Study On The Biological Function And Mechanism Of Hsa-miR-20a-5p In Head And Neck Squamous Cell Carcinoma Cells

Objective:Head and neck squamous cell carcinoma(HNSCC)is a malignant epidermal tumors,which is a serious threat to human life and health,with low 5-year-survival rate.The main factors affecting the prognosis of the patients are the local recurrence and lymph node metastasis.MiRNA is a class of about 22 nucleotides,endogenous,nonprotein encoded small RNA molecules?It is involved in many cellular processes,such as cell cycle,apoptosis,signal transduction,and so on.It exerts its effects by inhibiting target mRNA translation at the transcriptional or post transcriptional level.Hsa-miR-20 a is one of the miR-17-92 gene cluster in the human genome and hsa-miR-20a-5p is a mature sequence.A recent study found that abnormal expression of miR-20 a in many malignant tumors,as oncogene or anti-oncogene involved in the occurrence and development of tumor.It has been reported that miR-20 a was highly expressed in head and neck squamous cell carcinoma,but the effect of miR-20a-5p on the occurrence and development of head and neck squamous cell carcinoma have not been reported.TNFRSF21,also known as DR6,is a person engaged in family of tumor necrosis factor receptor superfamily.It is a transmembrane receptor with four cysteine rich.The gene is widely expressed in vivo,such as lymph nodes,heart,and it is even expressed in cultured cells.Abnormal expression of DR6 in some cell lines can promote apoptosis,activation of nuclear factor kappa-B and proliferation of protein kinase 8(also known as c-Jun N-terminal kinase 1).CCR7,the chemokine receptor 7,is closely related to the occurrence and development of many kinds of tumors.Most scholars think that the mechanism of lymph node metastasis in CCR7 positive tumor cell is similar to lymphocyte chemotaxis homing.Study found that CCR7 is highly expressed in head and neck squamous cell carcinoma metastasis cells,which is associated with cervical lymph node metastasis.And compared with head and neck squamous cell carcinoma cells with low expression of CCR7,high expression of CCR7 in head and neck squamous cell carcinoma cells wasmore aggressive and NF-kB activity increased.In previous research of our research group,CCR7 was able to promote the invasion and chemotaxis of head and neck squamous cell carcinoma cells by a number of signaling pathways.But there is no literature reports about the relationship between mir-20a-5p and CCR7.Methods:Using real-time fluorescence quantitative PCR method to detect the expression of hsa-miR-20a-5p in head and neck squamous cell carcinoma cell lines PCI-37 A and PCI-37B(PCI-37 A derived from primary tumor,and PCI-37 B derived from lymph node metastasis from the same HNSCC patients).Synthesis of hsa-miR-20a-5p overexpression mimics plasmid and inhibitor fragments,with lipofectamine 2000,using transient transfection techniques,PCI-37 B were transfected.The experiment was divided into five groups,blank control group(PCI-37B),overexpression positive group(PCI-37 B mimics),negative control of overexpression group(PCI-37 B mimics-NC),inhibitor positive group(PCI-37 B inhibitor),negative control of expression inhibited group(PCI-37 B inhibitor-NC).After transfection,the expression of miR-20a-5p in each group was detected by real-time fluorescence quantitative PCR.Proliferation changes was observed in PCI-37 B through CCK8 experiments;apoptosis change was observed by apoptosis experiments;migration and invasion was observed through scratches assay and transwell experiment.Using bioinformatics software such as Targetscan,MirBase and Pic Tar,target genes of miR-20a-5p was predicted jointly.By luciferase reporter and Western blot assays,the relationship between miR-20a-5p and the predicted target genes was verified.Pre-experiments of our research group show that CCR7 can significantly promote the migration and invasion of PCI-37 B,so using Western Blotting assay to verify whether there is a relationship between CCR7 and miR-20a-5p.Results:Mi R-20a-5p was expressed both in PCI-37 A and PCI-37 B,but the expression in PCI-37 B was higher than that in PCI-37 A,and the difference was statistically significant(P <0.001).After transient transfection,RT PCR was used to detect miR-20a-5p expression in each group.As a result,the expression amount of miR-20a-5p in PCI-37 B mimics group was significantly higher than that in PCI-37 B mimics-NC group and PCI-37 B group,and the difference was statistically significant(P<0.01).The