Abstract
Microcystin-LR (MC-LR) exists widely in polluted food and water in humid and warm areas, and facilitates the progression of colorectal cancer (CRC). However, the molecular mechanism associated with the MC-LR-induced CRC progression remains elusive. The purpose of this study is to explore the role of the hub genes associated with MC-LR-induced CRC development at the molecular, cellular and clinical levels through bioinformatics and traditional experiments. By utilizing R, we screened and investigated the differentially expressed genes (DEGs) between the MC-LR and the control groups with the GEO, in which, HOXB4 highly expressed in MC-LR-treated group was identified and further explored as a hub gene. With the aid of TCGA, GEPIA, HPA, UALCAN, Cistrome, and TIMER, the increased mRNA and protein levels of HOXB4 in CRC tissue were found to be positively associated with high tumor stage and poor prognosis, and were linked to immune infiltration, especially tumor-associated macrophages and cancer-associated fibroblasts. Cox regression analysis and nomogram prediction model indicated that high HOXB4 expression was correlated to poor survival probability. To elucidate the mechanism of high HOXB4 expression induced by MC-LR, we overlapped the genes involved in the MC-LR-mediated CRC pathways and the HOXB4-correlated transcription genes. Importantly, C-myc instead of PPARG and RUNX1 promoted the high expression of HOXB4 through experiment validation, and was identified as a key target gene. Interestingly, C-myc was up-regulated by HOXB4 and maintained cell cycle progression. In addition, MC-LR was proved to up-regulate HOXB4 expression, thus promoting proliferation and migration of Caco2 cells and driving the cell cycle progression. In conclusion, MC-LR might accelerate CRC progression. In the process, MC-LR induced C-myc augmentation elevates the high expression of HOXB4 through increasing the S phase cell proportion to enhance Caco2 cell proliferation. Therefore, HOXB4 might be considered as a potential prognostic biomarker for CRC.
Highlights
Microcystins (MCs) are the largest and most diverse group of cyanotoxins [1]
We find that the Homeobox B4 (HOXB4) messenger RNA (mRNA) expression was significantly higher in tumor tissue of colon adenocarcinoma (COAD) (P=0.045) than in normal tissue in the The Cancer Genome Atlas (TCGA) (Figure 2A)
The results indicated that no noteworthy difference was found in associations of HOXB4 mRNA expression levels with gender and pathologic metastasis stage (M stage), the higher HOXB4 mRNA expression level was observed in patients in the advanced tumor stage (T stage) and lymph node stage (N stage) in overall survival (OS), disease-specific survival (DSS) and progression free interval (PFI) events (Figures 2E–J)
Summary
Of over 270 structural variants of MCs, microcystin-LR (MC-LR) is the most studied congener and considered as one of the most potent cyanobacterial toxins based on previous toxicity studies [1, 2]. Regular water treatment technology, such as filtration and chlorination disinfection, and conventional heating (100°C, 30min) cannot effectively remove MCs [3], and chronic environmental exposure to MCs, either directly through drinking water or indirectly through food-chain and dermal absorption, has been a health concern worldwide. Previous studies on the toxicity of MC-LR mainly concentrate on its impact on liver and kidney [10], but its effect on the gut, which is the primary target organ for MC-LR from both direct (drinking water) and indirect (food chain) exposure routes, is less studied [11]. To identify specific molecular markers for effects of MC-LR exposure on CRC progression may help understanding MC-LR-mediated carcinogenic activity and be in favor of CRC treatment
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