?0076710 on the proliferation ability of PTC cells was clarified by CCK8,plate clone formation and Ed U experiments.The effect of hsa?circ?0076710 on the migration ability of PTC cells was verified by wound healing assay and Transwell experiments.4.After establishing a stable cell line overexpressing hsa?circ?0076710,the effect of hsa?circ?0076710 on the proliferation ability of PTC cells was determined by CCK8,plate clone formation and Ed U experiments.The effect of hsa?circ?0076710 on the migration ability of PTC cells was verified by wound healing assay and Transwell experiments5.The pathway was screened by Western-blot(WB)experiment,and the sponge molecular mi RNA of the target gene was predicted by Targetscan and Starbase database.mi RNA of hsa?circ?0076710 was predicted by circBank database,and the intersection of the three datas was used to determine the candidate mi RNA.It was further verified by the dual luciferase reporter gene experiment.6.Using CCK8,plate clone formation,Ed U,wound healing assay,Transwell,WB experiments,si-hsa?circ?0076710 was rescued by mi R-214-3p inhibitor.7.Using CCK8,plate clone formation,Ed U,wound healing assay,Transwell,WB experiments,OE-hsa?circ?0076710 was rescued by mi R-214-3p mimics.8.203 cases of PTC and 20 cases of benign thyroid were included,and q RT-PCR was used to detect tumor tissues,tumor-distant normal tissues,ultrasound-guided fine needle aspiration biopsy(FNAB),peripheral serum of PTC and benign patients,respectively.Using the area under the curve(AUC)of the receiver operator characteristic curve(ROC)to analyze the predictive value of hsa?circ?0076710 for the diagnosis and prognosis of PTC.The correlation between the expression of hsa?circ?0076710 and the invasive pathological characteristics of PTC was further analyzed by logistic regression.Results.1.Screening and structural verification of hsa?circ?0076710(1)Taking |fold change|>2 and P<0.05 as the standard,a total of 85 differentially expressed cicr RNA were screened,of which 44 were up-regulated and 41 were downregulated.The top ten up-regulated molecules were verified in the PTC cell line TPC-1,and the verification results were consistent with the sequencing results.In vitro cell experiments showed that hsa?circ?0076710 was highly expressed in PTC cell lines TPC-1,K1,and BCPAP(P<0.01).(2)Sanger sequencing has confirmed that hsa?circ?0076710 is reverse spliced and the circularization site was correct.(3)Both convergent primers and divergent primers can amplify products in c DNA,but only convergent primers can amplify products in g DNA.(4)After digestion with RNase R,the abundance of m RNA was significantly reduced(P<0.01),while the abundance of hsa?circ?0076710 had no significant change(P>0.05).(5)m RNA can be reverse transcribed using Oligo(d T)and random hexamer primers at the same time,and there is no statistical difference in the yield between the two(P>0.05).While hsa?circ?0076710 can only be reverse transcribed by random hexamer primers to obtain effective c DNA.2.Biological function and mechanism exploration of hsa?circ?0076710(1)Interfering with hsa?circ?0076710 significantly could inhibit the proliferation and migration of TPC-1 and K1 cells(P<0.01).Overexpression of hsa?circ?0076710could significantly promoted the proliferation and migration of TPC-1 and K1 cells(P<0.01).(2)After interfering with hsa?circ?0076710,the results of RNA sequencing showed that the NOTCH pathway was significantly inhibited,and WB was used to confirmed that JAG1,LFNG,NOTCH2,and HES1 were significantly down-regulated.(3)The dual-luciferin reporter gene results showed that mi R-214-3p was a common sponge molecule of hsa?circ?0076710 and JAG1.mi R-214-3p inhibitor and mi R-214-3p mimics were treated in thyroid cancer cells,and the results confirmed that the former could promote the proliferation and migration of PTC cells,while the latter inhibited the proliferation and migration of PTC cells.(4)After co-transfection of si-hsa?circ?0076710 and mi R-214-3p inhibitor,mi R-214-3p inhibitor could rescue the inhibitory effect of si-hsa?circ?0076710 on cell proliferation and migration(P<0.01).After co-transfection of OE-hsa?circ?0076710and mi R-214-3p mimics,it was found that mi R-214-3p mimics could restore the promoting effect of OE-hsa?circ?0076710 on cell proliferation and migration(P<0.01).(5)After interfering with hsa?circ?0076710 and mi R-214-3p at the same time,WB results showed that mi R-214-3p inhibitor could rescue the expression of JAG1,NOTCH2 and HES1,which were up-regulated compared with only interfering with hsa?circ?0076710(P < 0.01).After co-transfection with OE-hsa?circ?0076710 and mi R-214-3p mimics as a combination,WB results showed that mi R-214-3p mimics could rescue the expression of JAG1,NOTCH2 and HES1 in cells,which was downregulated compared with overexpression of hsa?circ?0076710 only(P < 0.01).3.Clinical value analysis of hsa?circ?0076710(1)Compared with tumor-distant normal tissues,the expression of hsa?circ?0076710 in thyroid tumor tissues was significantly up-regulated,AUC=0.851(95% CI: 0.816-0.887),and the sensitivity and specificity were 0.887 and 0.645,respectively.(2)Compared with benign thyroid nodules,the expression of hsa?circ?0076710in fine needle aspiration biopsy(FNAB)in thyroid nodules was significantly upregulated,AUC=0.838(95% CI 0.774-0.902),and the sensitivity and specificity were0.951,0.583 respectively.(3)Compared with benign patients,the expression of hsa?circ?0076710 in serum of PTC patients was significantly up-regulated,AUC=0.769(95% CI: 0.655-0.883),and the sensitivity and specificity were 0.837 and 0.650,respectively.(4)The expression level of hsa?circ?0076710 was significantly increased in patients with tumors >1cm,extraglandular invasion,and lymph node metastasis.Regardless of gender and age,the expression of hsa?circ?0076710 was significantly increased in larger tumors(diameter>1cm),extrathyroidal extension,and lymph node metastasis(P<0.01),but inconsistent expression in different BMI groups and different thyroid functions.Conclusion.1.The expression of hsa?circ?0076710 is up-regulated in PTC tissues and cells,and it is a potent molecule with a closed ring structure.2.hsa?circ?0076710 can promote the proliferation and migration of PTC cells,and play a role in promoting cancer in PTC.3.hsa?circ?0076710 competitively could bind to mi R-214-3p and regulate the JAG1/NOTCH2/HES1 pathway to promote PTC progression.4.hsa?circ?0076710 has important value for clinical diagnosis of PTC. |
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