Expression And Significance Of PD-L1 Regulated By Hsa-miR-224-3p In Ameloblastoma

Objective: Ameloblastoma is one of the most common odontogenic tumors in oral and maxillofacial region.Although it is classified as benign tumor,its growth and postoperative recurrence is easy to make it the focus and difficulty of scholars’ research.At present,surgical resection is the main treatment method for ab.although this treatment method can reduce the postoperative recurrence rate significantly,the maxillofacial defects caused by this treatment are seriously affected the quality of life of patients,It is the more significant effect,less side effects of treatment that matters.Programmed cell death 1 ligand 1(PD-L1)is one of the ligands of programmed cell death protein 1,PD1,which can inhibit the activation and transduction pathway of normal T cells after binding with PD-L1.It is very important to maintain the stability of immune function.Tumor cell rely on PD-L1 pathway to evade the immune monitoring of the body and mediate the related adverse biological behaviors.it is feasible to study its expression and significance in ab for less studied in ab.Micro RNA(mi RNA)widely exists in the body,its expression is important to control the abnormal growth and development of the body.Its abnormal expression can directly or indirectly affect the development of the disease.At present,the research on mi RNA has been extended to the field of diagnosis and treatment.At the same time,related drugs have been continuously carried out clinical trials and applied in tumor treatment,showing good curative effect.After preliminary screening,our group decided to select hsa-mir-224-3p as a possible upstream,and then verify the relationship between hsa-mir-224-3p and PD-L1 to explore the relationship between hsa-mir-224-3p and ab.Methods :(1)Human mRNA microarray was used to screen downstream genes.(2)Quantitative real-time PCR(QRT PCR)and Western blot(WB)were used to detect the expression of PD1 / PD-L1 in AB and NOM tissues.(3)Immunohistochemical technique(IHC)was used to detect the expression of PD1 / PD-L1 in AB paraffin and NOM paraffin,and the related clinical data were analyzed.(4)Hsa-mir-224-3p was selected as the next experimental object.(5)The expression of hsa-mir-224-3p in AB and NOM was detected by QRT PCR.(6)Luciferase report experiment was used to confirm whether hsa-mir-224-3p and PD-L1 have targeted regulatory relationship.(7)The expression level of target gene was verified by transfection of hsa-mir-224-3p mimics into immortalized AB cell line.The wound healing assay and transwell test was used to verify whether the transfection of hsa-mir-224-3p mimics could affect tumor invasion Results: the result of Human mRNA chip suggested that the abs expression of PD-L1 was evidently up-regulated(P < 0.05).(2)The results of QRT PCR showed that the relative expression of PD-L1 in AB tissues was significantly up-regulated(P < 0.05),but the difference of PD1 expression was not significant(P > 0.05);WB results showed that PD-L1 was expressed in both AB and NOM tissues,and the expression level of PD-L1 in AB tissues was significantly higher than that in NOM tissues(P < 0.05),PD1 was expressed in both AB and NOM(P > 0.05),and the difference of PD1 expression was not significant.(3)IHC results showed that the expression of PD-L1 was mainly positive in AB tissues and negative in NOM tissues,with significant difference(P < 0.05).Combined with clinicopathological data,the results showed that the expression of PD-L1 in ameloblastoma was not significantly different in gender,age,pathological classification and recurrence(P > 0.05).Compared with other sites,the expression rate of PD-L1 in mandibular ameloblastoma was significantly higher than that in other sites(P <0.05).There was no significant difference in the expression of PD1 in AB tissues(P >0.05).(4)Combined with the public bioinformatics website,hsa-mir-224-3p may be the upstream regulation of PD-L1,which can be further studied.(5)QRT PCR results showed that the expression of hsa-mir-224-3p in AB was undoubtedly decreased(P <0.05).(6)Dual luciferase validation report showed that hsa-mir-224-3p had binding sites with PD-L1,which could target PD-L1.(7)QRT PCR results showed that after hsa-mir-224-3p mimic transfection,the expression of PD-L1 in HAM-1 and HAM-3decreased prominently(P < 0.05);the expression of PD-L1 in HAM-2 transfected by mimic decreased inconspicuously(P > 0.05);WB results showed that the expression of PD-L1 in HAM-1 and HAM-3 groups decreased significantly(P < 0.05),and no effective protein was detected in HAM-2;wound healing assay test results showed that the expression of PD-L1 in HAM-1 and HAM-3 groups was significantly lower than that in the control group(P < 0.05),The migration rate of cells in HAM-3 and HAM-1 mimic transfection group was decreased than that in NC group(P < 0.05),and the migration rate of cells had little change between the HAM-2 mimic group and HAM-2 NC group(P > 0.05).The transwell show that number of cells in HAM-3 and HAM-1mimic transfection group was decreased than that in NC group(P < 0.05),and the number of cells had little change between the HAM-2 mimic group and HAM-2 NC group(P > 0.05).Conclusions:(1)There were significant differences in the expression of PD-L1 and hsa-mir-224-3p between AB and NOM tissues,which can be used as a molecular marker for ab diagnosis.(2)The expression of PD-L1 and hsa-mir-224-3p in AB was correlated closely,hsa-mir-224-3p can target PD-L1 and affect AB invasive growth.