UVB-induced MiR-486-3p Promotes The Progression Of Cutaneous Squamous Cell Carcinoma By Targeting FLOT2

BackgroundCutaneous squamous cell carcinoma(cSCC),which arises from epidermal keratinocytes,is the second most co mmon type of non-melanoma skin cancer.According to the Global Cancer Database 2020,the number of new cases of nonmelanoma skin cancer worldwide is approaching 1.2 million.cSCC is associated with a variety of factors,including UV radiation exposure and chronic photoaging,age,sex,i mmunosuppression,smoking and genetic factors,of which long-term UV radiation exposure is the most co mmon and extremely important factor.MicroRNAs(miRNAs)are a class of endogenous non-coding single stranded small molecule RNA.MiRNA regulates the expression of target genes by binding to the 3’-end non-coding region of mRNA of target genes through base complementary pairing.A large number of studies have shown that the abnormal regulation of miRNA is involved in the pathological process of a variety of cancers,therefore miRNA is considered as a potential biomarker for diagnosis,treatment and prognosis.MiR-486-3p has low expression level in oral cancer,bladder cancer,lung cancer and other malignant tumors,and plays a role as a tumor suppressor.However,there has been no relevant study on MiR-486-3p and cSCC so far.More and more evidence indicates that FLOT2 has been proved to show high expression level in various malignant tumors such as nasopharyngeal cancer,cervical cancer and breast cancer,and may play a key role in the occurrence and development of human malignant tumors.However,the expression and clinicopathological significance of FLOT2 in cSCC have not been reported at home and abroad.This study aims to explore the biological role of miR-486-3p and its downstream target gene FLOT2 in cSCC,further explore the molecular mechanism of miR-486-3p affecting the progression of cSCC by regulating FLOT2,and provide theoretical basis for clinical diagnosis and treatment of cSCC.MethodsThe expression of miR-486-3p in normal skin and cSCC was detected by fluorescence in situ hybridization(FISH)and qPCR.The effects of miR-486-3p on malignant behaviors of cSCC cell lines were detected by CCK-8,colony formation and migration assay,after cell models of silenced or overexpressed miR-486-3p were constructed in cSCC cell lines.The effect of Mir-486-3p on the tumorigenicity of cSCC was detected by subcutaneous tumorigenicity model in nude mice.Subsequently,the direct targeted binding between miR-486-3p and FLOT2 was verified by bioinformatics website prediction and double luciferase report assay.Meanwhile,after regulation of miR-486-3p,the expression of FLOT2 was detexted by qPCR and Western blot.The expression level of FLOT2 in normal skin and cSCC was detected by qPCR and western blot.After the regulation of FLOT2 and miR-486-3p,the proliferation and migration of FLOT2 in cSCC cell lines was verified by ccK-8,colony formation and migration assay.Results1.Compared with normal skin tissues and HaCaT cell,the expression level of miR-486-3p in cSCC tissues and cSCC cell lines was higher.2.Bioinformatics predicted that FLOT2 was the downstream target gene of miR486-3p.The direct targeting relationship between miR-486-3p and FLOT2 was verified by dual luciferase reporting assay.3.Upregulation of miR-486-3p or silencing of FLOT2 enhanced the proliferation and migration of HSC-1 and HSC-5 cells,and the tumor-for ming ability in tumor xenograft mouse model.However,downregulation of miR-486-3p or overexpression of FLOT2 inhibited the proliferation and migration of cutaneous squamous cell carcinoma cells,as well as the tumorigenesis ability in tumor xenograft mouse model.4.Recovery experiments confirmed that FLOT2 could inhibit the promoting effect of overexpression of miR-486-3p in cSCC cells.ConclusionUVB radiation-sensitive miR-486-3p,which is highly expressed in cSCC,can promote the progression of malignant behavior of cSCC.There is a targeting relationship between miR-486-3p and FLOT2.FLOT2 is underexpressed in cSCC.FLOT2 can inhibit the malignant behavior of cSCC,and reverse the promoting effect of miR-486-3p overexpression on malignant behavior of cSCC.