Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer.

BACKGROUND: Although gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro. METHODS: Immunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178. RESULTS: We found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan-Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters. CONCLUSIONS: Our results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

miRNome of Child A hepatocellular carcinoma in Egyptian patients.

Introduction: Hepatocellular carcinoma (HCC) has different etiologies that contribute to its heterogeneity. In regards to the number of HCC patients, Egypt ranks third in Africa and fifteenth worldwide. Despite significant advancements in HCC diagnosis and treatment, the precise biology of the tumor is still not fully understood, which has a negative impact on patient outcomes. Methods: Advances in next-generation sequencing (NGS) have increased our knowledge of the molecular complexity of HCC. Results & discussion: In this research, 16 HCC and 6 tumor adjacent tissues (control) of Child A Egyptian patients were successfully profiled for the expression profile of miRNAs by NGS. Forty-one differentially expressed miRNAs (DEMs) were found by differential expression analysis, with 31 being upregulated and 10 being downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was then conducted on these differentially expressed miRNAs revealing that Sensitivity and specificity analysis showed that hsa-miR-4488, hsa-miR-3178, and hsa-miR-3182 were unique miRNAs as they are expressed in HCC tissues only. These miRNAs were all highly involved in AMPK signaling pathways. However, hsa-miR-214-3p was expressed in control tissues about eight times higher than in cancer tissues and was most abundant in "pathways in cancer and PI3K-Akt signaling pathway" KEGG terms. As promising HCC diagnostic markers, we here suggest hsa-miR-4488, hsa-miR-3178, hsa-miR-3182, and hsa-miR-214-3p. We further urge future research to confirm these markers' diagnostic and prognostic potential as well as their roles in the pathophysiology of HCC.

hsa-miR-3177-5p and hsa-miR-3178 Inhibit 5-HT1A Expression by Binding the 3'-UTR Region in vitro.

Abnormal expression of the 5-HT1A receptor, which is encoded by the HTR1A gene, leads to susceptibilities to neuropsychiatric disorders such as depression, anxiety, and schizophrenia. miRNAs regulate gene expression by recognizing the 3'-UTR region of mRNA. This study evaluated the miRNAs that might identify and subsequently determine the regulatory mechanism of HTR1A gene. Using the HEK-293, U87, SK-N-SH and SH-SY5Y cell lines, we determined the functional sequence of the 3'-UTR region of the HTR1A gene and predicted miRNA binding. Dual luciferase reporter assay and Western Blot were used to confirm the effect of miRNA mimics and inhibitors on endogenous 5-HT1A receptors. In all cell lines, gene expression of the -17 bp to +443 bp fragment containing the complete sequence of the 3'-UTR region was significantly decreased, although mRNA quantification was not different. The +375 bp to +443 bp sequence, which exhibited the most significant change in relative chemiluminescence intensity, was recognized by hsa-miR-3177-5p and hsa-miR-3178. In HEK-293 and U87 cells, hsa-miR-3177-5p significantly inhibited the 5-HT1A receptor expression, while a hsa-miR-3178 inhibitor up-regulated HTR1A gene expression in SK-N-SH and SH-SY5Y cells. By constructing the pmirGLO-vector with the mutated HTR1A gene, we further confirmed that hsa-miR-3177-5p recognized the HTR1A gene tgtacaca at +377 bp to +384 bp, and the +392 bp to +399 bp fragment cgcgccca was identified by hsa-miR-3178. hsa-miR-3177-5p and hsa-miR-3178 had significant inhibitory effects on expression of the HTR1A gene and 5-HT1A receptor and may directly participate in the development of neuropsychiatric diseases.