Supplementary Material for: The Long Non-Coding RNA CRNDE Promotes Colorectal Carcinoma Progression by Competitively Binding miR-217 with TCF7L2 and Enhancing the Wnt/β-Catenin Signaling Pathway

<i>Background/Aims:</i> The long non-coding RNA colorectal neoplasia differentially expressed (CRNDE) contributes to the proliferation and migration of tumors. However, its molecular mechanism underlying gastric cancer remains unknown. In the present study, we investigated whether CRNDE was involved in the development of colorectal cancer via the binding of microRNA (miR)-217 with transcription factor 7-like 2 (TCF7L2) to enhance the Wnt signaling pathway. <i>Methods:</i> Quantitative polymerase chain reaction was used to detect CRNDE, miR-217 and TCF7L2 in colorectal cancer tissues and cells. The CCK-8 assay, wound healing assay, and Transwell assay were used to detect cell proliferation, migration and invasion, respectively. Western blotting and luciferase activity assays were used to identify CRNDE and TCF7L2 as one of the direct targets of miR-217. The activity of the Wnt/β-catenin signaling pathway was analyzed by the TOPflash assay, and the subcellular localization of β-catenin and TCF7L2 was analyzed by western blotting and confocal microscopy. <i>Results:</i> In this study, we found that high expression of CRNDE is negatively correlated with low expression of miR-217 in colorectal cancer tissue and colorectal cancer cells. The dual luciferase reporter analysis showed that miR-217 is bound to CRNDE and TCF7L2 and negatively regulate their expression. CRNDE down-regulation inhibited the cell proliferation, migration and invasion <i>in vitro</i> and <i>in vivo</i> and the inhibitions were both completely blocked after miR-217 inhibition or TCF7L2 overexpression. Finally, TOPflash analysis showed that the activity of Wnt/β-catenin signaling is inhibited by CRNDE down-regulation and rescued by TCF7L2 over-expression. Consistently immunostaining and western blotting analysis showed that the expression of b-catenin and TCF7L2 in the nucleus was significantly decreased by CRNDE down-regulation and was rescued by TCF7L2 over-expression. <i>Conclusions:</i> The present study suggest that CRNDE involves in the cell proliferation, migration and invasion of colorectal cancer cells via increasing the expression of TCF7L2 and activity of Wnt/β-catenin signaling through binding miR-217 competitively

Supplementary Material for: Dietary Protein and Fiber Affect Gut Microbiome and Treg/Th17 Commitment in Chronic Kidney Disease Mice

Background: Patients with chronic kidney disease (CKD) have dysbiosis, dysmetabolism, and immune dysregulation. Gut microbiome plays an important role shaping the immune system which is an important modulator of CKD progression. Methods: We compared the effect of a diet low in protein and high in fiber (LP-HF; n = 7) to that of diet rich in protein, but low in fiber (HP-LF; n = 7) on gut microbiome and T-cell commitment in male CKD (Alb/TGF-β1) mice. The gut microbiomes of these mice were subjected to 16S rRNA taxonomic profiling at baseline, 6 weeks and 12 weeks of the study. Results: The LP-HF diet was associated with an increase in Butyricicoccus pullicaecorum BT, a taxon whose functions include those closely related to butyric acid synthesis (Kendall’s W statistic = 180 in analysis of microbiome composition). HP-LF diet was associated with increased abundance of two predominantly proteolytic bacterial strains related to Parabacteroides distasonis (W statistic = 173), Mucispirillum schaedleri, and Bacteroides dorei (W statistic = 192). Pathway analysis suggested that the LP-HF diet induced carbohydrate, lipid, and butyrate metabolism. As compared with HP-LF mice, LP-HF mice had 1.7-fold increase in CD4+Foxp3+Treg cells in spleen and 2.4-fold increase of these cells in peripheral blood. There was an 87% decrease in percentage of CD4+ Th17 + cells in spleen and an 85% decrease in peripheral blood, respectively, in LP-HF mice compared to the HP-LF mice. Conclusion: The LP-HF diet promotes the proliferation of saccharolytic bacteria and favors T-cell commitment toward Treg cells in a CKD mouse of model. Clinical significance of the finding needs to be further investigated