Characterization of the small RNA transcriptomes of cell protrusions and cell bodies of highly metastatic hepatocellular carcinoma cells via RNA sequencing.

Abstract

Increasing evidence suggest that hepatocellular carcinoma (HCC) HCCLM3 cells initially develop pseudopodia when they metastasize, and microRNAs (miRNAs/miRs) and circular RNAs (circRNAs) have been demonstrated to serve important roles in the development, progression and metastasis of cancer. The present study aimed to isolate the cell bodies (CBs) and cell protrusions (CPs) from HCCLM3 cells, and screen the miRNAs and circRNAs associated with HCC infiltration and metastasis in CBs and CPs. The Boyden chamber assay has been confirmed to effectively isolate the CBs and CPs from HCCLM3 cells via observation of microtubule immunofluorescence, DAPI staining and nuclear protein H3 western blotting. Following high-throughput sequencing of the successfully isolated CBs and CPs, 64 pairs of miRNAs, including 23 pairs of upregulated genes and 41 pairs of downregulated genes, and 260 sets of circRNAs, including 127 upregulated genes and 133 downregulated genes, were significantly differentially expressed, using the following criteria: HP/HB ratio, fold change ≥|1.5|, P<0.05). PCR analysis verified that changes in the expression levels of hsa-let-7a-5p, hsa-let-7c-3p, hsa-miR-30c-5p, hsa_circ_0059580, hsa_circ_0067475, hsa_circ_0002100 and hsa_circ_00072309 were consistent with the sequencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze the functions and roles of the differentially expressed miRNAs and circRNAs. The interaction maps between miRNAs and circRNAs were constructed, and signaling pathway maps were analyzed to determine the molecular mechanism and regulation of the differentially expressed miRNAs and circRNAs. Taken together, the results of the present study suggest that the Boyden chamber assay can be used to effectively isolate the somatic CBs and CPs of HCC, which can be used to screen the miRNAs and circRNAs associated with invasion and metastasis of HCC.