title: Regulation of colorectal cancer metastasis by modulating mitochondrial miRNAs
Creators: name: Ebrahimi, Faeza
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Colorectal cancer
subject: Mitochondrial respiration
subject: miR-126
subject: miRNAs
subject: miR-15a
subject: miR-335
subject: miR-145

description: Background Cancer is one of the major contributors to mortality and morbidity worldwide. Current literature demonstrates that by the age of 85 years, 1 in 2 males and 1 in 3 females in Australia will have been diagnosed with cancer at some stage in their life. Cancer occurs due to external and or internal stimuli to cells causing them to respond by dividing uncontrollably and growing aberrantly. This abnormal development later forms into solid malignant tumours, which gain the ability to invade and destroy adjacent tissue and disseminate to distant organs of the body via the general circulatory or lymphatic system of the host. While there are many treatment modalities that exist to treat cancers, complete cure may or may not occur due to high tendency of cancer recurrence. This study sets emphasis on the regulation of colorectal cancer (CRC) metastasis by modulating mitochondrial microRNAs. Cancer of the colon and rectum is the most prevalent cancer in Australia after breast and prostate cancer in both genders and the second most frequent cancer-related cause of death. Numerous literatures indicate that specific microRNAs are aberrantly expressed in various cancers including colorectal cancer. Micro-ribonucleic acids (miRNAs) are small (17–25 bases) single-stranded, evolutionarily conserved, non-coding RNAs that play a key role in the regulation of gene expression during essential cellular processes like cell differentiation, cell cycle progression, stress response and apoptosis. Mitochondrial miRNAs (Mitomirs) are those pre-miRNAs that mature in the mitochondria to regulate mRNAs related to mitochondrial function. While some mature miRNAs also translocate to and function in the mitochondria. Earlier studies have shown that mitochondrial dysfunction is linked with cancer pathogenesis. This can be controlled by overexpression of tumour suppressor miRNAs via targeting specific messenger RNAs (mRNAs). This study has focused on four key microRNAs (miR-126, miR-15a, miR-145, and miR-335), which has a significant role in regulating mitochondrial function. The role of these miRNAs in the modulation of mitochondrial function in colorectal cancer has not been previously studied. For the first time the expression and clinicopathological parameters of miR-335 and miR-145 have been investigated in colorectal cancer. Aims and hypothesis This research is proposed to investigate the functions of mitochondrial miRNAs miR-126, miR-15a, miR-145, and miR-335 in metastatic colorectal cancer tissues and cell lines, to determine their role in regulating of colorectal cancer. The hypotheses to be tested were: (1) miRNA expression will be altered in different stages of colorectal cancer tissues and cell lines compared to control groups; (2) expression of miRNAs will have a correlation with the clinicopathological features of patients with colorectal cancer (3) miRNA expression levels will differ in different stages of colorectal cancer; (4) in-vitro studies will show tumour suppressive roles of these miRNAs; (5) expression of tumour suppressor miRNA will target genes to regulate colorectal cancer; (6) mitochondrial respiration will be increased by the ectopic expression of these miRNAs compared to control groups. Materials and methods Tissue samples were recruited from patients at Gold Coast University Hospital in Queensland, Australia. Ethical approval for this study was obtained from the Griffith University Human Research Ethics Committee (GU Ref No: MSC/17/10/HREC). Colorectal tissues from 163 primary adenocarcinomas, 96 matched metastatic lymph node, 48 distant metastatic, 40 adenocarcinomas and 42 non-neoplastic tissues were recruited from 389 patients and used to check the expression of miR-126. Post histopathological analysis, 182 tissue samples from 124 patients (37 non-neoplastic, 87 primary adenocarcinomas, 50 matched metastatic lymph node, and 8 distant metastatic) samples were used to examine the expression of miR-15a; a total of 185 tissue samples comprising of 37 non-neoplastic, 90 primary adenocarcinomas, 50 matched metastatic lymph node, and 8 distant metastatic