MiR-29b-3p Suppresses Migration And Invasion Of Oral Squamous Cell Carcinoma Cells Via IL32-pAKT Pathway

Objectives: Oral Squamous Cell Carcinoma(OSCC)is aggressive and easy to metastasize to regional lymph nodes accompanied with distant metastasis in late stage.Our laboratory has confirmed that miR-29b-3p was downregulated in OSCC invasion front cells.The objectives of this study are investigating how miR-29b-3p regulate the migration and invasion of OSCC invasion front cells and exploring the mechanism.Methods: The OSCC cell line UM-SCC6 was a kind gift of Peking Union Medical University.UM-SCC6-M cells were obtained from invasion front of UM-SCC6 cells on a microfluidic chip in vitro.The morphologies of UM-SCC6 and UM-SCC6-M were observed under light microscopy and the migration and invasion abilities of two cells were investigated by wound healing and transwell invasion assay.The expressions of ?-CATENIN,AKT,pAKT and other migration-associated protein markers were detected by western blot and the expression of miR-29b-3p was detected by RT-qPCR.How miR-29b-3p regulate migration and invasion abilities of UM-SCC6-M and UM-SCC6 was investigated after transfecting miR-29b-3p mimic/inhibitor in two cells and verifying transfection efficiency by RT-qPCR.Based on the results of western blot,AKT inhibitor MK-2206 was added in UM-SCC6 and UM-SCC6-M,as well as UM-SCC6 transfected with miR-29b-3p inhibitor,to investigating the influence on migration and invasion.In order to have an insight into UM-SCC6 and UM-SCC6-M and the mechanism of miR-29b-3p.The whole-genome profiles were analyzed and the downstream target genes of miR-29b-3p were predicted.Based on the sequencing results,the expression of IL32 were examined at mRNA and protein level in UM-SCC6 and UM-SCC6-M and how miR-29b-3p regulating IL32 was conformed at mRNA and protein level by RT-qPCR and western blot.Then,the effects of IL32 on UM-SCC6 and UM-SCC6-M in migration and invasion were investigated after transfecting IL32 overexpression and knockdown plasmids.The transfection efficiency was detected by western blot and RT-qPCR.The effect of MK-2206 on IL32 overexpression plasmid transfected UM-SCC6 in migration and invasion was also examined.Meanwhile,the alteration of AKT and pAKT were investigated by western blot after IL32 plasmids transfectings.Finally,co-transfection of miR-29b-3p inhibitor/mimic and IL32 plasmids was performed in two cells and the alterations of AKT and pAKT in each group were detected by western blot.The recovery of IL32 on miR-29b-3p was investigated by wound healing and transwell invasion assay.Results: UM-SCC6 cells were "paving stone".The cells were closely connected and formed into a mass.However,UM-SCC6-M cells were "spindle" with loose connections and interstitial cell characteristics.Wound healing and transwell invasion assay revealed that UM-SCC6-M showed greater migration and invasion abilities then UM-SCC6.Western blot results confirmed that SNAI1 and pAKT were upregulated,whereas pMEK was downregulated in UM-SCC6-M.?-CATENIN,TWIST1,MMP2 and AKT were similar at protein level between the two cells.Meanwhile,RT-qPCR results verified that miR-29b-3p was downregulated in UM-SCC6-M compared with UM-SCC6.We confirmed that miR-29b-3p mimic suppressed migration and invasion of UM-SCC6-M and miR-29b-3p inhibitor had an opposite effect after verifying the transfection efficiency.MK-2206 suppressed the migration and invasion of UM-SCC6 and UM-SCC6-M and inhibited the regulation of miR-29b-3p inhibitor.The whole-genome profiles analysis showed that IL32 was a target gene of miR-29b-3p affecting OSCC migration and invasion.RT-qPCR and western blot showed that IL32 was upregulated in UM-SCC6-M at mRNA and protein level and miR-29b-3p could regulate the expression of IL32 at the post-transcriptional level.We confirmed that IL32 promoted migration and invasion of UM-SCC6 after verifying the transfection efficiency of IL32 overexpression and knockdown plasmids.The addition of the AKT inhibitor MK-2206 demonstrated that IL32 played a role by AKT pathway.Finally,co-transfection of miR-29b-3p inhibitor/mimic and IL32 confirmed that IL32 could attenuate the effect of miR-29b-3p on OSCC cells migration and invasion and pAKT protein expression.Conclusions: The present study showed that miR-29b-3p was downregulated in OSCC invasion front and miR-29b-3p suppressed migration and invasion of OSCC cells via IL32-pAKT pathway.