| Background: Cutaneous squamous cell carcinoma(cSCC)is the second most common skin cancer in humans,accounting for 20% of skin cancer deaths,second only to cutaneous melanoma.cSCC occurs most frequently in people over 60 years of age.Long-term UV exposure is the primary risk factor for cSCC,tends to occur on the face and neck.It usually develops from a precancerous lesion called Actinic Keratosis.AKs progress to cSCC at a rate estimated between 0.025% and 16% for an individual lesion per year,but the exact cause of AK to cSCC transformation remains unknown.At present,the common treatment methods for cSCC include surgical excision,photodynamic therapy and radiotherapy.In the late stage,single drug or combination therapy,such as chemotherapy,immunotherapy and targeted therapy,are mainly used.However,the percentage of recurrence or metastasis within 5 years is 8% and5% respectively.Although currently immunotherapy has been used partially successful,about half of patients do not respond well,and many patients have developed resistance to immunotherapy.Therefore,exploring the molecular mechanism of the occurrence and development of cSCC and the progression of AK to cSCC will be helpful for its diagnosis and treatment.As the most abundant post transcriptional modification of RNA in eukaryotic cells,N6-methyladenosine(m6A)is an important component of epigenetics.N6-methyladenosine(m6A)is involved in RNA maturation,nuclear output,translation,and splicing,thereby playing a key role in cell pathophysiology and disease processes.Circular RNAs(circRNAs)are a class of non-coding RNAs with a covalent closed loop structure.Due to their conservation and stability,circRNAs can participate in many physiological and pathological processes through unique pathways.m6A modified circRNA plays a crucial role in the progression and treatment of various tumors.Currently,m6A has been found in various tumors to regulate the multiple functions of circRNA by modifying it,revealing its mechanism in tumor genesis and development,providing potential targets for tumor treatment,and laying the foundation for future research.Objective: To preliminarily reveal the change of m6A modification level of circRNAs in cSCC,and to analyze the circRNAs with significant differences in transcription level and post-transcriptional modification level in combination with the change of circRNAs in transcriptome;To explore the effects of m6A methylated circRNA on the expression of downstream molecules and the specific regulatory effects of upstream m6A modified molecules on circRNA,laying a foundation for finding potential targets for diagnosis and treatment of cSCC.Methods: In this study,Me RIP sequencing and RNA sequencing were combined,and circRNAs with significantly different in m6A modification level and transcription level were screened for GO analysis and KEGG analysis,respectively.Time series analysis was used to analyze the pathway enrichment of continuously up/down regulated mRNAs in the progression of NS/AK/cSCC and to analyze the corresponding regulatory relationship between continuously up/down regulated miRNAs.The cyclic structure of circ＿0128829was verified by Sanger sequencing and RNase R experiments.Dual luciferase report assay was used to verify circ＿0128829 downstream targeted binding molecules.After transfecting HSC-5 cells with circ＿0128829 overexpressed recombinant lentivirus vector,ALKBH5 knockout recombinant lentivirus vector and miR-139-5p mimics plasmid,Real-time quantitative PCR and Western blot were used to verify the expression changes of each molecule at gene and protein levels.The proliferation and invasion ability of HSC-5 cells transfected with recombinant lentivirus vector overexpressed circ＿0128829 and recombinant lentivirus vector knocked out ALKBH5 were verified by CCK-8 assay and Transwell assay.Results: Part Ⅰ: Transcriptomic sequencing showed that compared with normal skin,591 circRNAs were significantly up regulated and 611 circRNAs were significantly down regulated in cSCC,mainly concentrated in ubiquitin-mediated protease degradation pathway and ERB signaling pathway.Me RIP sequencing showed that compared with normal skin,809 circRNAs m6A methylation levels were up regulated in cSCC,and 714 circRNAs m6A methylation levels were down regulated,mainly concentrated in m TOR signaling pathway,Foxo signaling pathway,Rap1 signaling pathway,etc.By joint analysis,113 circRNAs were significantly changed at both transcription level and m6A modification level(60 crir RNAs were significantly up regulated at m6A modification level,and 53 circRNAs were significantly down regulated at m6A modification level).Among them,circ＿0128829 was significantly down regulated at m6A methylation modification level,circ＿0128829 was also significantly down regulated at transcription level,and ranked the fourth among circRNAs at decreased transcription level according to p values,and the top three circRNAs had no significant difference in m6A modification level compared with the control group.By time series analysis of mRNA and miRNA continuously up/down regulated in NS/AK/cSCC,it was found that the up regulated mRNA was mainly enriched in herpes simplex virus-1infection,endocytosis and endoplasmic reticulum processing proteins.The continuously down regulated mRNA mainly concentrated in the neural ligand-receptor interaction pathway,CAMP signaling pathway and cytokine ligand-receptor interaction pathway,and21 pairs of continuously up/down regulated miRNA/mRNA relationship pairs were found.Part two: The cyclic structure of circ＿0128829 is verified;The expression of circ＿0128829and HOXA9(Homo sapiens homeobox A9)decreased in cSCC tissues and corresponding cell lines.Moreover,circ＿0128829 can bind and negatively regulate the expression of miR-139-5p,and miR-139-5p can also bind and negatively regulate the expression of HOXA9.Overexpression of circ＿0128829 significantly decreased the expression level of miR-139-5p,and significantly increased the expression level of HOXA9.Overexpression of circ＿0128829 significantly decreased the proliferative and invasive ability of HSC-5 cell line.Part Three: The expression of demethylating enzyme ALKBH5(Alk Bhomologue5,alkylation repair homoloe5)was increased in cSCC tissues and corresponding cell lines,and the methylation level of circ＿0128829 was significantly increased after ALKBH5 was knocked down.It also affected the expression of circ＿0128829/miR-139-5p/HOXA9 axis.After ALKBH5 was knocked down,the proliferation ability of HSC-5 cells had no significant change,but the invasion ability was significantly decreased.Conclusions: In this study,the differentially expressed circRNAs at the transcriptional level and m6A modification level in cSCC were identified,and the time series analysis was used to analyze the pathway enrichment of the continuously up regulated and down regulated mRNAs and miRNA/mRNA regulatory relationship in the progression of NS/AK/cSCC.circ＿0128829 was found to regulate the expression of miR-139-5p/HOXA9 axis,and overexpression of circ＿0128829 could inhibit the proliferation and invasion of tumor cells.In addition,after knocking down the demethylase ALKBH5,the methylation level of circ＿0128829 was increased,and the expression of circ＿0128829/miR-139-5p/HOXA9 axis was affected,and the invasion ability of tumor cells was significantly inhibited.Therefore,circ＿0128829 and ALKBH5 may be potential diagnostic and therapeutic targets for cSCC. |
PDF Full Text Request