Study On The Construction Of CeRNA Network And The Role Of PAK4 In Human Head And Neck Squamous Cell Carcinoma

Objective: Head and neck squamous cell carcinoma（HNSCC）are the malignant tumor that most seriously threatens health;however,the molecular mechanism underlying its invasion and metastasis remains unclear.MicroRNAs（miRNAs）are 17-25-nt-long RNAs with principal roles in their mRNA targets translational inhibition and mRNA degradation by binding to partially complementary sequences in the 3′-untranslated region（UTR）of the mRNA targets.Changes in miRNA expression are essential for malignant transformation of tumors by regulating the expression of vital invasion and metastasis target genes.Then,Circular RNAs（circRNAs）are a class of RNAs,which is play important roles in cancer progression.Although the functional role of circRNAs is under increasing scrutiny,developing an effective strategy in circRNAs investigation is difficult.Our previous researches confirmed that circRNA₁02411 promoted target gene CCR7 expression by inhibiting microRNA let-7b-5p activity and enhanced the cell proliferation,migration and invasion in HNSCC.Recent studies have revealed a potential link between circRNA/miRNA/mRNA-associated-ceRNA networks.However,few genome-wide studies have identified the potential circRNA/miRNA/mRNA-associatedceRNA pairs involved in HNSCC.To this end,we compared circRNA and miRNA expression profiles between cancer tissues and para-non-tumorigenic tissues in 5 cases of HNSCC.We found that hsa_circRNA₄02089/ hsa-miR-218-2-3p/PAK4-related ceRNA pairs may be involved in the invasion and metastasis of HNSCC.We took the target gene PAK4 as the main object of study to explore the mechanism of PAK4 in HNSCC invasion and metastasis.Methods: We used a circRNA/miRNA microarray to explore the expression profil e of HNSCC.The HNSCC circRNA/miRNA/ mRNA-associated-ceRNA network was constructed based on the miRDB,miRTarBase,and TargetScan databases.We applied miRwalk3.0 online tools for prediction of HNSCC circRNA/miRNA/mRN A-associated-ceRNA network.The genes expression of hsa_circRNA₄02089/ hsamiR-218-2-3p/ PAK4 were confirmed by qRT-PCR in HNSCC tissues.Transwell a nd wound healing experiments was performed to explore the functions of PAK4 in HNSCC invasion and migration.We investigated the expression of PAK4 in HN SCC tissue by Western blotting and immunohistochemistry essays.Then,the clinic al impact of PAK4 on HNSCC was conducted by using Kaplan-Meier survival an alysis.Results: In this study,we systematically explored the circRNA/miRNA/mRNA-associated-ceRNA mechanism in cancer tissues and para-non-tumorigenic tissues in 4cases of HNSCC through microarray.We obtained 433 significantly dysregulated circRNA transcripts and 63 significantly dysregulated miRNAs.By combining the sets of data we proposed a ceRNA network included 162 mRNAs,36 circRNAs,and 7 miRNAs.hsa_circRNA₄02089/ hsa-miR-218-2-3p/ PAK4 genes with possible involvement in HNSCC invasion and metastasis.We verified hsa_circRNA₄02089 high expression and hsa-miR-218-2-3p low expression in HNSCC tissues.The expression of the upregulated mRNA,PAK4,was confirmed by qRT-PCR in both HNSCC cancer cells and HNSCC tissues.Importantly,over-expression of PAK4 remarkably promoted the migration and invasion of SCC9 cell lines.A higher level of PAK4 was significantly correlated with poorer prognosis of patients with HNSCC.Conclusion: Taken together,hsa_circRNA₄02089/ hsa-miR-218-2-3p/ PAK4 axis may serve as a novel prognostic marker and therapeutic target for HNSCC.