lncRNA PAARH impacts liver cancer cell proliferation by engaging miR­6512­3p to target LASP1.

Long non-coding (lnc)RNAs serve a pivotal role as regulatory factors in carcinogenesis. The present study aimed to assess the involvement of the lncRNA progression and angiogenesis-associated RNA in hepatocellular carcinoma (PAARH) in liver cancer, along with the associated underlying mechanism. Through the use of reverse transcription-quantitative (RT-q)PCR, differences in the expression levels of PAARH in HepG2, HEP3B2.1.7, HCCLM3, Huh-7 and MHCC97-H liver cancer cell lines and THLE-2 epithelial cell lines were evaluated. The liver cancer cell line with the greatest, significantly different, level of expression relative to the normal liver cell line was selected for subsequent experiments. Using ENCORI database, the putative target genes of the microRNA (miR) miR-6512-3p were predicted. Cells were then transfected with lentiviruses carrying short-hairpin-PAARH to interfere with PAARH expression. Subsequently, HepG2 liver cancer cells were transfected with a miR-6512-3p mimic and an inhibitor, and the expression levels of miR-6512-3p and the LIM and SH3 domain protein 1 (LASP1) in cells were assessed using RT-qPCR analysis. Cell proliferation was subsequently evaluated using colony formation assays, and immunofluorescence and western blotting were used to assess the expression level of LASP1 in transfected cells. The binding interaction between miR-6512-3p and LASP1 was further evaluated using a dual-luciferase reporter gene assay. Liver cancer cells were found to exhibit higher expression levels of PAARH compared with normal liver cells. Following PAARH interference, the expression level of miR-6512-3p was significantly increased, whereas that of LASP1 was significantly decreased, resulting in a reduction in cell proliferation. In liver cancer cells, miR-6512-3p overexpression led to a significant reduction in the LASP1 level and reduced proliferation, whereas suppressing miR-6512-3p led to a significant increase in LASP1 levels and increased proliferation. Additionally, the inhibition of miR-6512-3p caused the states of low LASP1 expression and reduced cell proliferation to be reversed. LASP1, a recently identified target gene of miR-6512-3p, was demonstrated to be suppressed by miR-6512-3p overexpression, thereby inhibiting liver cancer cell proliferation. Taken together, the findings of the present study demonstrate that the lncRNA PAARH may enhance liver cancer cell proliferation by engaging miR-6512-3p to target LASP1.

METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

N6 -methyladenosine-modified FAM111A-DT promotes hepatocellular carcinoma growth via epigenetically activating FAM111A.

As an epitranscriptomic modulation manner, N6 -methyladenosine (m6 A) modification plays important roles in various diseases, including hepatocellular carcinoma (HCC). m6 A modification affects the fate of RNAs. The potential contributions of m6 A to the functions of RNA still need further investigation. In this study, we identified long noncoding RNA FAM111A-DT as an m6 A-modified RNA and confirmed three m6 A sites on FAM111A-DT. The m6 A modification level of FAM111A-DT was increased in HCC tissues and cell lines, and increased m6 A level was correlated with poor survival of HCC patients. m6 A modification increased the stability of FAM111A-DT transcript, whose expression level showed similar clinical relevance to that of the m6 A level of FAM111A-DT. Functional assays found that only m6 A-modified FAM111A-DT promoted HCC cellular proliferation, DNA replication, and HCC tumor growth. Mutation of m6 A sites on FAM111A-DT abolished the roles of FAM111A-DT. Mechanistic investigations found that m6 A-modified FAM111A-DT bound to FAM111A promoter and also interacted with m6 A reader YTHDC1, which further bound and recruited histone demethylase KDM3B to FAM111A promoter, leading to the reduction of the repressive histone mark H3K9me2 and transcriptional activation of FAM111A. The expression of FAM111A was positively correlated with the m6 A level of FAM111A-DT, and the expression of methyltransferase complex, YTHDC1, and KDM3B in HCC tissues. Depletion of FAM111A largely attenuated the roles of m6 A-modified FAM111A-DT in HCC. In summary, the m6 A-modified FAM111A-DT/YTHDC1/KDM3B/FAM111A regulatory axis promoted HCC growth and represented a candidate therapeutic target for HCC.

M2 macrophage-derived extracellular vesicles facilitate CD8+T cell exhaustion in hepatocellular carcinoma via the miR-21-5p/YOD1/YAP/ß-catenin pathway.

Hepatocellular carcinoma (HCC) is a common malignancy. CD8+ T cell-mediated immune response is critical for the inhibition of HCC progression. M2 macrophages participate in HCC progression. This study set out to investigate the effect of M2 macrophage-derived extracellular vesicles (EVs) on CD8+ T cell exhaustion in HCC. M2 macrophage-derived EVs were isolated and identified. The murine model of primary HCC was established through DEN/CCl4 induction, and model mice were injected with EVs. Peripheral blood mononuclear cells (PBMCs) were isolated from the mouse liver and CD8+ T cells were sorted. The expressions of immune checkpoint inhibitory receptors and effector cytokines on CD8+ T cells were detected, followed by the evaluation of CD8+ T cell proliferation and killing function. miR-21-5p expression in M2 macrophage-derived EVs was detected. The binding relationship between miR-21-5p and YOD1 was verified. The activation of the YAP/ß-catenin pathway was detected. Consequently, M2 macrophage-derived EVs promoted CD8+ T cell exhaustion in HCC mice. miR-21-5p expression was upregulated in M2 macrophage-derived EVs, and EVs carried miR-21-5p into HCC tissues. miR-21-5p targeted YOD1. Inhibition of miR-21-5p or overexpression of YOD1 annulled the promoting effect of EVs on CD8+ T cell exhaustion. YOD1 inactivated the YAP/ß-catenin pathway. In conclusion, M2 macrophage-derived EVs facilitated CD8+ T cell exhaustion via the miR-21-5p/YOD1/YAP/ß-catenin axis. This study may confer novel insights into the immunotherapy of HCC.