MicroRNA-141 Inhibits The Growth And Metastasis Of Head And Neck Squamous Cell Carcinoma By Targeting EGFR

Background and objective: Head and neck squamous cell carcinoma(HNSCC)is one of the six most common cancers in humans.More than 70% of patients have recurrence or metastasis.It is an epithelial-derived malignant tumor that seriously affects human health.Therefore,finding an effective treatment to treat HNSCC has become an urgent need.miRNAs are a class of endogenous single-stranded non-coding small RNAs of 20-24 nucleotides in length.They are regulated by protein binding to mRNA and exist in plants and animals.They are widely involved in the expression and regulation of post-transcriptional genes,such as cells differentiation,proliferation,apoptosis and cell cycle regulation.Currently,only a few miRNAs have been elucidated in their biological functions,and these miRNAs have been shown to be widely involved in various physiological and pathological processes in life processes.miRNA-141 is one of the miRNA-200 families and is a mature sequence.Recent studies have found that miRNA-141 can be used as an oncogene or tumor suppressor gene to express abnormalities in various cancers by regulating different signaling pathways,such as gastric cancer,hepatocellular carcinoma,colorectal cancer,bladder cancer and ovarian cancer.The proliferation,differentiation and migration of tumors affect the prognosis of patients.At present,the mechanism of action of miRNA-141 in the development of head and neck squamous cell carcinoma has not been clearly reported.EGFR,the epidermal growth factor receptor,is a widely distributed cell surface receptor.EGFR is a member of the conserved receptor family of ErbB,which has tyrosine kinase activity and plays an important role in the physiological processes of cell growth,metastasis and differentiation.EGFR can initiate nuclear related genes and promote cell division and proliferation.It is known that EGFR expression is increased in head and neck squamous cell carcinoma,breast cancer,gastric cancer and bladder cancer.There are no reports of the relationship between miR-141 and EGFR.CDK4,a cyclin-dependent kinase 4,is important for its normal expressionduring cell cycle progression.It is known that CDK4 is highly expressed in various cancers and promotes the proliferation of cancer cells,and is an oncogene.Bcl-2 is a known apoptosis-inhibiting gene,and its product bcl-2 protein can inhibit the apoptosis process of various malignant tumors and prolong the growth cycle of tumor cells.MMP2 can degrade polymorphism collagen,binding protein and elastin,participate in the degradation of basement membrane collagen and extracellular matrix,promote the invasion and destruction of cancer cells,and infiltrate and migrate to surrounding tissues.In this study,we will analyze the expression of miR-141 in head and neck squamous cell carcinoma and its relationship to clinical stage and prognosis of the disease.In addition,we will overexpress or inhibit miR-141 in head and neck squamous cell carcinoma cells FaDu and Cal-27,respectively.The effects of miR-141 on the expression of EGFR,CDK4,bcl-2 and MMP2 were examined by in vitro cell function assay and in vivo animal experiments to investigate the role of miR-141 in head and neck squamous cell carcinoma.To preliminarily elucidate the role and significance of miR-141 expression in head and neck squamous cell carcinoma,and provide a reference for the development of new therapeutic targets for the diagnosis and treatment of HNSCC.Method: The expression of miR-141 in head and neck squamous cell carcinoma and adjacent normal tissues was detected by real-time fluorescence quantification.The target gene EGFR of miR-141 was predicted by bioinformatics software MiRDB,and the relationship between EGFR and miR-141 was verified by luciferase assay.The expression of EGFR in head and neck squamous cell carcinoma and adjacent normal tissues was detected by RT-PCR.Western blot was used to detect the expression of EGFR protein in INOK,FaDu and Cal-27 cells.Real-time quantitative PCR was used to detect the expression of miR-141 and EGFR in each group of cells;the head and neck squamous cell carcinoma FaDu and Cal-27 cells were transfected with miR-141 mimics and miR-141 inhibitor fragments,respectively.After transfection,the expression of EGFR protein was detected by Western blot in each group,and the expression of miR-141 and EGFR in each group was detected by real-time fluorescent quantitative PCR.The regulation of miR-141 on theproliferation of FaDu and Cal-27 cells was observed by MTT assay.The expression of CDK4 was