The Effect Of MicroRNA-18a On ATM Gene Expression And Autophagy Process In HCT116Colon Cancer Cells

Autophagy is a cellular self-catabolic degradation process whichworks to optimize the bio-energetic cellular microenvironment bydegrading cytoplasmic components such as long-lived proteins anddamaged organelles. Autophagy is active at basal cellular growth levels towork as an endogenous cleaning system, and also can be triggered bydiverse stressful conditions, such as adaptation to starvation, oxidative orgenotoxic stress, and elimination of pathogens. And thus deregulatedautophagy has been observed in various physiological and patho-physiological processes, including aging, cancer and neurodegenerativediseases. The exact role of autophagy in carcinogenesis remains elusive.Autophagy shows dual roles during tumorigenesis as it can behave as atumor suppressor or oncogene depending on the cell context. A similarparadox is exhibited during tumor radio-or chemotherapy therapy,wherein autophagy has been reported to support cancer cell survival and thereby reduce efficacy of the cancer treatment. However, in othercontexts, induction of autophagy has been shown to contribute to cancercell death (also known as programmed cell death type–II) andconsequently enhance therapeutic efficacy of tumor radio-orchemotherapy. Therefore, achieving better molecular understanding ofautophagy and the discovery of specific autophagy modulators suitablefor in vivo use will help to dramatically improve cancer therapy. One ofthe major tumor suppressor genes that have been found recently tomodulate autophagy is ATM (Ataxia telangiectasia mutated). ATM is amulti-functional serine/threonine protein kinase which works to maintaingenomic stability by inducing cell cycle arrest, apoptosis, and DNA repairin response to DNA damage inducing agents particularly ionizingradiation (IR). Newly, ATM has been found to up-regulate autophagyprocess in response to genotoxic and oxidative stimuli. ATM suppressesthe activity of mammalian target of rapamycine complex1(mTORC1)through stimulating AMPK/LKB1/TSC2signaling pathway to ultimatelyinduce autophagy. Therefore, regulating ATM through chemical or genetic methods to modulate autophagy process has become an attractivepoint in cancer research. MicroRNAs are a short (~20–22nucleotideslong) single-stranded RNA molecules, non-coding, novel class of generegulators. They are synthesized from endogenous hairpin-shapedtranscripts (60–110nucleotide RNA precursor structures) in the nucleusthen exported to the cytoplasm. In the cytoplasm the mature microRNAstrand binds to the3’-untranslated region (3’-UTR) of the target mRNAto repress gene expression at the post-transcriptional level. In mammals,it is now predicted that microRNAs control the activity of more than60%of all protein-coding genes. The huge interest in the field of microRNAshas led to the conclusion that microRNAs take part in regulation ofalmost every cellular process known so far like development, growth,apoptosis, and the changes in expression of microRNAs are associatedwith many human disorders especially cancers. MicroRNAs have recentlybeen characterized as modulating the process of autophagy via thetargeting of cardinal autophagy-regulating genes. MiR-30a and miR-376bhave been demonstrated to target and inhibit Beclin-1activity, thereby blocking autophagy. Additionally, miR-101inhibits RAB5A, which actsin the early stages of autophagosome formation.Objective: Although the field of microRNA-autophagy interrelationshipis still in its infancy, however exploring this filed could help us tounderstand in depth the molecular pathways that control autophagy andoffer future therapeutic perspectives. In our study we were interested todiscover a microRNA that could target ATM efficiently, and then studythe impact of that microRNA on autophagy process (basal and IR-induced autophagy).Methods:(1) Bioinformatics analysis was implemented to find a microRNA thatcould target ATM (2) PCR was used to acquire partial3’UTR fragmentand construct luciferase expression plasmids (3) Dual-LuciferaseReporter Assay was used to demonstrate the binding of selectedmicroRNA and ATM target gene (4) X-ray generator was utilized to deliver radiation (5) Western blot was used to analyze ATM proteinexpression level (6) qRT-PCR was used to detect endogenous miRNAexpression (7) GFP-LC3localization assay and LC3-II western blotassay to were used to monitor autophagy process (8) Clonogenic assaywas performed to study the changes in radiosensitivity (9) Cells viabilitywas evaluated by Cell counting Kit-8(10) Student t-test and χ2test wereused to determine statistical significance. P<0.01and P<0.05wereconsidered significant statistically (11) Image J software was used tocalculate the western blot band values.Results:1-Chose miR-18a that could bind to the3′-UTR of ATMVia intersecting results from three microRNA target prediction programs(Miranda, Targetscan, and miRDB), miR-18a was found to be a potentcandidate in all three algorithms for targeting ATM. As a putative targetgene, ATM has an extremely conserved single miR-18a target site(position3481-3488of human ATM3’-UTR) in human and other mammalian species, including