| 1 Background and purposePapillary thyroid carcinoma(PTC)is the most common pathological type of thyroid cancer,accounting for about 80%-85% of thyroid cancer.It can occur at any age,and is mainly affectted by multiple factors including environmental and genetic factors.Although PTC is a less aggressive thyroid cancer,the incidence has been steadily and rapidly increasing in recent years.Recurrence and metastasis are the main causes of its poor prognosis and even death.Circular RNAs(circ RNA)are a type of non-coding RNAs that widely exists in eukaryotes.Circular RNAs are involved in many cellular processes including growth,development,apoptosis and migration by binding to 3’ untranslated region(3’UTR)of micro RNA and inhibiting its regulation of target m RNA.ci RS-7 has been reported to be involved in the development of malignancies including gastric cancer and colon cancer.ci RS-7 contains mi R-7(micro RNA-7)binding sites and can participate in the pathophysiological process of cells by regulating mi R-7.However,whether ci RS-7 in PTC sponge to mi RNAs and regulate the expression level of downstream genes remains unclear.Therefore,we firstly detected the expression of ci RS-7 in PTC by q RT-PCR and analyzed the relationship between ci RS-7 and clinicopathological factors.Then we overexpressed and inhibited the expression of ci RS-7 by plasmid transfection to analyze the functions of ci RS-7 in PTC biological behavior.In addition,we explored the mechanism of ci RS-7 in PTC.This study will clarify the mechanism ci RS-7regulating the proliferation,migration and invasion of PTC,so as to provide strong experimental evidence for targeted therapy of PTC.2 Material and methods2.1 The expression of ci RS-7 in papillary thyroid cancer and its clinical significanceWe randomly selected 17 cases of PTC tumor tissues and matched adjacent non-carcerous epithelium(NCE).The expression of ci RS-7 was detected by quantitative real-time PCR(q RT-PCR).Then its association with the clinicopathological features of human with papillary thyroid cancer was analyzed by statistical analysis.2.2 The effect of ci RS-7 on cellular biological behavior of papillary thyroid cancer cellIn this part,we overexpressed and inhibited the expression of ci RS-7 by plasmid transfection.Then we assayed the cell proliferation,migration and invasion ability by CCK8 assay,colony formation assay,Annexin V-FITC assay,Wound healing experiment and transwell assays.2.3 The mechanism of ci RS-7 on cellular biological behavior of PTC cell2.3.1 The relationship between ci RS-7 and mi R-7 in regulating cell proliferation,migration and invasionq RT-PCR was used to detect the expression of mi R-7 after ci RS-7 was downregualted.Then we used ci RS-7 si RNA to retransfect the mi R-7 downregulated PTC cells to investigate whether ci RS-7 promoted proliferation,migration and invasion of PTC cells by modulating mi R-7.2.3.2 The targeting relationship between mi R-7 and EGFRPublicly available Bioinformatics tools were used to identify target gene of mi R-7.Luciferase reporter assay was used to demonstrate their interaction.2.3.3 ci RS-7 regulates cell proliferation,migration and invasion through EGFRWestern blot was used to detect expression of EGFR when ci RS-7 was downregulated.Then we retransfected ci RS-7 downregulated plasmid into EGFR overexpressed PTC cells to investigate whether ci RS-7 promoted proliferation,migration and invasion of PTC cells by modulating EGFR.2.4 Statistical analysisSPSS 21.0 statistical software was used to analyze the data.Chi-square test was used for a single factor analysis of variance,and t test was used for the quantitative data of two samples.P<0.05 means the difference is statistically significant.3 Result3.1 The expression of ci RS-7 in papillary thyroid cancer and its clinical significanceCi RS-7 levels were higher in PTC tissues and cell lines than normal thyroid tissues and thyroid epithelial cell line(P<0.01).In addition,the overexpression of ci RS-7 was significantly correlated with large tumor size(P < 0.05)and lymph metastasis(P<0.05).3.2 The effect of ci RS-7 on cellular biological behavior of papillarythyroid cancer cellTo explore the function of ci RS-7,its expression was upregulated and downregulated by plasmid transfection.CCK8 and colony formation assay results showed that ci RS-7 downregulation significantly inhibited the in vitro proliferation.Annexin V-FITC assay showed that ci RS-7 downregulation significantly promoted apoptosis.Decreased invasion and migration in vitro was observed in ci RS-7-downregulated PTC cells using wound healing assay and transwell assays.Oppositely,overexpression of ci RS-7 promoted the proliferation,migration,invasion and inhibited apoptosis in PTC cell lines.3.3 The mechanism of ci RS-7 on cellular biological behavior of PTC cell3.3.1 The relationship between ci RS-7 and mi R-7 in regulating cell proliferation,migration and invasionq RT-PCR revealed that mi R-7 was increased after ci RS-7 was downregulated.Downregulation of ci RS-7 reduced the ability of proliferation,migration,and invasion in mi R-7 inhibited cells.3.3.2 EGFR gene was the direct target gene of mi R-7Using publicly available Bioinformatics tools,we identified that there was a recognition site of mi R-7 on the 3’non-coding region(3’UTR).The dual luciferase reporter assay result further demonstrated their direct interaction.3.3.3 ci RS-7 regulates proliferation,migration,and invasion of PTC by targeting mi R-7/EGFR axisWestern blot results showed that the expression of EGFR decreased in PTC cells transfected by ci RS-7 si RNA.Downregulation of ci RS-7 reduced the ability of proliferation,migration and invasion in EGFR up-regulated cells.4 Conclusion1 ci RS-7 may play an important role in PTC.2 mi R-7 mediated the proliferation,migration and invasion of ci RS-7 in PTC cells;EGFR may be a direct target of mi R-7.3 ci RS-7 may regulate cell proliferation,migration and invasion through mi R-7/EGFR. |
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