Down-regulation of EVA1A by miR-103a-3p promotes hepatocellular carcinoma cells proliferation and migration.
EVA1A (Eva-1 homolog A), a novel protein involved in autophagy and apoptosis, functions as a tumor suppressor in some human primary cancers, including hepatocellular carcinoma (HCC). While it is consistently downregulated in several cancers, its involvement in hepatocarcinogenesis is still largely unknown.
We first detected the expression of EVA1A in HCC tissues and cell lines using RT‒qPCR, immunohistochemistry and western blotting and detected the expression of miR-103a-3p by RT‒qPCR. Then, bioinformatics prediction, dual-luciferase reporter gene assays and western blotting were used to screen and identify the upstream microRNA of EVA1A. After manipulating the expression of miR-103a-3p or EVA1A, wound healing, invasion, proliferation, colony formation, apoptosis, autophagy, mitosis and mitochondrial function assays, including mitochondrial membrane potential, ROS and ATP production assays, were performed to investigate the functions of miR-103a-3p targeting EVA1A in HCC cells. Apoptosis-related proteins were assessed by RT‒qPCR (TP53) or western blotting (TP53, BAX, Bcl-2 and caspase-3). Autophagy level was evaluated by observing LC3 puncta and examining the protein levels of p62, Beclin1 and LC3-II/I.
We found that EVA1A expression was decreased while miR-103a-3p expression was increased in HCC tissues and cell lines and that their expression was inversely correlated in HCC patients. The expression of miR-103a-3p was associated with HCC tumor stage and poor prognosis. miR-103a-3p could target EVA1A through direct binding to its 3'-UTR and suppress its expression. Overexpression of miR-103a-3p significantly downregulated the expression of EVA1A, TP53 and BAX, upregulated the JAK2/STAT3 pathway and promoted HCC cell migration, invasion and proliferation, while repression of miR-103a-3p dramatically upregulated the expression of EVA1A, TP53, BAX and cleaved-caspase-3, inhibited HCC cell migration, invasion and proliferation, and caused mitochondrial dysfunction and apoptosis. Overexpression of EVA1A significantly attenuated the cancer-promoting effects of miR-103a-3p in HCC cells, while knockdown of EVA1A alleviated the mitochondrial dysfunction and apoptosis caused by miR-103a-3p inhibition. Overexpression of EVA1A did not induce significant changes in autophagy levels, nor did it affect G2/M transition or mitosis.
These findings indicate that the downregulation of the tumor suppressor EVA1A by miR-103a-3p potentially acts as a key mediator in HCC progression, mainly by inhibiting apoptosis and promoting metastasis. The miR-103a/EVA1A/TP53 axis provides a new potential diagnostic and therapeutic target for HCC treatment.
Xu Q
，Liao Z
，Gong Z
，Liu X
，Yang Y
，Wang Z
，Yang W
，Hou L
，Yang J
，Song J
，Liu W
，Wang B
，Hua J
，Pu M
，Li N
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Midazolam inhibits proliferation and accelerates apoptosis of hepatocellular carcinoma cells by elevating microRNA-124-3p and suppressing PIM-1.
Recently, the impact of microRNAs (miRNAs) has been identified in hepatocellular carcinoma (HCC), this study was designed to assess the effects of miR-124-3p and midazolam (MDZ) in HCC with the involvement of PIM-1.
HepG2 human HCC cells were selected for our study, which were treated with different concentrations of MDZ. The gain- and loss-of-function experiments were performed to elucidate the migration, invasion, proliferation, colony formation ability, cell cycle, and apoptosis of HepG2 cells upon treatment of MDZ, miR-124-3p mimics, or miR-124-3p inhibitor. The expression levels of miR-124-3p, PIM-1, Bax, Bcl-2, P21, and Ki-67 in HepG2 cells were assessed by reverse transcription quantitative polymerase chain reaction and western blot analysis. Moreover, HepG2 cell growth in vivo was measured by subcutaneous tumorigenesis in nude mice, and the target relation between miR-124-3p and PIM-1 was evaluated using dual luciferase reporter gene assay.
We have found that after treated with overexpression of miR-124-3p and MDZ, there exhibited elevated miR-124-3p, declined expression of PIM-1, attenuated migration, invasion, proliferation and colony formation ability, and promoted apoptosis of HepG2 cells. Additionally, it could be observed that the tumor volume and weight were all reduced upon treatment of overexpression of miR-124-3p and MDZ. Meanwhile, the results in the HepG2 cells that treated with down-regulated miR-124-3p were the opposite. Furthermore, PIM-1 was found to be a target gene of miR-124-3p.
Our study found that MDZ could inhibit proliferation and accelerate apoptosis of HCC cells by elevation of miR-124-3p and suppressing PIM-1, which may be an effective method in the treatment of HCC.
Qi Y
，Yao X
，Du X
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ResearchGate
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钛学术
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