Transcriptome Profiling Of Cervical Squamous Cell Carcinoma By RNA Sequencing

Part Ⅰ:Analysis of differentially expressed genes of cancer and adjacent non-tumor tissues from cervical squamous cell carcinoma patients by RNA sequencingAbstract:Objective:Cervical cancer is the third most common cancer and the fourth leading cause of cancer deaths among women in the world. It has been demonstrated that HPV infection alone is insufficient for cervical cancer and the abnormal host genes are critical in the development of cervical cancer. The transcription is the base and starting point of gene function. In this study, we are aimed to thoroughly analyze the transcriptome of cervical squamous cell carcinoma(CSCC) and matched adjacent non-tumor (ATN) tissue, to screen out the differentially expressed genes (DEGs) among CSCC and ATN tissues, the interaction of DEGs, the related signal pathways, and relationship between clinicopathological parameters of CSCC and DEGs.The discovery of vital diagnostic and therapeutic markers against CSCCwould provide research and theoretical basis for diagnosis and treatment of CSCC.Methods:Firstly, cDNA library was prepared, RNA sequencing was performed to screen the DEGs of 3 pairs of CSCC and ATN tissues. The GO analysis was used to uncover the biological functions of DEGs. The KEGG pathway enrichment analysis was used to find out the related signal pathways. Protein interaction network was carried out to reveal interaction of DEGs. Quantitative real-time PCR was conducted to validate the expression of DEGs. Immunohistochemistry was used to detect the relationship between clinicopathological parameters of CSCC and DEGs.Results:There were a total of 347 significantly common DEGs in the 3 paired samples, including 104 consistent upregulated and 148 consistent downregulated DEGs. The 347 DEGs were categorized into 73 functional categories by GO analysis.The 104 consistent upregulated DEGs were categorized into 34 functional categories, and the 148 consistent downregulated DEGs were categorized into 80 functional categories. The KEGG pathway analysis suggested 6 significant signal pathways, including cytokine-cytokine receptor interaction, complement and coagulation cascades, retinol metabolism, chemokine signaling pathway, metabolism of xenobiotics by cytochrome P450, and melanogenesis. The protein interaction network uncovered three important DEGs, including RDH12, UBD, and SAA1.We found that RDH12 expression was decreased in 74.5% of CSCC tissues, and was negatively associated with tumor size and depth of cervical invasion. The UBD gene was overexpressed in 61.7% of CSCC tissues, and was positively related with tumor size and lymphatic metastasis. The SAA1 gene was overexpressed in 57.4% of CSCC tissues, and was positively related with tumor size, lymphatic metastasis and depth of cervical invasion.Conclusion:In this study, the DEGs of CSCC and ATN tissues were thoroughly analyzed for the first time.There were a total of 347 significantly common DEGs in 3 paired samples, and enriched on 6 significant signal pathways. The RDH12, UBD, and SAA1 genes were associated with clinicopathological parameters of CSCC, and may be used as molecular diagnostic markers and therapeutic targets in CSCC, while further research is needed to demonstrate the mechanism of these genes in CSCC progression.Part Ⅱ:The landscape analysis of alternative splicing in the cervical squamous cell carcinoma by RNA sequencingAbstract:Objective:Alternative splicing (AS) is a key regulatory mechanism in protein synthesis and proteome diversity. Abnormal AS events were closed related with tumorigenesis, and were involved in cell cycle, metabolism, and various cell signal pathways. In this study, we are aimed to identify AS events in cervical squamous cell carcinoma(CSCC) and adjacent non tumor tissues(ATN) using RNA sequencing, and found out the AS genes, the related signal pathways, and relationship between clinicopathological parameters of CSCC and AS genes, and provide experimental foundation and theoretical basis of diagnosis and treatment of cervical cancer.Methods:The transcripts of the four paired samples of CSCCand matched ATNtissue were thoroughly analyzed by RNA sequencing. SpliceMap software was used to detect the splicing junctions. Each segment is mapped to the human genome with Bowtie software.The AS events presented only in the CSCC or in ATN amples were detected.KEGG pathway analysis was conducted to detect the AS genes related signal pathways. The AS genes KLHDC7B and SYCP2 were choosed according to the mapped reads, and their expressions in CSCC were validated by RT-PCR, the relationship between the two AS genes and clinicopathological parameters of CSCC was analysed.Results:There were 35 common AS genesin the four CSCC samples, they were novel and CSCC specific.16 pathways were significantly enriched (p<0.05), including metabolic pathways, endocytosis, ras signaling pathway. One novel 5’AS site in KLHDC7B gene, and an exon skipping site in SYCP2 gene were validated by RT-PCR. The KLHDC7B gene with 5’AS was found in 66.0%(31/47) of CSCC, and was significantly related with tumor size. The exon skipping of SYCP2 gene was found in 38.2%(18/47) of CSCC, and was significantly related with depth of cervical invasion.Conclusion:In the study, the AS events of CSCC and ATN tissues were thoroughly analyzed for the first time.16 pathways were significantly enriched, and 35 novel AS genesof CSCC were detected. The AS genes KLHDC7B and SYCP2 might involve in the development and progression of CSCC and could be used as biomarkers in the diagnosis and prognosis of CSCC, however, the detailed mechanism needs further research.