Defining the LINC00152-modulated ceRNA network in human hepatocellular carcinoma

Large intergenic noncoding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes; particularly, they regulate gene expression at the level of post-transcriptional processing such as protein synthesis, RNA maturation, and transport. To date only few lncRNAs have been functionally characterized and some have already proven to exert protumorigenic functions. Using methylome analysis and integrative profiling of human hepatocellular carcinoma (HCC) we observed differential hypomethylation of the LINC00152 promoter in human HCCs compared to normal liver, which was associated with LINC00152 overexpression in the tumor tissue. Recently, LINC00152 has been proposed to exert tumorigenic functions by acting as competing endogenous RNA (ceRNA) in gastric cancer. Therefore we hypothesized that LINC00152 may function as sponge for miRNAs, modulating the bioavailability of the interacting miRNAs and their target mRNAs in human HCC.

In silico analysis (http://www.mircode.org) revealed that the LINC00152 splice variant 3, which is the most abundantly expressed one in human HCC, contains binding sites for members of 24 miRNA families. In our cohort of human HCC samples the expression of 22 miRNAs was detectable by array analyses. In order to model the potential LINC00152-mediated ceRNA network, we applied a meta-approach Starbase (http://starbase.sysu.edu.cn/) to identify potential miRNA target genes. In total, 2664 genes were predicted to be potentially involved in this ceRNA network by at least two independent mirRNA target prediction algorithms. Next, we performed an integrative analysis by evaluating the correlation of mirRNA targets and LINC00152 expression in our human HCC cohort and TGCA. We were able to select putative miRNAs and target genes involved in the LINC00152 ceRNA network. The top scoring candidates were miRNAs miR.23a.3 p, miR.125a.5 p miR.125b.5 p, miR.223.3 p and miR.143.3 p, and their potential target genes STK39, FAM60A, FUT4, PALLD and MAP3K1. Furthermore, we could detect decreased expression of these target genes in HuH7 cells deficient for LINC00152 when compared to the corresponding control cell line. To proof that LINC00152 acts as a sponge in HCC, we performed RNA IP and we were able to detect LINC00152 as a component of ribonucleoprotein complexes (miRNPs), assigning to LINC00152 a potential role in the control of miRNA bioavailability.

All together our data demonstrate that LINC00152 may function as a part of ceRNA network in human HCC and further analyses are needed to investigate its precise tumorigenic function by acting as a miRNA sponge.