Exploring long non-coding RNAs and metabolic proteins as potential biomarkers for colorectal cancer

Abstract

Colorectal cancer (CRC) has been ranked as the third most diagnosed malignancy and the second leading cause of cancer-related death. According to the World Health Organization, 1.8 million new cases, and 0.86 million deaths were recorded in 2018. Locally, the incidence of CRC continued to rise, and it became the most common cancer representing 17% of total cancer diagnosed and ~15% of total cancer-related death in 2017. The carcinogenesis of CRC starts from genetic or epigenetic mutation and goes through a cascade of regulatory signaling pathways such as epithelial-mesenchymal transition (EMT) autophagy, PI3K/AKT, MAPK, p53, and mTOR signaling resulting in the tumour. Despite the implementation of screening programmes, and advanced treatment modalities, the outcomes are not satisfactory due to the development of metastasis/chemo-resistance, or the absence of predictive/prognostic biomarkers. The aim of this project was to explore epigenetic and metabolic factors as potential biomarkers for CRC. We hypothesize that long non-coding RNAs (lncRNAs) and specific metabolic proteins may be differentially expressed in CRC. The objectives are (i) to identify novel oncogenic autophagy-modulating lncRNAs in CRC cells and elucidate their molecular mechanism in carcinogenesis using an in vitro model; and (ii) to determine the expression of specific metabolic proteins in mouse xenograft CRC tumours that responded differentially after radiotherapy. In the first study, we identified a group of differentially expressed autophagy-modulating lncRNAs in CRC cells after RNA-sequencing. Among the 32 isolated differentially expressed autophagy­modulating lncRNAs, we validated the most differentially expressed lncRNA, cancer susceptibility candidate 9 (CASC9) expression profiles and explored its molecular mechanisms in CRCWe validated the expression of CASC9 in two publicly available datasets and the results showed that CASC9 is upregulated in CRC samples compared to their adjacent normal tissues, and higher CASC9 expressions reduced the overall survival of patients. We also demonstrated that the expression of CASC9 is higher in CRC cell lines (HCT-116, SW480, HT-29, and DLD-1) than normal colon cell line (CCD-112CoN). To evaluate the biological and physiological functions of CASC9 in CRC cells, we performed the Dicer-substrate mediated siRNA of CASC9 (Dsi-CASC9) gene silencing in HCT-116 and SW480 cells. The results indicated that effective and consistent silencing of CASC9 significantly reduced cell proliferation, migration, and colony formation in both cell lines. The key molecular pathways associated with carcinogenesis were explored using western blotting (WB). The results confirmed that the silencing of CASC9 significantly increased cellular self-degradation process autophagy. Besides, Dsi-CASC9 treated cells significantly phosphorylated the AMP-activated protein kinase (AMPK), downregulated protein kinase B (AKT) and the mammalian target of rapamycin (mTOR) in HCT-116 and SW480 cells. Furthermore, CASC9 silencing significantly alters the expression of EMT marker proteins E-cadherin, N­cadherin, and vimentin. Using the same techniques above, we explored the role of a newly identified lncRNA called RNA associated with metastasis-11 (RAMS11). RAMS11 was reported to be upregulated in metastatic CRC and it promotes aggressive phenotype in vitro and in vivo. We are novel in exploring the molecular mechanisms and demonstrated that silencing of RAMS11 significantly promote autophagy and apoptosis, downregulate AKT/mTOR signaling via the AMPK pathway, and inhibit EMT markers and transcription factors in CRC cells. Both CASC9 and RAMS11 might be used as potential biomarkers and silencing these lncRNAs may be used for personalized CRC management. To achieve the second objective of this study, we used the CRC tumours previously generated from a mouse xenograft model which received 15 Gy of irradiation and tumour size measured daily. The tumours were then collected and divided into unirradiated (control), poor responders and good responders based on their tumour size changes. We selected 3 samples from each group for high through-put proteomics analysis. Nine metabolic proteins which belong to some essential biochemical pathways such as glycolysis (PGK1, PGAM1, ENO1, PKM, TKT), ammonia detoxification (GLUD1), carcinogenesis (LDHA, GAPDH), and drug responses (MDH2) were being shortlisted. To warrant our findings, we further validated the expressions of PGK1, GLUD1, LDHA and GAPDH in the tumours by WB. Our results suggested that these proteins may be used as potential biomarkers for radiotherapy response in CRC. Overall, this thesis demonstrated the oncogenic roles of CASC9 and RAMAS11 in CRC. We showed that CASC9 and RAMS11 were upregulated in CRC cell lines and silencing of these lncRNAs reduced CRC cells proliferation, viability, and migration through mTOR-dependent autophagy and EMT pathways. Both CASC9 and RAMS11 might be used as potential biomarkers. We also proposed 9 metabolic proteins as potential RT biomarkers. Further investigation is needed to translate our findings into clinical practice and enhance the treatment outcomes of CRC by personalized medicine and radiotherapy.