| BackgroundCutaneous squamous cell carcinoma(cSCC)is the second most common skin cancer,which derives from keratinocytes of epidermal and its appendages and carries the risk of metastasis.According to statistics,the incidence and the death toll of cSCC have been increased over time.Therefore,it is necessary to further study the mechanism of cSCC development.MiRNAs are a large class of small endogenous non-coding RNAs that transcriptional regulate gene expression by directly binding to their 3’UTR region according to the principle of complementary base pairing.miRNAs are involved in many biological processes,including cell differentiation,cell survival and apoptosis.Accumulating evidence shows that miRNAs play an important role in the tumorigenesis,progression and metastasis of various cancers,which have become promising targets or tools for cancer treatment.miR-27a is aberrantly expressed in many types of cancers,but the role of miR-27a in cSCC progression has not been elucidated.APE1(Human Apurinic/Apyrimidinic Endonuclease 1)is a multifunctional protein involved in many biological processes,such as DNA repair,transcription factor regulation and RNA metabolism.APE1 has been extensively studied in various cancers,but no research has elucidated the function and regulatory mechanism in the progression of cSCC.This study was aimed to investigate the biological role of miR27a and its downstream,APE1 cSCC progression,to clarify its molecular mechanism and to provide a new theoretical basis for clinical treatment of cSCC.Methods(1)HaCaT cells upon UVB irradiation were detected by miRNAs sequencing.qPCR was used to verify the differentially expressed miR-27a in the sequencing results and to detect the expression of miR-27a in the cSCC cell line.(2)Western blot was used to detect the expression of proteins in the NF-κb signaling pathway after up-or down-regulation of miR-27a.(3)CCK-8,clone formation,migration and invasion experiments were performed after regulating miR-27a to test the effects of miR-27a on the proliferation,migration and invasion of cSCC cells in vitro.At the same time,a nude mouse subcutaneously transplanted tumor model was used to detect the effect of miR-27a on cSCC cell proliferation in vivo experiments.(4)The bioinformatics method was used to predict the target genes of miR-27a and the direct targeting relationship between miR-27a and APE1 was verified by double luciferase experiments.qPCR and western blot were used to detect and regulate the expression level of APE1 after miR-27a.(5)qPCR and western blot were used to detect the expression of APE 1 in HaCaT cells and cSCC cells after UVB irradiation.(6)CCK-8,clone formation and migration experiments,and recovery experiments were used to test the proliferation and migration of cSCC cells.The effects of EMT on key proteins were detected by western blot.Results(1)miRNAs sequencing and qPCR confirmed that UVB irradiation downregulated the expression of miR-27a,and qPCR results showed that miR-27a was lowly expressed in HSC-1 and HSC-5.(2)miR-27a inhibits NF-κB signaling pathway,including IKK,p-p65 and p-IκB.(3)Up-regulation of miR-27a can inhibit the proliferation,migration and invasion of HSC-1 and HSC-5 cells.Subcutaneous tumor formation experiments have confirmed that miR-27a can inhibit the growth of subcutaneous tumors in mice.(4)A double luciferase reporting experiment confirms that APE1 is a target gene of miR-27a,and qPCR and western blot confirm that upregulating miR-27a can inhibit APE1 expression.(5)APE1 was highly expressed in cSCC cells,clinical tumor samples and UVB-irradiated HaCaT cells.(6)APE1 promotes the proliferation and migration of cSCC cells and induces the expression of EMT marker proteins.(7)The results of rescue experiment show that APE1 can restore the inhibition of cSCC cell proliferation and migration and the expression of EMT markers caused by miR-27a overexpression.ConclusionThis study revealed that APE1 as a direct target of miR-27a improved cell proliferation and migration to promote the progression of cSCC. |
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