Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Currently, an increasing evidence showed that circular RNAs (circRNAs) play important roles in tumor progression. However, the effects and underlying mechanisms of circRNAs in CRC progression remain unclear. In the present study, through circRNA high-throughput sequencing and quantitative real-time polymerase chain reaction, we identified that hsa_circ_0136666 was significantly overexpressed in CRC tissues and cell lines. High hsa_circ_0136666 expression was associated with poor overall survival of patients with CRC. In vitro function assays showed that hsa_circ_0136666 inhibition suppressed CRC cell proliferation, migration, invasion, and arrested CRC cells in the G0/G1 phase. Furthermore, we showed that hsa_circ_0136666 inhibition reduced CRC cell growth in vivo. Mechanistically, we revealed that hsa_circ_0136666 could increase SH2B1 expression via competitively binding miR-136 in CRC cells. In addition, SH2B1 overexpression could reverse the effects of hsa_circ_0136666 inhibition on CRC cell progression. In conclusion, our data suggested that hsa_circ_0136666 could promote CRC cell progression via the miR-136/SH2B1 axis, elucidating a novel approach to improve the effectiveness of CRC treatment. K E Y W O R D Scolorectal cancer (CRC), hsa_circ_0136666, miR-136, SH2B1

Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3′untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway. K E Y W O R D Smelanoma, miR-145-5p, the nuclear factor kappa B (NF-κB) pathway, toll-like receptor 4 (TLR4)

Key points• Voltage-gated H + channels help to compensate for the pH and voltage disturbances generated by production of reactive oxygen species.• In this study, we investigated how changes in the intracellular pH levels control H + channel activity in macrophage-lineage cells, osteoclasts and microglia.• An increase in intracellular pH decreased the numbers of H + channels available at the plasma membrane through facilitation of dynamin-dependent endocytic internalization.• This inhibitory regulation mechanism for H + channels is novel.• The results help us to understand better the significance of the intracellular pH levels in membrane dynamics and H + channel availability, which, in turn, may modulate natural immunity.Abstract Voltage-gated proton channels (H + channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pH i ) in regulating the availability of H + channels in osteoclasts and microglia. In whole-cell clamp recordings, the pH i was elevated after exposure to NH 4 Cl and returned to the control level after washout. However, the H + channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H + channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H + channel conductance were probably due to the decreases in H + channels available at the plasma membrane. The decreases in the H + channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH 4 Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pH i increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H + channels, pH i increases by these H + -transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H + channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pH i increases in osteoclasts and microglia decrease the numbers of H + channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.

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