detected by Western blot and real-time fluorescent quantitative PCR.The effect of miR-141 on apoptosis of FaDu and Cal-27 cells was observed by JC-1 assay.The effect of miR-141 on bcl-2 was detected by Western blot and real-time fluorescent quantitative PCR.The effect of miR-141 on the migration of FaDu and Cal-27 cells was detected by transwell assay.Western blot and real-time fluorescent quantitative PCR were used to detect the regulation of miR-141 on MMP2 expression.The liver metastasis model of head and neck squamous cell carcinoma in nude mice was constructed.The Fa Du and control Fa Du cells overexpressing miR-141 were injected into the tail vein.The liver colonization of head and neck squamous carcinoma cells was observed by HE staining.EGFR was detected by Western blot and real-time fluorescent quantitative PCR.Expression of CDK4,bcl-2 and MMP2.The liver metastasis model of head and neck squamous cell carcinoma in nude mice was constructed.The Fa Du and control Fa Du cells overexpressing miR-141 were injected into the tail vein.The liver colonization of head and neck squamous carcinoma cells was observed by HE staining.Expression of EGFR,CDK4,bcl-2 and MMP2 were detected by Western blot and real-time fluorescent quantitative PCR.Results: The expression of miR-141 was analyzed in 30 pairs of tissues we collected,and it was found that miR-141 expression in HNSCC tissues was relatively lower than that in adjacent tissues,and it was associated with the occurrence and development of HNSCC.It was also found that EGFR is highly expressed in HNSCC tissues.Patients with higher miR-141 expression have longer survival times than patients with lower miR-141 expression.In HNSCC,mi R-141 was negatively correlated with EGFR expression.It was found by miRDB prediction software that miR-141 can be combined with the 3'-UTR of EGFR.Luciferase assay showed that luciferase activity was inhibited when FaDu and Cal-27 cells were co-transfected with EGFR and miR-141;when EGFR mut or miR-141 antisense was transfected into cells,no inhibition disappears.In addition,there is higher EGFR expression and lower miR-141 expression in HNSCC cells.When miR-141 is overexpressed in cells,the expression of EGFR is decreased,and when miR-141 is inhibited,the expression of EGFR is increased.It isindicated that EGFR is a direct target of miR-141.MTT experiments showed that overexpression of miR-141 inhibited proliferation of HNSCC cells.In contrast,when miR-141 was inhibited,proliferation of HNSCC cells was promoted.The study also found that overexpression of miR-141 inhibits CDK4 expression,downregulation of miR-141 promotes CDK4 expression,and CDK4 is also a downstream regulatory protein of EGFR.Description miR-141 inhibits the proliferation of HNSCC cells.The JC-1 assay showed that transfection of miR-141 promoted apoptosis of FaDu and Cal-27 cells compared to the control group.Western blot and real-time PCR showed that this may be due to the inhibition of bcl-2 by miR-141,which also the downstream regulatory protein of EGFR.This indicates that miR-141 promotes apoptosis of HNSCC cells.Transwell assays showed that transfection of miR-141 inhibited migration and invasion of FaDu and Cal-27 cells,whereas inhibition of miR-141 promoted cell metastasis.Western blot and real-time PCR showed that overexpression of miR-141 inhibited MMP2 expression,downregulation of miR-141 promoted MMP2 expression,and MMP2 is a protein regulated by EGFR.This indicates that miR-141 inhibits migration and invasion of HNSCC cells.In animal experiments,liver metastases in nude mice after injection of head and neck squamous cell carcinoma with high expression of miR-141 were significantly reduced,and the tumor volume was also small.At the same time,Western blot and RT-PCR were used to detect the decrease of target gene EGFR expression,and the expression of EGFR-regulated downstream genes CDK4,bcl-2 and MMP2 was also decreased.Conclusion: 1.The expression of Has-miR-141 in head and neck squamous cell carcinoma was lower than that in adjacent normal tissues.The expression of EGFR in head and neck squamous cell carcinoma was higher than that in adjacent normal tissues.2.Has-miR-141 regulates the role of EGFR in tumor suppressor gene through targeted binding.3.Has-miR-141 can inhibit the proliferation and migration of head and neck squamous cell carcinoma.4.Has-miR-141 is negatively correlated with EGFR,CDK4,bcl-2 and MMP2.miR-141 may regulate CDK4,bcl-2 and MMP2 byregulating EGFR,inhibiting the proliferation and migration of head and neck squamous carcinoma cells,and promoting apoptosis of head and neck squamous cancer cells.