LINC00152 regulates KLC2 via sponging of miR143/KLC2 thereby promoting proliferation of human liver cancer cells

Long non-coding RNAs (lncRNAs) play pivotal roles in multiple cellular processes by regulating gene transcription and protein turnover. In particular, lncRNAs can work as sponge for miRNAs, thereby indirectly regulating miRNA target gene expression. We have previously identified a methylation-dependent upregulation of LINC00152 in human hepatocellular carcinoma (HCC) by integration of methylome and gene expression profiling data. Here, we explored the competing endogenous RNA (ceRNA) network driven by LINC00152 in human HCC.

An in silico approach predicted 22 miRNAs potentially binding to the LINC000152 RNA sequence. In the next step, 2664 genes were predicted by at least two algorithms to be targets of the identified miRNAs. The association between the expression of the candidate miRNAs and their putative mRNA candidates was analyzed in a human HCC cohort (n=40) to derive the components of the LINC00152-driven ceRNA network. As an experimental validation, the presence of the top 10 ranked miRNAs was confirmed in LINC00152 ribonucleoprotein complexes (miRNPs) by RNA immunoprecipitation of HuH7 and HepG2 cells. Furthermore, quantitative RT-PCR revealed that the expression level of predicted genes was significantly downregulated in LINC00152 knock-out HuH7 cell clones. One of the most significantly altered genes was Kinesin Light Chain 2 (KLC2), which was upregulated in HLE cells overexpressing LINC00152. Analyses of the 3´UTR of KLC2 showed a potential binding site for miR143-3p, which was also found enriched in the miRNPs with LINC00152. As expected, transfection of the specific miRNA mimics led to significant decrease in KLC2 mRNA and protein levels in HuH7 cells when compared to control cells. Additionally, siRNA-mediated knockdown of KLC2 revealed reduced cell viability of liver cancer cell lines compared to scramble siRNA transfected cells; in line with this similar results were observed in LINC00152-deficient cell lines. Furthermore, KLC2 expression was upregulated in human HCCs compared to non-tumor liver tissues in the TCGA sample cohort and was significantly associated with LINC00152 expression, thereby independently validating our human findings.

In summary, our data demonstrate that LINC00152 drives a ceRNA network in human HCC, in which KCL2 is upregulated via sponging and thus reducing the bioavaibility of miR-143-3p, which promotes proliferation of HCC cells.

Publication History

Article published online:
04 January 2021