Hsa-let-7e-5p Inhibits The Proliferation, Migration And Invasion Of Head And Neck Squamous Cell Carcinoma By Targeting Chemokine Receptor 7

Objective:Head and Neck Squamous Cell Carcinoma(Head and Neck Squamous Cell Carcinoma,HNSCC)mainly originates from the upper respiratory tract and upper gastrointestinal mucosa,mainly involving in the areas such as the oral cavity,pharynx and larynx and accounting for more than 90% of head and neck cancer.It is one of the most common malignant epidermal tumors in head and neck cancer,which seriously threatening the life and health of human.Though the treatment has been improved in recent years,the 5-year survival rate remained in 50-60%.Being small,endogenous non-coding RNAs consisting of 20-24 nucleotides,microRNAs(miRNAs)repress gene expression post-transcriptionally via binding to the complementary nucleotides in the 3'untranslated regions(3'UTRs)of target gene mRNAs and regulate many physiological activities.So far,a great number of evidences have shown that deregulated miRNAs have involed in not only the growth and development in animals and plants but also the occurrence,development as well as the metastasis of cancer.Recent studies have found that the there was deregulated expression of hsa-let-7e-5p in lots of cancers,but it has not been reported that how it effects on the occurrence and development of head and neck squamous cell carcinomas.In recent years,the studies have found that the high expression of chemokine receptor 7 not only on immune cells but also on a variety of tumor cells to different degree and it has closely related to the deregulated proliferation,invasion,migration and poor prognosis.Our team has found that when the CCR7 was able to promote the proliferation,invasion and migration in head and neck squamous cell carcinoma cells by a number of signaling pathways.But the mechanism of upstream regulatory is still unclear.Therefore,the main research goal of this paper is to explore what role hsa-let-7e-5p plays in head and neck squamous cell carcinomas and whether CCR7 is invloved in the regulation hsa-let-7e-5p mediated in HNSCC.Methods:Real time PCR was uesed to examined the expression level of hsa-let-7e-5p in HNSCC cell lines(HNSCC cell lines PCI-37 B and PCI-37 A,which are respectively derived from the metastatic lymph node and primary tumor of the same HNSCC patient,were kindly donated by the University of Pittsburgh Cancer Institute.The PCI-37 B cell expressing much more CCR7 is characterized of high invasive and migrational ability,however,the PCI-37 A cell expressing less CCR7 is characterized of low invasive and migrational ability)and in the primary tumors and matched matched adjacent normal tissues of 15 HNSCC patients.Next,PCI-37 B and PCI-37 A cells were transfected with hsa-let-7e-5p mimic plasmid(Mimic)and inhibitor fragments(Inhibitor),respectively,using liposome 2000 The experiment was divided into two groups.Each group was then divided into three groups according to the metastatic ability of HNSCC cells,named Blank group(PCI-37 B without transfection),NC group(PCI-37B+NC),Mimic group(PCI-37B+hsa-let-7e-5p mimic),Blank group(PCI-37 A without transfection),NC group(PCI-37A+NC),Inhibitor group(PCI-37A+hsa-let-7e-5p inhibitor).After transfection,the expression level of hsa-let-7e-5p in each group was detected by real-time PCR.Changes of proliferation were examined in HNSCC PCI-37 B and PCI-37 A cells by CCK8 assay.Changes of invasion were examined in HNSCC PCI-37 B and PCI-37 A cells by transwell invasion assay.Changes of migaration were examined in HNSCC PCI-37 B and PCI-37 A cells by scratches assay and transwell migrational assay.Bioinformatics softwares,for exmaple,Targetscan,Miranda,Pic Tar were used to predict the target gene of hsa-let-7e-5p.Next,the relationship between hsa-let-7e-5p and its ownstream gene was detected by western blot assay and dual-luciferase reporter assay.At last,the effect of hsa-let-7e-5p on tumor growth was observed in vivo.Results:The expression level of hsa-let-7e-5p was relative lower in PCI-37 B cells and relative higher in in PCI-37 A