The miR-561-5p/CX₃CL1 Signaling Axis Regulates Pulmonary Metastasis in Hepatocellular Carcinoma Involving CX₃CR1⁺ Natural Killer Cells Infiltration

Abstract

Natural killer (NK) cell can inhibit tumor initiation and regulates metastatic dissemination, acting as key mediators of the innate immune response. Intrinsic factors modulating NK cells infiltration and its anticancer activity remain poorly characterized. We investigated the roles of dysregulation of micro(mi)RNAs and NK cells in progression of hepatocellular carcinoma (HCC). Methods : Small RNA sequencing were used to detect the miRNA profiles of tumor tissues from HCC patients with (n=14) or without (n=13) pulmonary metastasis and HCC cell lines with different pulmonary metastatic potentials. Chemokine expression profiling and bioinformatics were used to detect the downstream target of candidate target. In gain- and loss-of-function assays were used to investigate the role of miRNA in HCC progression. Different subsets of NK cells were isolated and used for chemotaxis and functional assays in vivo and in vitro . In situ hybridization and immunohistochemical analyses were performed to detect the expression of miRNA in tumor tissues from 242 HCC patients undergoing curative resection from 2010. Results : Three miRNAs (miR-137, miR-149-5p, and miR-561-5p) were identified to be associated with pulmonary metastasis in patients with HCC. miR-561-5p was most highly overexpressed in metastatic HCC tissues and high-metastatic-potential HCC cell lines. In gain- and loss-of-function assays in a murine model, miR-561-5p promoted tumor growth and spread to the lungs. Yet, miR-561-5p did not appear to affect cellular proliferation and migration in vitro . Bioinformatics and chemokine expression profiling identified chemokine (C-X 3 -C motif) ligand 1 (CX 3 CL1) as a potential target of miR-561-5p. Furthermore, miR-561-5p promoted tumorigenesis and metastasis via CX 3 CL1-dependent regulation of CX 3 CR1 + NK cell infiltration and function. CX 3 CR1 + NK cells demonstrated stronger in vivo anti-metastatic activity relative to CX 3 CR1 - NK cells. CX 3 CL1 stimulated chemotactic migration and cytotoxicity in CX 3 CR1 + NK cells via STAT3 signaling. Blockade of CX 3 CL1, CX 3 CR1, or of pSTAT3 signaling pathways attenuated the antitumor responses. Clinical samples exhibited a negative correlation between miR-561-5p expression and levels of CX 3 CL1 and CX 3 CR1 + NK cells. High miR-561-5p abundance, low CX 3 CL1 levels, and low numbers of CX 3 CR1 + NK cells were associated with adverse prognosis. Conclusion ...

Expression profile of SYNE3 and bioinformatic analysis of its prognostic value and functions in tumors

“…However, the function of miR-149-5p depends on specific tumor types. MiR-149-5p can help to disable the invasion and proliferation of medullary thyroid carcinoma cells [23] and suppress the development of non-small cell lung cancer [24] and hepatocellular carcinoma [25,26]. Combined with our survival analysis, tumor-promoting functions of miRNAs displayed more obvious in KIRC, CESC, LUAD and HNSC.…”

“…First, we screened cancer types whose immune infiltration was correlated with SYNE3 expression. Accordingly, we found that SYNE3 expression was significantly correlated with dendritic cell, neutrophil, CD4 + T cell, macrophage, CD8 + T cell and B cell in 30,30,27,26,25,25 and 22 types of cancer respectively.…”

Highly efficient serum-free manipulation of miRNA in human NK cells without loss of viability or phenotypic alterations is accomplished with TransIT-TKO

“…Analysis of miRNA transcriptomes from NK cells derived from peripheral blood, cord blood, and uterine decidua reveal significant differences in miRNA profiles, suggesting that miRNA may help to calibrate and tailor NK cell effector functions. Manipulation of miRNA profiles may complement ongoing engineering by allowing control of intracellular networks to buffer environmental signals and allow persistent NK cell migration, reactivity or regulation [18].…”

“…The high stability of miRNA, and that it is released and available in body fluids make it attractive candidates as biomarkers or for targeted therapies [16][17][18]. Although transfection techniques are numerous, efficient transfection of primary NK cells without introducing offtarget effects or skewing the NK cell population remains challenging.…”

“…The broad regulation of cellular processes by miRNA make them intriguing as agents to modulate NK cells for immunotherapy and to monitor ongoing processes. Already, miRNA species have been implicated as biomarkers for identifying and characterizing diseases or monitoring treatment efficacy [16][17][18]. Noteworthy, one miRNA-based treatment, miravirsen, is being tested against hepatitis C (clinicaltrials.gov # NCT02508090).…”