ISG20L2 as a Novel Prognostic Biomarker Facilitates the Progression of Pancreatic Cancer Via Glycolysis

15 Background : Longstanding type 2 diabetes mellitus (T2DM) is an increased risk of 16 pancreatic cancer (PC) in western populations, and PC is also a cause of T2DM. 17 However, the association of glucose metabolism between T2DM and PC remains 18 unclear. 19 Methods: Differentially expressed genes (DEGs) were identified by bioinformatic 20 analysis from Gene Expression Omnibus (GEO) datasets GSE20966 and GSE16515, 21 respectively. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes 22 (KEGG) pathway, Gene Set Enrichment Analysis (GSEA), the Kaplan-Meier (KM) 23 Plotter and Tumor Immune Estimation Resource (TIMER) database were applied. 24 Pancreatic cancer cell lines and primary PDAC samples were used. Cell culture, 25 immunohistochemistry (IHC), siRNA transfection, Western blot, RT-PCR, and migration assay, animal xenograft model studies and statistical analysis were performed 27 in this study. We identified 64 DEGs in GSE20966 of T2DM, and 296 DEGs were identified in 30 GSE16515 of pancreatic cancer, respectively. T2DM-DEGs were mainly enriched in 31 synaptic vesicle cycle, protein export. KEGG pathways in pancreatic cancer included 32 spliceosome, RNA transport. Here, ISG20L2 was identified as only a co-expressed gene 33 between T2DM and PDAC. We found that the expression of ISG20L2 was associated 34 with tumor immune cell infiltration. ISG20L2 was significantly upregulated in PDAC 35 and associated with prognosis of PDAC patients. Moreover, ISG20L2 expression was 36 regulated by GLUT1 , HK2 , LDHA , PKM1 and PKM2 related with glycolysis in PDAC. 37 ISG20L2 promoted PDAC cell proliferation and migration both in vitro and in vivo. 38 Conclusion: This study showed that ISG20L2 promoted the progression and ISG20L2 39 may be a potential therapeutic strategy in PDAC. 40


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Pancreatic cancer (PC) is the seventh leading cause of cancer death in the world, one of 43 the most fatal malignancies 1 . While pancreatic cancer therapy may be differentiated, 44 the overall 5-year survival of pancreatic cancer is less than 5 % 2 and remains one of 45 the lowest in all cancers. Increasing evidence suggested that type 2 diabetes mellitus 46 (T2DM) was a risk factor for pancreatic cancer. Previous study suggested that T2DM 47 is associated with hyperglycemia and a risk to develop pancreatic ductal 48 adenocarcinoma (PDAC) 3 . The relationship between T2DM and pancreatic cancer is 49 complex. This causal relationship between T2DM and PDAC remains unclear. 50 Glucose metabolism is a critical element in T2DM and PDAC. Skytte, M. J. et al. 51 reported that carbohydrate restriction affected glucose metabolism in T2DM 4 . Mason,52 I. C. et al. found that glucose metabolism in T2DM was regulated by the circadian 53 system and impaired insulin sensitivity 5 . Interestingly, glucose metabolism is also 54 associated with cancer growth and progression, especially in PDAC 6 . 55 In this study, we firstly identified differentially expressed genes (DEGs) of T2DM and 56 PC. Secondly, we elucidated molecular functions of T2DM-related DEGs (T2DM-57 DEGs) and pancreatic cancer-related DEGs (PC-DEGs). Thirdly, ISG20L2 was 58 identified a co-expressed gene and we performed a bioinformatic analysis of ISG20L2. 59 Finally, ISG20L2 was a prognostic biomarker promoting proliferation and migration in 60 PDAC associated with glycolysis. Taken together, ISG20L2 may be a potential 61 mechanism and therapeutic target of pancreatic cancer. The Study flowchart was shown in Fig.1. 66 Selecting GEO database mRNA expression array, a total of 64 DEGs were identified 67 including 33 down-regulated genes and 31 up-regulated genes in GSE20966 68 ( Fig.2A,2B). Beta-cells were acquired from pancreatic tissue sections using the laser 69 capture microdissection technique 7 . We next analyzed DEGs in PDAC. In pancreatic 70 cancer data GSE16515, a total of 296 DEGs were screened out with 71 genes 71 overexpression and 225 genes low expression (Fig.2C, 2D).