cells(P<0.01).The expression level of hsa-let-7e-5p was significantly decreased in 10/15 HNSCC tissues as compared to their matched adjacent normal tissues(P<0.01).However,the expression level of hsa-let-7e-5p was significantly increased in 4/15 HNSCC tissues as compared to their matched adjacent normal tissues(P<0.01).The expression level of hsa-let-7e-5p in one patient was no significant difference as compared to their matched adjacent normal tissues(P>0.05).These data suggest that down-regulation of hsa-let-7e-5p may lead to the initiation and progression of HNSCC.In the study of proliferation assay,OD value at 450 nm of each group was respectively examined in different time periods(0h,24 h,48h,72 h,96 h).OD value was basically identical at 0h of each group.In the PCI-37 B group,the OD value of Mimic group was much more significantly decreased than that of the control group,and the difference was statistically significant(P<0.01).On the contrary,in the PCI-37 A group,the OD value of Mimic group was much more significantly increased than that of the control group,and the difference was statistically significant(P<0.01).The difference between NC group and Blank group was not statistically significant(P>0.05).These data suggested that hsa-let-7e-5p has an inhibitive effects on HNSCC cell proliferation.In the study of migration assay,both scratches assay and transwell migrational assay showed that over-expression of hsa-let-7e-5p markedly reduced the migratory ablity of cells in group of PCI-37B(P<0.01),whereas hsa-let-7e-5p inhibitor significantly increased the migratory ablity of cells in group of PCI-37A(P<0.01).The difference between NC group and Blank group was not statistically significant(P>0.05),indicating that hsa-let-7e-5p has an inhibitive effects on HNSCC cell migration.In the study of invasion assay,the number of cells that invaded the matrigel was calculated in each group.The result showed that over-expression of hsa-let-7e-5p markedly reduced the number of invasive cells in group of PCI-37B(P<0.01),whereas hsa-let-7e-5p inhibitor significantly increased the number of invasive cells in group of PCI-37A(P<0.01).The difference between NC group and Blank group was not statistically significant(P>0.05),indicating that hsa-let-7e-5p has an inhibitive effects on HNSCC cell invasion.The results of bioinformatics softwares showed that HMGA2,CCND1,STAT3,LIN28,WNT1 and CCR7 may be the target genes of hsa-let-7e-5p.Because our previous studies have shown that CCR7 and its downstream molecules can promote the survival,chemotaxis,migration and invasion of the head and neck squamous cell carcinomas.So we determined to regard CCR7 to be the downstream target gene of hsa-let-7e-5p for subsequent experiments.Next,western bolt assay showed that over-expression of hsa-let-7e-5p markedly reduced the expression of CCR7 in group of PCI-37B(P<0.01,whereas hsa-let-7e-5p inhibitor significantly increased the expression of CCR7 in group of PCI-37A(P<0.01).The difference between NC group and Blank group was not statistically significant(P>0.05),indicating that the hsa-let-7e-5p was negatively correlated with CCR7.Further dual-luciferase reporter assay showed that the luciferase activity was significantly downregulated only in the group of PCI-37 B cells co-transfected with hsa-let-7e-5p mimic compared to other group(P<0.01).The difference between the other group was not statistically significant(P>0.05),indicating that CCR7 is the target gene of hsa-let-7e-5p in HNSCC PCI-37 B cell.The in vivo assay showed that nude mice xenografts with hsa-let-7e-5p mimic significantly reduced volume compared to the control group after epitopic over-expression of hsa-let-7e-5p.Conclusions:1.The expression level of hsa-let-7e-5p is relative lower in PCI-37 B cells and relative higher in in PCI-37 A cells.2.Hsa-let-7e-5p is downregulated in HNSCC tissues.3.Hsa-let-7e-5p inhibits the proliferation,migration as well as invasion of HNSCC cells at least via negatively regulateing CCR7.4.Hsa-let-7e-5p 5.Ectopic over-expression of hsa-let-7e-5p inhibits the growth of xenograft tumor in vivo.