| Background and ObjectiveIn the past few decades,thyroid cancer is the most common endocrine malignancy,and its incidence is increasing rapidly.Papillary thyroid carcinoma（PTC）is the main pathological subtype of thyroid cancer,accounting for more than 80%of all cases.Although most PTC are inert,with a 10-year survival rate greater than 90%,some aggressive characteristics of PTC are associated with poor prognosis,such as older age,larger tumor size,extrathyroidal extension,lymph node and distant metastases,advanced TNM stage,and certain subtypes.These invasive PTC usually require aggressive treatment,such as bilateral thyroidectomy and extensive lymph node dissection,as well as post-operative high-dose thyroid hormone therapy or radioactive iodine therapy.But aggressive treatment can also lead to more complications and poorer quality of life,as well as a higher financial burden on PTC patients.Therefore,it is of great significance to find new biomarkers,improve the diagnostic level of PTC,and early detection of patients with high risk of invasion.In recent years,with the large-scale application of next-generation sequencing technology and bioinformatics methods,a large number of experimental data show that only about 2%of the genes in the whole human genome are protein-coding genes,while most of the genes are non-coding genes.Among non-coding genes,circRNAs are a class of non-coding RNAs widely found in mammals.Most circRNAs are derived from exon regions of genes,and only a small part is formed by intron cleavage.CircRNA is generated by the selective splicing of upstream splicing receptor and downstream splicing donor during the reverse splicing of pre-mRNA transcript,forming a covalently closed continuous loops with neither a 5’-to-3’polarity nor a poly-adenylated tail.Moreover,due to their closed structure and insensitivity to ribonuclease digestion,circRNAs are more conserved and stable in nature.Therefore,circRNA is likely to be a promising biomarker technically suitable for cancer diagnosis and may serve as a molecular target for cancer therapy.In this study,we found a circRNA hsacirc0002111（circBase ID:62 hsacirc0002111）located on chromosome 8p22 through bioinformatics analysis,which is a transcription product of PSD3 gene.According to the data of GEO dataset,hsacirc0002111 is one of the circRNAs with the highest expression level in PTC tissues.Compared with matched normal tissues,the expression level of hsacirc0002111 is significantly up-regulated in PTC tissue samples.Therefore,in this study,we attempted to investigate the expression of hsacirc0002111 and its clinical significance in PTC.Material and Method1.The circRNAs differentially expressed in papillary thyroid carcinoma and adjacent normal tissues were screened by bioinformatics method,and 4 samples of tumor tissues and adjacent normal tissues resected by thyroid surgery were collected for pre-experiment to select the appropriate circRNAs for further study..2.The expression of hsacirc0002111 in normal thyroid cell lines Nthy-ori 3-1 and thyroid papillary cancer cell lines TPC-1 and B-CPAP was detected by qRT-PCR assay.3.The small interference RNA with the target gene hsacirc0002111 was packaged into lentivirus,and the lentivirus with the package number was transfected into TPC-1 and B-CPAP cell lines,and the stable passage cell lines TPC-1-si-circ0002111 and B-CPAP si-circ0002111 were obtained,and the transfection effect was verified by qRT-PCR.4.The effect of hsacirc0002111 on the proliferation of TPC-1 and B-CPAP tumor cell lines was verified by colony formation assay,CCK-8 assay,flow cytometry and qRT-PCR assay.The effects of hsacirc0002111 on invasion and migration of tumor cells were detected by Transwell assay and cell scratch assay.The apoptotic rate was detected by flow cytometry to evaluate the effect of hsacirc0002111 on the apoptotic ability of tumor cells.5.82 pairs of tumor and adjacent normal tissue specimens removed by thyroid surgery were collected.The expression of hsacirc0002111 gene in cancer and adjacent normal tissues was detected by qRT-PCR,and the correlation between hsacirc0002111 gene and clinicopathological characteristics was analyzed6.SPSS 25.0,ImageJ and Prime7.0 were used for statistical analysis and mapping of the obtained experimental data.Four-grid table chi-square test was used for comparison of the two sample rates.One-way ANOVA was used for comparison of multiple local mean,and paired t-test was used for analysis of the two quantitative samples.P<0.05 was considered statistically significant.Result1.Through the analysis of bioinformatics results and the results of pre-experiment,it is concluded that hsacirc0002111 hsa a high expression level in PTC,which hsa the potential for further research.2.The expression of hsacirc0002111 in thyroid papillary carcinoma cell lines TPC-1 and B-CPAP was significantly higher than that in normal thyroid cell lines Nthy-ori 3-1（P<0.05）.The expression of hsacirc0002111 in cell lines TPC-1-si-circ0002111 and B-CPAP-si-circ0002111 was significantly decreased after transfection with packaged virus（P<0.05）.3.CCK-8 method and plate colony formation assay were used to detect cell growth.After interfering with the expression of hsacirc0002111 in cells,the ability of colony formation and cell proliferation rate were significantly reduced（P<0.05）.4.Flow cytometry analysis showed that after interfering with the expression of hsacirc0002111 in TPC-1 and B-CPAP cells,the proportion of cells in the G1 phsae in TPC-1 cells was significantly decreased,while the proportion of cells in the S phsae was increased（P<0.05）.The number of cells in the G1 phsae in B-CPAP cells was significantly decreased,and the number of cells in the G2 phsae was significantly increased（P<0.05）.We also evaluated the effect of hsacirc0002111 expression on apoptosis by flow cytometry,and the results showed that the apoptosis rate did not significantly increase after the interference of hsacirc0002111 expression（P>0.05）.5.The expression level of hsacirc0002111 in PTC tissues was higher than that in adjacent tissues in 69 of 82 tissue samples,with statistical significance（P<0.05）.The expression of hsacirc0002111 was correlated with TNM stage（P=0.040）and lymph node metastasis（P=0.013）.However,there was no significant correlation between the expression of hsacirc0002111 and the age,sex and tumor size of the patients（P>0.05）.6.Transwell assay and cell scratch assay showed that after the expression of hsacirc0002111 was interfered with,the number of cells passing through the micelle was significantly reduced,the speed and number of cells migrating to the scratch were significantly decreased,and the ability of cell invasion and migration were significantly decreased（P<0.05）.Conclusion1.The expression level of hsacirc0002111 in thyroid papillary carcinoma cell line TPC-1 and B-CPAP was significantly higher than that of normal thyroid cells Nthy-ori 3-1;2.Interference with hsacirc0002111 expression can significantly inhibit the proliferation,invasion and migration ability of TPC-1 and B-CPAP of thyroid papillary carcinoma cells,and promote cell apoptosis;3.The expression of hsacirc0002111 gene in papillary thyroid carcinoma samples obtained after surgical resection of the tumor focus was significantly higher than that in paracancerous tissues,and its high expression was correlated with tumor TMN stage and lymph node metastasis of the corresponding patients;4.hsacirc0002111 may be a potential diagnostic biomarker and therapeutic target for papillary thyroid carcinoma. |