the chimpanzee, rhesus monkey, mouse,rat, dog and rabbit.2-miR-18a targets and upregulates ATM gene expressionDuring luciferase assay, surprisingly, in HCT116colon cancer cells, miR-18a mimic increased the expression of the reporter gene with the ATM3’-UTR tag to almost90%(**P<0.01) as compared to30%in negativecontrol (NC) transfected cells. Almost similar results (*P<0.05) wereobtained when luciferase assay was repeated using lentiviral PCDH-vector expressed miR-18a (PCDH-miR-18a). Western blot results showedthat miR-18a mimic led to1.6folds increase in ATM protein level innon-irradiated and1.7folds increase in4Gy irradiated HCT116cellsrelative to NC transfected cells. Moreover, inhibition of miR-18a led toalmost50%decrease in ATM expression level in irradiated and non-irradiated cells. 3-Endogenous miR-18a expression was up-regulated by ionizingradiationq-RT-PCR results showed that expression level of endogenous miR-18adramatically increased to more than200folds one hour after exposure ofHCT116to IR. Seven hours after exposure to IR, miR-18a expressionlevel was just more than five folds above the basal level, indicating thatmiR-18a expression changes overtime after exposure to IR. Such resultssuggest that miR-18a could play role in IR-induced cellular events likeautophagy.4-miR-18a overexpression promotes autophagy:MiR-18a mimic significantly increased percentage of GFP-LC3puncta-positive cells to15%(**P <0.01) as compared to8%in NC transfectedcells and mock. Further more; the percentage of puncta-positive cellswas further increased up to25%when miR-18a mimic was combined with IR (**P <0.01), relative to cells treated with IR alone (15%).Consistent with GFP-LC3immunofluorescence, miR-18a mimic led tosharp increase in the LC3-II protein expression level in non-irradiated (2folds) and irradiated (3.5folds) HCT116cells as compared to NCtransfected counterparts. P62/SQSTM1is a poly-ubiquitin bindingprotein that was found to bind directly to ATG8/LC3and localize toautophagosomes to ultimately be degraded during autophagy. Therefore,the level of P62reflects the autophagic turnover. In the HCT116cellstransfected with miR-18a mimic, the expression level of theP62/SQSTM1protein was markedly reduced by almost50%in non-irradiated and irradiated cells as compared with the NC transfected cells.Consequently, such findings demonstrate that miR-18a overexpressionenhances the autophagic flux.5-MiR-18a overexpression inhibits mTORC1activityATM has been demonstrated to upregulate autophagy via inhibition ofmTORC1. We hypothesized that miR-18a positively regulated ATM, inhibiting mTORC1and inducing autophagy. To examine whether miR-18a regulated mTORC1, we measured P70S6K phosphorylation atThr389as a typical readout of mTORC1activity by western blotting.MiR-18a mimic markedly decreased the phosphorylation of P70S6K atThr389in non-irradiated and irradiated HCT116cells (which indicatedmTORC1activity inhibition) as compared NC-transfected cells.6-miR-18a overexpression enhances the radiosensitivity of HCT116cell lineCologenic survival study showed that miR-18a mimic decreased thesurvival fraction of4,6, and8Gy irradiated HCT116cells (*P<0.05,*P<0.05and**P<0.01respectively) compared to NC transfected cells.7-Ectopic miR-18a overexpression inhibits the growth of colon cancercellsCCK-8assay surprisingly showed that miR-18a mimic decreased the cell viability to almost70%in HCT116and SW116colon cancer cells(**P<0.01) as compared to NC transfected cells. On the other hand,inhibition of miR-18a increased cell viability (**P<0.01) of HCT116cellline.Conclusions:1-ATM is a novel target gene for miR-18a, and ectopic miR-18aoverexpression uniquely promotes ATM gene expression in HCT116colon cancer cells (pre and post-IR exposure). Such phenomena could beprobably through the direct impact of miR-18a on ATM3’UTR segment,which could be cell line-dependent phenomena.2-Ectopic miR-18a overexpression results in strong induction of basaland IR-induced autophagy in HCT116cells, probably and at leastpartially through regulation of the expression of the known autophagyactivator ATM. This could be partially supported by finding that miR-18a overexpression could suppress mTORC1activity in HCT116cells, as indicated by decreased phosphorylation of P70S6K at Thr389in miR-18amimic transfected cells.3-Exogenous overexpression of miR-18a enhances the radiationsensitivity of HCT116cells.4-MiR-18a is a novel tumor suppressor microRNA in colon cancer. MiR-18a belongs to oncogenic miR-17-92cluster which encodes sixmicroRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1. All other members of this cluster have been previously reported toenhance colon cancer growth especially miR-92. This suggests that miR-18a opposes the oncogenic effect of miR-17-92cluster in colon cancer.In summary, results of the present study pertaining to the role of miR-18ain regulating ATM gene expression and autophagy process in coloncancer cells revealed a novel function for miR-18a in a critical cellularevent and on a crucial gene with significant impacts in cancerdevelopment, progression, treatment and in other diseases. Therefore,miR-18a is a good candidate for novel epigenetic colon cancer therapy.