expression amount of miR-20a-5p in PCI-37 B inhibitor group was significantly lower than that in PCI-37Binhibitor-NC group and PCI-37 B group,and the difference was statistically significant(P<0.01).The results show that the constructed vector is effective and can be carried out in the following experiments.In the experiment of CCK8,OD values in each group are basically the same at 0h,and 24 h,48h,72 h,96h later,the OD value in PCI-37 B mimics group was significantly higher than that in PCI-37 B mimics-NC group and PCI-37 B group,and the difference was statistically significant(P<0.05).OD values in PCI-37 B inhibitor group was significantly lower than that in PCI-37 B inhibitor-NC group and PCI-37 B group and the difference was statistically significant(P<0.01).So mir-20a-5p can promote the proliferation of PCI-37 B cell line.In the annexin V-FITC/PI double staining apoptosis experiment,the number of apoptotic cells in PCI-37 B mimics group was less than that in PCI-37 B mimics-NC group and PCI-37 B group,and the difference was statistically significant(P<0.01).In PCI-37 B inhibitor group the number of apoptotic cells was significantly higher than that in PCI-37 B inhibitor-NC group and PCI-37 B group,and the difference was statistically significant(P<0.01).The apoptosis of PCI-37 B cells could be inhibited by miR-20a-5p.In wound-healing migration assay,the wound width of each group was basically the same at 0h.12-24 h later,the wound width in PCI-37 B mimics group was significantly narrower than that in PCI-37 B mimics-NC group and PCI-37 B group and there was a significant difference(P<0.01).In mi R-20a-5p inhibitor group,cell migration was inhibited and wound width change is not obvious,and significant difference was exsisted between miR-20a-5p inhibitor group and PCI-37 B inhibitor-NC group and PCI-37 B group(P < 0.01).MiR-20a-5p can promote the migration of PCI-37 B cells.In transwell migration assay,the number of migration cells in miR-20a-5p mimics group was significantly more than that in PCI-37 B group mimics-NC and PCI-37 B group(P < 0.01).the number of migration cells in miR-20a-5p inhibitor group was less than that in PCI-37 B inhibitor-NC group and PCI-37 B group(P<0.01).All these showed that mir-20a-5p can promote PCI-37 B cell migration.In the transwell invasion assay,the number of cells through Matrigel in miR-20a-5p mimics group was significantly higher than that in the PCI-37 B mimics-NC group and PCI-37 B group(P < 0.01),and the number of cells through Matrigel in miR-20a-5p inhibitor group was significantly lessthan that in PCI-37 B inhibitor-NC group and PCI-37 B group(P < 0.01).All these indicated that miR-20a-5p can promote the invasion of PCI-37 B cells.Through bioinformatics prediction software,target genes TNFRSF21 was screened.Western Blot experiments show that in mi R-20a-5p mimics group,TNFRSF21 protein expression is lower than that in PCI-37 B mimics-NC group and PCI-37 B group(P < 0.05);While in miR-20a-5p inhibitor group,the TNFRSF21 protein expression was significantly higher than that in PCI-37 B inhibitor-NC group and PCI-37 B group(P<0.05).The luciferase reporter assay showed that luciferase activity was down-regulated by approximately 40% in the WT group transfected with miR-20a-5p mimics compared with that in the control group(p<0.05).However,there was no obvious alteration of luciferase activity in the MT group(p>0.05).In addition,in Western blot,CCR7 protein expression amount in miR-20a-5p mimics group was higher than that in PCI-37 B mimics-NC group and PCI-37 B group(P < 0.05);CCR7 protein expression amount in miR-20a-5p inhibitor group was lower than that in PCI-37 B inhibitor-NC group and PCI-37 B group(P < 0.05).The results illustrate that TNFRSF21 was negatively correlated with miR-20a-5p,which may be a downstream target genes of miR-20a-5p.CCR7 expression was positively correlated with miR-20a-5p expression,which may be a downstream target genes or target gene downstream target of miR-20a-5p,and can be regulated by mi R-20a-5p directly or indirectly.Conclusions:1?Hsa-mi R-20a-5p expression level is higher in HNSCC cell line PCI-37 B than that in HNSCC cell line PCI-37 A.2?Hsa-miR-20a-5p can promote the proliferation?migration and invasion of head and neck squamous cell,with the inhibition of apoptosis.3?Hsa-miR-20a-5p plays a role as oncogene through targeting of TNFRSF21 to promote proliferation and inhibit apoptosis.4?CCR7 may be a downstream target of miR-20a-5p target genes,direcct or indirect regulated by miR-20a-5p.