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* List gives the number of target gene transcripts that are hit by one or more of the miRNAs in order of decreasing number of miRNAs hitting them. **List gives the number of target gene transcripts that are hit by those miRNAs that are over-expressed in the crypt bottom in addition to hsa-mir-25 and hsa-mir-007

'Legends for Supplementary Figures'

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. ©2017 AACR.

List of 83 miRNAs which were differentially expressed in CRC vs normal colon.

Lists of predicted gene targets of miRNAs in the upper crypt. List of predicted gene targets in the crypt bottom. GO term analysis of the top 200 predicted gene targets for both the lists.

'Fig S1. Validation of miRNA chip results by quantitative PCR analysis. Fig S2. Analysis of differentially expressed miRNAs using target predictions. Fig S3. The relative abundance of the common predicted targets in relation to miRNA levels in CRC cell lines. Fig S4. The effect of miRNAs on mRNA expression levels of their predicted targets. Fig S5. Relative expression of four candidate microRNAs in HT29 ALDH+ cells as compared to ALDH- cells. Fig S6. miRNA23b targets 3' UTR of LGR5 and LRIG1 mRNA. Fig S7: miRNA23b affects cell proliferation. Fig S8: CaCo2 data on miRNA23b precursor and antimer effect on sphere forming ability as well as 5FU response.'

Autism spectrum disorder (ASD) has been associated with mitochondrial dysfunction but few studies have examined the relationship between mitochondrial function and ASD symptoms. We measured Complex I and IV and citrate synthase activities in 76 children with ASD who were not receiving vitamin supplementation or medication. We also measured language using the Preschool Language Scales or Clinical Evaluation of Language Fundamentals, adaptive behavior using the Vineland Adaptive Behavioral Scale, social function using the Social Responsiveness Scale and behavior using Aberrant Behavior Checklist, Childhood Behavior Checklist and the Ohio Autism Clinical Impression Scale. Children with ASD demonstrated significantly greater variation in mitochondrial activity compared to controls with more than expected ASD children having enzyme activity outside of the normal range for Citrate Synthase (24%), Complex I (39%) and Complex IV (11%). Poorer adaptive skills were associated with Complex IV activity lower or higher than average and lower Complex I activity. Poorer social function and behavior was associated with relatively higher Citrate Synthase activity. Similar to previous studies we find both mitochondrial underactivity and overactivity in ASD. This study confirms an expanded variation in mitochondrial activity in ASD and demonstrates, for the first time, that such variations are related to ASD symptoms.

Treatment for mitochondrial dysfunction is typically guided by expert opinion with a paucity of empirical evidence of the effect of treatment on mitochondrial activity. We examined citrate synthase and Complex I and IV activities using a validated buccal swab method in 127 children with autism spectrum disorder with and without mitochondrial disease, a portion of which were on common mitochondrial supplements. Mixed-model linear regression determined whether specific supplements altered the absolute mitochondrial activity as well as the relationship between the activities of mitochondrial components. Complex I activity was increased by fatty acid and folate supplementation, but folate only effected those with mitochondrial disease. Citrate synthase activity was increased by antioxidant supplementation but only for the mitochondrial disease subgroup. The relationship between Complex I and IV was modulated by folate while the relationship between Complex I and Citrate Synthase was modulated by both folate and B12. This study provides empirical support for common mitochondrial treatments and demonstrates that the relationship between activities of mitochondrial components might be a marker to follow in addition to absolute activities. Measurements of mitochondrial activity that can be practically repeated over time may be very useful to monitor the biochemical effects of treatments.

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. ©2017 AACR.

Aim: Mitochondrial function studies in autism spectrum disorders (ASD) have detected skeletal muscle mitochondrial enzyme deficiencies in respiratory complex (RC) activities. As a muscle biopsy is expensive and invasive, we assessed RC-I and RC-IV activities in buccal swabs. Methods: 92 children with ASD and 68 controls were studied with immunocapture for RC-I and microspectrophotometry for RC-IV. Results: Significant RC activity deficiencies were found in 39 (42%) ASD patients (p < 0.01) and more prevalent in more severe cases. Aberrant RC overactivity was seen in 9 children. RC-I/RC-IV activity ratio was significantly increased in 64% of the entire ASD cohort including 76% of those more severely affected (p < 0.05). Conclusion: Buccal swab analysis revealed extensive RC abnormalities in ASD providing a noninvasive biomarker to assess mitochondrial function in ASD patients.

Phelan-McDermid Syndrome (PMS), which is defined by a deletion within 22q13, demonstrates significant phenotypic variation. Given that six mitochondrial genes are located within 22q13, including complex I and IV genes, we hypothesize that mitochondrial complex activity abnormalities may explain phenotypic variation in PMS symptoms. Complex I, II, II + III and IV activity was measured in 51 PMS participants. Caretakers completed questionnaires and provided genetic information through the PMS foundation registry. Complex activity was abnormal in 59% of PMS participants. Abnormalities were found in complex I and IV but not complex II + III and II activity, consistent with disruption of genes within the 22q13 region. However, complex activity abnormalities were not related to specific gene deletions suggesting a “neighboring effect” of regional deletions on adjacent gene expression. A specific combination of symptoms (autism spectrum disorder, developmental regression, failure-to-thrive, exercise intolerance/fatigue) was associated with complex activity abnormalities. 64% of 106 individuals in the PMS foundation registry who did not have complex activity measured also endorsed this pattern of symptoms. These data suggest that mitochondrial abnormalities, specifically abnormalities in complex I and IV activity, may explain some phenotypic variation in PMS individuals. These results point to novel pathophysiology mechanisms and treatment targets for PMS patients.