Upregulation of miR-205-3p expression in liver cancer vascular endothelial cells
Liver cancer vascular endothelial cells were discovered to have upregulated miR-205-3p expression levels. Additionally, the miR-205-3p levels expressed in liver cancer vascular endothelial cells and adjacent normal cells from patient specimens were evaluated using Real-Time Fluorescence Quantitative PCR (RT-qPCR). In ten samples of liver cancer vascular endothelial cells, miR-205-3p was observed to be significantly augmented. (Fig. 2A). Given these outcomes, RT-qPCR was conducted to further probe the potential alterations in miR-205 expression in HUVECs and among vascular endothelial cells (HepG2, HepG3B, and huh7) in varying liver cancer transplantation tumors. Consequently, the miR-205-3p levels were considerably elevated in vascular endothelial cells of liver cancer compared to those in HUVECs (Fig. 2B).
Five patients with advanced stages (stages III and IV) and five patients with early stages (stages I and II) were examined to understand the changes in miR-205-3p expression. Our findings illustrated a significant upregulation of miR-205-3p expression in the advanced stages relative to the early stages (Fig. 2C).
Screening for target genes of miR-205-3p.
The candidate miR-205-3p target genes were screened utilizing the miRNA targeting gene screening technique, and the colony PCR result map (Fig. 3A) was obtained using Total RNA extraction, and RT-PCR. Each band represented a potential miR-205-3p gene target. Luciferase reporter and TargetScan were applied to validate the candidate miR-205-3p target genes. Subsequent to this, specific sequences from the candidate miR-205-3p target genes were extracted, leading to the selection of HINT1, the target gene correlated with liver cancer. (Fig. 3B). By analyzing the miR-205-3p sequence and the 3’UTR region of HINT1, the complementary binding site (Fig. 3C) was determined. Hence, miR-205-3p has the potential to suppress HINT1 expression by interacting with the 3’UTR.
Identifying HINT1 as a miR-205-3p target gene.
It should be noted that miR-205-3p specifically targets HINT1 as its gene of interest. MiRNAs suppress the biological functioning of their gene targets by regulating their expression in cells [25]. Furthermore, a single miRNA has the capability to target numerous genes simultaneously, potentially reaching thousands of gene targets. In addition, based on our findings, there is a possibility that HINT1 and miR-205-3p interact with one another at the 3’UTR region, as depicted in Figure 4A. To confirm our hypothesis, we employed a dual luciferase assay by inserting the wild-type (WT) or mutant (MUT) 3’UTR of HINT1 downstream of the firefly luciferase gene (Figure 4B). After 48 hrs, luciferase expression data were extracted from HepG2 and HepB3 cells post-transfection with MiR-205-3p. These cells were then co-transfected with either the WT or MUT 3’UTR of the HINT1 plasmid. Remarkably, when contrasted to the miR-NC group, the luciferase activity of the WT 3’UTR was considerably elevated by the miR-205-3p inhibitor. However, there was no significant variation between the miR-NC and MUT 3’UTR groups (Figure 4B). Moreover, as illustrated in Figure 4C, after transfecting HepG2 and HepB3 cells with an inhibitor of miR-205-3p, the levels of mRNA and protein expression of HINT1 were observed to be elevated. These data strongly imply that HINT1 potentially represents a direct miR-205-3p target in vascular endothelial cells derived from liver cancer tissues.
The impact of miR-205-3p knockdown on the proliferative capacity of ECs derived from HepG2 and HepB3 xenografts.
We sought to investigate the impact of miR-205-3p knockdown on the proliferative capacity of vascular endothelial cells (ECs) derived from liver cancer. To accomplish this, to probe the impact that miR-205-3p has on the proliferative capacity of HepG2 and Hep3B cells, we used an miR-205-3p inhibitor. These cell lines were specifically chosen for their upregulated miR-205-3p expression in liver cancer xenograft tumor vessels.
When contrasted to the group that was treated with miR-NC, the level of miR-205-3p was remarkably reduced in the group that had been transfected with an inhibitor of miR-205-3p (Fig. 5A). We applied MTT tests to determine whether or not miR-205 had any effect on the proliferative capacity of HepG2 and Hep3B cells. As depicted in Fig. 5B, at 72 hours post-transfection, cell viability was remarkably reduced due to the miR-205-3p inhibitor-induced downregulation of miR-205-3p expression. Furthermore, we conducted colony formation experiments to determine whether knocking down miR-205-3p affected cellular proliferation. The data confirmed a reduction in the total count of clones in the miR-205-3p inhibitor group as opposed to the control group (Fig. 5C).
Inhibition of miR-205-3p inhibits the proliferative capacity of liver cancer cells and enhances apoptosis in mouse xenograft models.
The tumor growth inhibition and apoptosis promotion effects of miR-205-3p antagomir were investigated in mouse xenograft models of liver cancer. To assess the mid-term tumor suppression activity of miR-205-3p, HepG2 cells were subcutaneously injected into mice before treatment using miR-205-3p antagomir (Fig. 6A and Fig. 6B). Notably, a reduction in tumor weight and size was observed in the miR-205-3p antagomir treatment group as opposed to the antagomir NC treatment group. Moreover, miR-205-3p antagomir treatment significantly suppressed tumor cell proliferation, as evidenced by a decrease in Ki67 expression (Fig. 6C).
Preceding research revealed that in liver cancer, HINT1 suppresses the β-catenin protein’s functionality[26], a crucial protein entity within the Wnt signaling pathway. The translocation of this protein from the cytoplasm to the nucleus signifies the activation of the Wnt signaling pathway and its corresponding function. In the cell, the cytoplasmic destruction complex, which includes β-catenin, APC, Axin, GSK-3β, and CK1, is involved in facilitating the degradation of β-catenin at a reduced level. Upon the destruction of this complex, the accumulation of β-catenin is accompanied by its movement into the nucleus, where it activates its target genes. Previous research suggests that around 70% of liver cancer cases exhibit cytoplasmic accumulation of β-catenin.[27]. Precise regulation of cell invasion and migration is critical to prevent cancer cells from acquiring high invasiveness and metastatic potential. In this process, β-catenin serves dual roles. Firstly, it acts as a crucial linker in adherens junctions, facilitating cell-cell adhesion. Moreover, the AJ or tight junction complex, mediated by E-cadherin, maintains the adhesive function of E-cadherin as a key component in the Wnt/β-catenin pathway. Additionally, β-catenin is involved in the adherens junction, AJ, or tight junction complex, where it acts as a bridge between E-cadherin and the cytoskeleton, thereby preserving the adherence function of E-cadherin. Furthermore, β-catenin functions as a pivotal effector molecule in the Wnt/β-catenin pathway, translocating into the nucleus and promoting the expression of tumor cell metastasis-related genes [28]. Hence, the invasion and migration processes are regulated by the intracellular levels of β-catenin [29]. Effectively suppressing tumorigenesis, progression, invasion, and migration can be achieved by reducing intracellular levels of β-catenin and attenuating its capacity to translocate into the nucleus [30].