| BackgroundCutaneous squamous cell carcinoma(CSCC)is the cocond common skin malignant tumor,and the incidence follows basal cell carcinoma.Ultraviolet(UV),including UVB,is one of the main causes of CSCC.Due to the more and more serious situation of atmospheric ozone depletion the incidence of CSCC increases in recent years.The exact mechanism of CSCC is still unclear.Therefore,it is of great significance to explore the mechanism and develop a more effective therapeutic target to explore a newstrategy for CSCC treatment.MicroRNAs are non-coding small RNAs that evolutionary conserved in eukaryotic cells,which are widely involved in various cellular processes through post-translational regulation of their target genes.Our group is aimed at the regulation of microRNAs in radiation damage and radiation related diseases,such as cancer.Previous study has shown that miR-365 is the most sensitive miRNA responsitive to UVB irradiation and upregulation of miR-365 in human keratinocyte HaCaT cells induce the formation of subcutaneous tumor,which reveals miR-365 may promote the initiation of CSCC as an"onco-miR".This study aims to predict and verify target genes of miR-365,understand the role of the target genes in the development of CSCC,and further explore the molecular mechanism that miR-365 promotes the initiation and development of CSCC through regulating the target genes to provided research foundation and a new possibility for the development of new drug or treatment for CSCC treatment.Methods(1)Bioinformatics predicted the target genes of miR-365 and verified the direct targeting of HOXA9 by luciferase reporter gene assay.Expression of HOXA9 was detected in clinical tumor samples and tumor cell lines of CSCC using western blot and qPCR.(2)By CCK-8 assay,transwell migration assay,transwell invasion assay and flow cytometry,proliferation,migration,invasion and apoptosis were respectively measured in HOXA9 up-regulated or down-regulated A431 cells,and nude mice tumor models were established to observe the tumor growth.(3)Transcriptome sequencing revealed the transcriptome expression changes by clustering analysis in siHOXA9 treatmed A431 cells and identified downstream target genes regulated by HOXA9,which were verified by qPCR and western blot to dectect mRNA and protein expression level.(4)By seahorse extracellular metabolic flux,the energy metabolism phenotypes of HOXA9-regulated CSCC cells were determined.By 18F-FDG PET/CT,18F-FDG was observed in HOXA9-regulated nude mice tumor.(5)By ChIP-PCR and EMSA,the bindings of HOXA9 and glycolysis related genes were detected.(6)Co-factors which interacted HOXA9 were explored by Co-IP and identified by western blot,transwell assay,etc.Results(1)Bioinformatics predicted HOXA9 as one of the target genes of miR-365 and verified by luciferase reporter gene assay.Expression of HOXA9 in clinical tumor samples and tumor cell lines of CSCC were lower than normal.(2)Silencing HOXA9 promoted proliferation,invasion and migration of A431cell,but inhibited apoptosis.(3)Transcriptome sequencing revealed down-regulated HOXA9 may promote glycolysis,which was verified in A431 cells and nude mice models.(4)OCR decreased and ECAR increased in siHOXA9-treated A431 cells;OCR increased and ECAR decreased in HOXA9-overexpressed A431 cells.Uptake of 18F-FDG in siHOXA9-treated nude mice tumor was significantly higher than control group,while uptake of 18F-FDG in HOXA9-overexpressed nude mice tumor was less than control group.(5)The binding of HOXA9 were at promoter regions of genes involved in glycolysis.(6)CRIP2 interacted HOXA9 directly to inhibit glycolytic pathway.Conclusion miR-365-targeted HOXA9 inhibits the progression of CSCC through regulating glycolytic pathway. |