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Identification of ISG20L2 as a prognostic biomarker 86 In the present study, ISG20L2 was identified only one co-expressed DEG in GSE20966 87 and GSE16515 datasets using online database VENNY 2.1 (Fig.3A). To explore the 88 regulatory role of ISG20L2, we firstly analyzed its expression. ISG20L2 was 89 significantly overexpressed in T2DM and PDAC (Fig.3B, 3C). Further exploring 90 revealed that the expression of ISG20L2 was overexpressed in PDAC from TCGA 91 database (Fig.3D). The Human Protein Atlas (HPA) (http://www.proteinatlas. org/) 8 92 was applied to explore ISG20L2 expression in PDAC tissues (Fig.3E). To explore the 93 relationship between the expression of ISG20L2 and overall survival, we found that 94 high expression of ISG20L2 associated with poor overall survival (Fig.3F).

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ISG20L2 is overexpressed in PDAC as an independent prognostic factor 96 Besides, we found that the ISG20L2 mRNA level in PDAC cells was also significantly 97 increased compared to HPDE6-C7 (Fig.3G). Moreover, ISG20L2 was notably increased 98 in PDAC patient tissues relative to the adjacent normal tissue (Fig. 3H) prognostic factor for patients with pancreatic cancer. As shown in Table 1. To explore the potential function of ISG20L2 in PDAC, GSEA software was performed 113 to find KEGG pathways enriched in the 89 highly-expressed samples. ISG20L2 mainly 114 enriched in "Rig-I-like receptor signaling pathway", "spliceosome", "aminoacyl tRNA 115 biosynthesis", "ubiquitin mediated proteolysis", "erbb signaling pathway" and 116 "pancreatic cancer" in this study (P < 0.05) (Fig.4A-4G). The above results 117 demonstrated that ISG20L2 exerted the relationship with metabolism in pancreatic 118 cancer.

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To evaluate the relationship between immune cell infiltration and ISG20L2 expression,  Matrigel invasion assays showed that ISG20L2 downregulation and upregulation 139 significantly inhibited and improved the migratory and invasive capabilities of PDAC 140 cells, respectively (Fig. 6A, 6C). This study indicated that PDAC cells proliferation was 141 significantly inhibited in si-ISG20L2 compared to si-NC cells (Fig. 6B). Collectively, 142 these results showed that ISG20L2 facilitated the proliferation, migration, and invasion 143 of PDAC cells.

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To elucidate the oncogenic role of ISG20L2 in PDAC in vivo, we performed 145 subcutaneous tumorigenesis experiment using si-NC and si-ISG20L2 SW1990 cells. 146 The data suggested that the ISG20L2 knockdown effectively reduced compared to si-147 NC group in tumor weight and volume ( Fig.6D-6F). Next, qPCR analysis was

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In the present study, we found that a total of 64 DEGs were identified in GSE20966 of 162 T2DM and a total of 296 DEGs were identified in GSE16515 of pancreatic cancer, 163 respectively. The result showed that ISG20L2 was identified as only one common gene 164 in GSE20966 and GSE16515. 165 We investigated the biological role of ISG20L2 in pancreatic ductal adenocarcinoma 166 (PDAC). Then we found that ISG20L2 was highly expressed in PDAC tumor samples 167 compared with normal tissues, and its expression was associated with a poor prognosis. 168 Functionally, we showed that ISG20L2 could promote cell growth, proliferation, and 169 migration of pancreatic cancer cell lines.

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Biochemical evidence demonstrates here that ISG20L2 is also an exoribonuclease that 173 processes RNAs from their 3'-end to their 5'end 13 .

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To explore the molecular function of ISG20L2, we performed GSEA enrichment 175 analysis. In this study, we observed that hub genes in ISG20L2-high group were 176 statistically significant enriched in pancreatic cancer and metabolism pathways. This

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table1.pdf