Dysregulation of tissue microRNAs in colorectal cancer and their potential role as prognostic and predictive biomarkers

Abstract

Colorectal cancer (CRC) continues to remain as one of the leading global causes of cancer related mortality. Cancer recurrence or progression of disease are the key factors that hamper the overall management of this disease. Several pathological, clinical and molecular factors have been identified that provide vital information regarding the overall prognosis of CRC. Recently the role of small non-coding RNAs (ncRNAs) as potent molecules involved in the development and progression of CRC has been identified which are also known to serve as valuable biomarkers. The current project aimed towards the investigation of such predictive, prognostic small ncRNAs in CRC in order to gain a specific and a sensitive evaluation of the overall course of the disease to allow improved disease management. Small RNA expression profiles of 16 paired CRC tissue samples were analyzed by next generation sequencing that revealed subtle differences between the small RNA expression within the non-malignant tissues of the recurrent and non-recurrent patient subgroups. Subsequent analysis led to the identification of miR-509-3p which was found to have a significantly high expression within the adjacent normal tissues of the recurrent patients compared to the non-recurrent group. A high expression of miR-509-3p also correlated with an overall poor disease prognosis. Furthermore, the expression of miR-509-3p also positively correlated with the depth of tumor invasion and a larger tumor size as well as the presence of distant metastasis and a higher stage in recurrent patients. Functional studies involving the modulation of the expression of miR-509-3p in CRC cells demonstrated an oncogenic role of the miRNA. Overexpression of miR-509-3p was associated with an induction in cell proliferation, cell survival and formation of larger tumors in mice. Also, increased expression of miR-509- 3p was found to promote a metastatic phenotype via decrease in the expression of epithelial marker E-cadherin and a concomitant increase in the expression of RhoA GTPase, both of which are associated commonly with the Epithelial- Mesenchymal-Transition pathway. A higher expression of miR-509-3p was also associated with a decrease in the mRNA levels of the tumor suppressor PH domain leucine-rich repeat-containing protein phosphatase 2 (PHLPP2), suggesting that PHLPP2 is a potential target of miR-509-3p. MiR-509-3p was also found to induce in vitro lateral oncogenesis. Exposure to culture media from miR-509-3p overexpressing CRC cell lines was found to cause a transient increase in the levels of the miRNA within CCD841CoN, a normal colon cell line. Also, co-culture of CCD841CoN with the culture media from miR-509-3p overexpressing CRC cell lines, caused an induction of oncogenesis accompanied with an increase in cell proliferation as well as the metastatic potential. However, no change was observed in the chemosensitivity of the CCD841CoN cells to Oxaliplatin and 5-fluorouracil, providing hints towards probable alternative mechanisms adopted by the transformed cell line to escape cell death induced by chemotherapy. Taken collectively, the study provides the first evidence of a role of miR-509- 3p in the development and induction of oncogenesis in CRC, and its potential as a prognostic biomarker to predict poor prognosis.