samples were used to examine expression of miR- 145 and finally a total of 101 tissue samples comprising of 16 non-neoplastic, 59 primary adenocarcinomas, 18 matched metastatic lymph node, 8 distant metastatic samples were used to examine expression of miR-335. Histopathological samples (non-tumour, pre-cancerous and cancerous lesions) were collected and the clinical characteristics were recorded with the help of clinicians. The miRNA of the selected samples was extracted by standard protocols. miRNA expressions were then studied by evaluating the quantitative amplification using quantitative real-time polymerase chain reaction (qRT-PCR). ΔΔCT and CT ratio methods were used to analyse gene expression. All miRNA expressions were correlated with clinical and pathological data. miRNA mimics and Hiperfect transfection reagents were used to identify post transfection alterations in colon cancer cell lines. Functional assays including MTT calorimetric, cell cycle analysis, apoptosis, colony formation, and basement membrane assays were performed with an exogenous microRNA mimic to detect any in-vitro changes in cancer cell biology. Western blot analysis was used to detect the target genes of Mitomirs. Targets such as p53, BCL-2, KRAS, TAB3, FAM134B, SOX2, and VEGF-A were tested. Mitochondrial respiration was also examined through the addition of mitochondrial inhibitors oligomycin, carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A, and analysed via Seahorse XF analyser serially 24 hours and 36 hours after transfection. Results Expression levels of miR-126, miR-15a, miR-145, and miR-335 in CRC tissues and cancer cell lines were significantly downregulated compared to normal tissues and cell lines. Ectopic expression of miRNAs: miR-126, miR-15a, miR-145, and miR-335 showed reduced cell proliferation, colony formation, and invasion compared to control groups. Target protein analysis showed that miR-126 reduced expression of BCL-2, while increased p53 and KRAS. On the other hand, miR-15a reduced expression of BCL-2, and SOX2 while TAB3, and FAM134B showed no changes. Overexpression of miR-145 reduced expression levels of p53 and VEGF-A, while no changes were noted in expression levels of SOX2 protein. miR-335 repressed expression of KRAS and VEGF-A. Mitochondrial functions evaluated through use of mitochondrial inhibitors, illustrated that oxygen consumption rate (OCR), extracellular acidification rate (ECAR), maximal respiratory capacity, spare respiratory capacity, ATP coupled respiration, and proton leak in SW480miR-126, SW480miR-15a, SW480miR-145, and SW480miR-335 cells, were elevated compared to scramble miRNA and wild-type as control post 24 hours of transfection. Post 36 hours of transfection we observed that the mitochondrial respiration in colon cancer cells with ectopic expression of miR-126, miR-15a, miR- 145, and miR-335 was reduced compared to their respective control groups. Conclusion To conclude, expression analysis of miRNAs (miR-126, miR-145, miR-335 and miR-15a) showed significant downregulation in colorectal adenocarcinomas with advanced pathological stages, indicating their roles in colorectal cancer pathogenesis. Moreover, in vitro studies implicated that the ectopic expression of miRNAs reduces CRCs proliferation, colony formation, and invasion and can induce apoptosis. Also, increased expression of miRNAs in colon cancer cells reduces the viability of cancer cells, potentially via suppression of growth promoting genes (BCL-2, KRAS, VEGF-A, and SOX2) and up-regulation of tumour suppressor genes (p53). Mitochondrial respiratory components such as OCR, ECAR, maximal respiratory capacity, spare respiratory capacity, ATP coupled respiration, and proton leak were notably increased while the metabolic potential was reduced in both miR-126 and miR-335 overexpressed colon cancer cells 24-hours after transfection. Moreover, the mitochondrial inhibition in colon cancer cells with ectopic expression of miRNAs post the 36 hours of transfection resulted in overall repressed mitochondrial respiration capacity. In summary, this study highlights that the dysregulation of miRNAs can modulate mitochondrial respiration and can regulate cancer cell biology via targeting various tumour-related proteins. This study in future will lead to the development of targeted therapies for patients with colorectal carcinomas with a specific focus on cancer cell mitochondria and miRNAs.