Mechanistic Study On The Inhibition Of The Development Of Cutaneous Squamous Cell Carcinomas By Tumor Suppressor HOXA9

BackgroundIn recent years,due to the inadvertent release of air pollutants by humans,the atmospheric ozone layer loophole has been intensified,and thus ultraviolet rays reaching the ground have increased,which causing an increase incidence of human cutaneous squamous cell carcinomas.Cutaneous squamous cell carcinomas(cSCC)is the second most common skin cancer.There are many basic researches on cutaneous squamous cell carcinoma.However,the exact pathogenesis of cutaneous squamous cell carcinoma remains unclear.Small interfering RNAs(miRNAs)have recently been discovered as a class of endogenous non-coding RNAs with a length of approximately 22 nucleotides.miRNAs are mainly found in the cytoplasm,and they are mainly involved in the regulation of cell proliferation,differentiation,and apoptosis.Changes in miRNAs expression have become one of the key features in cancer-related dysfunction.The development of a tumor is regulated by multiple miRNAs,and the role of a miRNA in different tumors may be completely different.Studies have found that miR-365 can inhibit ovarian cancer,glioma,malignant melanoma,colon cancer and non-small cell lung cancer.Another study found that miR-365 can promote the development of gastric cancer and pancreatic cancer.Our previous work found that miR-365 can promote the development of cutaneous squamous cell carcinoma,which suggesting that miR-365 plays an oncogene role in the development and progression of cutaneous squamous cell carcinoma.However,it is not clear for the endogenous of miR-365 to promote the cutaneous squamous cell carcinoma,so we further explored the downstream target genes of miR-365.By determining HOXA9 as a target gene for miR-365 in cutaneous squamous cell carcinoma,bio-functional experiments were carried out to verify the role of HOXA9 in the development of cutaneous squamous cell carcinoma.Methods①Expression of miR-365 and HOXA9 were detected in normal epidermal cells and skin squamous cell carcinoma cell lines with qPCR.Expression of HOXA9 protein level was detected with Western Blot.Correlation between the expression of HOXA9 anddifferentiation grade in cSCC tissue were tested by IHC.②)Bioinformatics predicted the target gene of miR-365 and verified that HOXA9 is the target gene of miR-365.③CCK-8 cell proliferation assay was used to detect the proliferative capacity of HSC-1 cells after silence and overexpression of HOXA9.④Transwell assays were used to detect capacity in migration and invasion of HSC-1 cells after silence and overexpression of HOXA9.⑤Flow cytometry was used to detect the apoptosis of HSC-1 cells after silence and overexpression of HOXA9.⑥By Seahorse extracellular metabolic flux,the energy metabolismphenotypes of HOXA9-regulated cSCC cells were determined.Results①Compared with normal skin cell lines,miR-365 is highly expressed in cutaneous squamous cell carcinoma cell lines,and HOXA9 is low expressed in gene and protein.The lower HOXA9 expression,the lower degree of differentiation of cSCC.②Proliferation increased of HSC-1 cell after si-HOXA9 treated(P<0.001).Proliferation reduced of HSC-1 cell after HOXA9 overexpression treated(P<0.001).③Migration and invasion of HSC-1 cells increased after si-HOXA9 treated(P<0.01).Migration and invasion of HSC-1 cells reduced after HOXA9 overexpression treated(P<0.01).④Apoptosis decreased of HSC-1 cells after si-HOXA9 treated(P<0.05).Apoptosis increased of HSC-1 cells after HOXA9 overexpression treated(P<0.05).⑤OCR decreased and ECAR increased in siHOXA9-treated HSC-1 cells;OCR increased and ECAR decreased in HOXA9-overexpressed HSC-1 cells.Conclusion HOXA9 can inhibit the proliferation,migration,invasion and regulation of glycolysis pathway of human cutaneous squamous cell carcinoma and promote it’s apoptosis.HOXA9 acts as a tumor suppressor in the progression of cutaneous squamous cell carcinoma.