MiR-199a-5p Targeted PD-L1 Affects The Biological Characteristics Of Follicular Thyroid Carcinoma

Objective1)To investigate the expression level of PD-L1 in follicular thyroid carcinoma.2)To explore the relationship between the expression of PD-L1 and the pathological characteristics of follicular thyroid carcinoma.3)To explore the effect of PD-L1 on the biological characteristics of follicular thyroid cancer cells.4)To explore microRNAs targeting PD-L1 in follicular thyroid cancer cells;5)To explore the regulatory effect of miR-199a-5p on PD-L1 in follicular thyroid cancer cells.Methods1)Postoperative tissue specimens and follow-up data of thyroid follicular carcinoma diagnosed in our hospital from 2015 to 2020 were collected;2)The expression level of PD-L1 in FTC cancer tissues was detected by immunohistochemistry.3)The relationship between PD-L1 expression level and FTC pathological characteristics was analyzed.4)The expression difference of PD-L1 in FTC cells and normal thyroid follicular epithelial cells was detected by qRT-PCR and Western blot.5)The expression of PD-L1 gene in FTC cells was silenced by transient transfection technique,and the silencing effect was verified by qRT-PCR and Western blot.6)To explore the effects of silencing PD-L1 on the proliferation,migration and invasion ability of FTC cells in vitro by CCK-8 assay and Transwell migration and invasion assay.7)To explore the effects of silencing PD-L1 on the expression Tight junction proteins.8)Micro RNAs targets of PD-L1 gene were searched through informobiology technology,and 4 miRNAs with conservative binding sites were selected.9)The expression differences of the four miRNAs in FTC cells and normal thyroid follicular epithelial cells were verified by qRT-PCR,and the miR-199a-5p with the highest reduced expression in FTC was screened out.10)The knockdown and overexpression of miR-199a-5p in FTC cells were performed by transient transfection technique,and the knockdown and overexpression effects were verified by qRT-PCR.11)To investigate the effects of knockdown and overexpression of miR-199a-5p on proliferation,migration and invasion of FTC cells in vitro by CCK-8 assay and Transwell migration and invasion assay.12)The effects of knockdown and overexpression of miR-199a-5p on PD-L1 expression in FTC cells were detected by qRT-PCR and Western blot;13)PD-L1 was verified to be a direct targeted regulation gene of miR-199a-5p by double luciferase gene reporting assay.14)qRT-PCR was used to verify the effect of miR-199a-5p expression on claudin-1 and Occludin expression.Result1)Among 60 patients with follicular thyroid carcinoma,the positive expression rate of PD-L1 in cancer tissues was 63.33%(38/60),and the paracancerous tissue was 11.67%(7/60).The expression of PD-L1 in cancer tissues was significantly higher than that in paracancer tissues(P < 0.05).2)The expression of PD-L1 was significantly correlated with tumor diameter,extrathyroid invasion,vascular invasion and recurrence(P < 0.05),but not with age,gender,number of tumor lesions,lymph node metastasis and TNM stage(P >0.05).3)Compared with normal thyroid cell Nthy-ori 3-1,the expression of PD-L1 in thyroid follicular carcinoma FTC-133 cell was significantly increased(P<0.05).4)The proliferation,migration and invasion abilities of FTC cells were significantly reduced after silencing PD-L1(P<0.05).5)Silencing PD-L1 could decrease the expression level of Claudin-1 and increase the expression level of Occludin in FTC cell.6)Compared with normal thyroid cell Nthy-ori 3-1,the expression of miR-199a-5p in FTC cells was significantly decreased(P<0.05).7)The overexpression of miR-199a-5p significantly inhibited the proliferation,migration and invasion of FTC cells(P<0.05),and decreased the expression of PD-L1 in cells(P < 0.05),inhibition of miR-199a-5p expression could significantly enhance the proliferation,migration and invasion of FTC cells(P<0.05)and the expression of PD-L1 in cells was significantly increased.8)Dual-luciferase reporter gene assay confirmed that miR-199a-5p could directly target the expression of PD-L1 gene;9)miR-199a-5p targeting PD-L1 affects the expression of claudin-1 and Occludin Conclusion1)PD-L1 is highly expression in follicular thyroid carcinoma.2)PD-L1 expression is associated with the increased tumor invasiveness.3)PD-L1 may promote the proliferation,migration and invasion of follicular thyroid cancer cell which may be related to promoting the expression of claudin-1 and occludin for Tight junction proteins.4)miR-199a-5p can affect the expression of Claudin-1 and Occludin by targeting PD-L1,and regulate the proliferation,migration and invasion ability of follicular thyroid carcinoma.