The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix specimens (313 CaCx, 78 controls) and CaCx cell lines, we explored relationships among Cx expression, prognostic variables and mechanisms that may link them. In CaCx specimens, Cx32 was upregulated and cytoplasmically localized, and three other Cx downregulated, relative to controls. Cx32 expression correlated with advanced FIGO staging, differentiation and increased tumor size. In CaCx cell lines, Cx32 expression suppressed streptonigrin/cisplatin-induced apoptosis in the absence of functional GJ. In CaCx specimens and cell lines, expression of Cx32 upregulated epidermal growth factor receptor (EGFR) expression. Inhibition of EGFR signaling abrogated the anti-apoptotic effect of Cx32 expression. In conclusion, upregulated Cx32 in CaCx cells produces anti-apoptotic, pro-tumorigenic effects in vivo and vitro. Abnormal Cx32 expression/localization in CaCx appears to be both a mechanism and biomarker of chemotherapeutic resistance.

MicroRNA (miRNA) holds promise as a novel therapeutic tool for cancer treatment. However, the transfection efficiency of current delivery systems represents a bottleneck for clinical applications. Here, we demonstrate that gap junctions mediate an augmentative effect on the antiproliferation mediated by miR-124-3p in U87 and C6 glioblastoma cells. The functional inhibition of gap junctions using either siRNA or pharmacological inhibition eliminated the miR-124-3p-mediated antiproliferation, whereas the enhancement of gap junctions with retinoic acid treatment augmented this miR-124-3p-mediated antiproliferation. A similar effect was observed in glioblastoma xenograft models. More importantly, patch clamp and co-culture assays demonstrated the transmission of miR-124-3p through gap junction channels into adjacent cells. In further exploring the impact of gap junction-mediated transport of miR-124-3p on miR-124-3p target pathways, we found that miR-124-3p inhibited glioblastoma cell growth in part by decreasing the protein expression of cyclin-dependent kinase 6, leading to cell cycle arrest at the G0 /G1 phase; moreover, pharmacological regulation of gap junctions affected this cell cycle arrest. In conclusion, our results indicate that the "bystander" effects of functional gap junctions composed of connexin 43 enhance the antitumor effect of miR-124-3p in glioblastoma cells by transferring miR-124-3p to adjacent cells, thereby enhancing G0 /G1 cell cycle arrest. These observations provide a new guiding strategy for the clinical application of miRNA therapy in tumor treatment.

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Micro RNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of micro RNA , which exerts biological effects on tumor cells. However, the length of micro RNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to micro RNA s consisting of 18‐27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the micro RNA s were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and sh RNA significantly decreased the transfer of these micro RNA s. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by micro RNA ‐34a. Additionally, these effects of micro RNA ‐34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of micro RNA by transfer of micro RNA to neighboring cells. Our data are the first to clarify the pattern of micro RNA transmission through gap junctions and provide novel insights to show that antitumor micro RNA s should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.

Tumour necrosis factor α (TNFα) and TNF‑related apoptosis inducing ligand (TRAIL) usually trigger either survival or apoptosis signals in various cell types, and nuclear factor κB (NF‑κB) is a key factor that regulates their biological effects. Connexin 32 (Cx32) is a gap junction (GJ) protein that plays vital roles in tumourigenesis and tumour progression. Our previous study explored abnormal Cx32 expression in para‑nuclear areas, exacerbated prognostic parameters and suppressed streptonigrin/cisplatin-induced apoptosis in human cervical cancer (CaCx) cells. In this study, we investigated the role of Cx32 in the extrinsic apoptosis pathway of CaCx cells. In transgenic HeLa cells and C-33A cells, Cx32 expression was manipulated using doxycycline or Cx32 siRNA. GJ inhibitors or low density culturing was used to change the status of gap junction intracellular communication (GJIC). We found that apoptosis induced by TNFα and TRAIL was suppressed by Cx32 expression despite the presence or absense of GJIC. We also found that Cx32 upregulated the expression of nuclear NF‑κB and its downstream targets c-IAP1, MMP‑2, and MMP‑9 in HeLa‑Cx32 and C-33A cells. Following our previous study design, our clinical data showed that NF‑κB and MMP‑2 levels increased in human CaCx specimens with high Cx32 expression compared to levels in para‑carcinoma of cervical specimens. SC75741 and JSH-23, NF‑кB signalling pathway inhibitors, inhibited the anti-apoptotic effects of Cx32. In conclusion, Cx32 suppressed TNFα /TRAIL-induced extrinsic apoptosis by upregulating the NF‑κB signalling pathway. This study demonstrates a novel mechanism for Cx32's anti-apoptotic effect and provides a reasonable explanation for the pro-tumour effect of Cx32 in human CaCx cells.