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  • br Results br Identification of


    3. Results
    3.1. Identification of lncRNAs locally linked to BCAGs
    Literature review with keywords “breast carcinoma” in Malacard (Version 4.5) retrieved 337 breast cancer-associated genes (BCAGs). Neighboring lncRNAs were surveyed within the 15 kb up- and down-stream range from each BCAG, and a total of 121 lncRNAs were iden-tified from 94 BCAGs. The survey strategy is illustrated in Fig. 1A and BCAGs with neighboring lncRNAs are listed in Table S1. Most of the BCAGs have only one nearby lncRNA, but multiple lncRNAs were found for some BCAG regions. The FGF8 gene harbors 4 lncRNAs and each of the regions surrounding IGF1, GATA3, KDM5B and NTRK3 harbors 3 lncRNAs (Fig. 1B). In order to determine direct association, only 73, one to one paired lncRNA and BCAGs, were analyzed further.
    3.2. BCAG and neighboring lncRNA AMG 925 in breast cancer-related cell lines
    We investigated lncRNA expression in MCF7, and MDA-MB-231 human breast cancer cell lines, as well as MCF10A normal control cell lines. Out of 73 pairs of BCAG and lncRNAs, only 20 pairs were vali-dated by RNA expression in the breast cancer-related cell lines The expression patterns of these BCAGs in the breast cancer related cell lines were diverse. MTOR, RAD51B, JUN, PELP1, IL6, TGFBR2, HEATR6, RAD51C and CDC25B showed high expression in breast cancer cell lines, but BRMS1, BARD1, KMD5B, HIF1A, BACH1, and MTA1 maintained a comparable gene expression level between normal and breast cancer cell lines. On the other hand, RAD51, PTK6, STAT1 and SMAD4 showed lower gene expression levels in cancer cell lines than the normal cell line. Very little CCNG1 expression was detected in all 3 breast cell lines (Fig. 2A).
    Furthermore, the expression levels and patterns of neighboring lncRNAs were very varied. Eighteen lncRNAs validated in this study were up-regulated at least in one cancer cell line. Three lncRNAs, LOC105377700 (CCNG1), LOC105370547 (RAD51B), and LINC00189 
    3.3. Correlation in the expression of BCAGs and neighboring lncRNAs
    Quantitative realtime PCR was employed to confirm a correlation in RNA expression between BCAGs and their adjacent lncRNAs in 5 human breast cancer related cell lines (Fig. 3). The most interesting patterns and correlations were found in three pairs of BCGs (RAD51C, BRMS1 and HEATR6) and their adjacent lncRNAs respectively. The pair of RAD51C v.4 and RAD51C mRNA as well as that of LOC105371849 and HEATR6 mRNA were abundantly expressed only in MCF7. Correlation in RNA expression was also found in the LOC102724064 and BRMS1pair, which tended to not be dependent on normal versus cancer, but rather on cell types (Fig. 3A). However, no correlation was found in RNA expression patterns between lnRNAs LOC101559451, LINC01135 and LOC541472 and nearby BCAGs PELP1, JUN and IL6, respectively (Fig. 3B). The genomic structures of these pairs of genes and lncRNAs are line-drawn in Fig. 3C. HEATR6 and BRMS1 have an anti-sense lncRNA 378 bp and 2866 bp upstream of TSS, respectively, but RAD51C and RAD51C v.4 share RNA structures, TSS and exons.
    We tested the concept that closely located lncRNAs and BCAGs may be functionally linked to each other. The LOC105371849 lncRNA and HEATR6 (HEAT repeat containing 6) gene are located on chromosome 17(17q23) in opposite directions. Since the lncRNA (LOC105371849) is 378 bp upstream from the TSS of HEATR6 and covers most of the HEATR6promoter region, the expression of either mRNA or antisense lncRNA, might influence the other. Although association of HEATR6 to breast cancer has been reported, the regulation of its gene expression and its function in both normal and cancer breast tissues are unknown (Fig. 4A). Thus, to knockdown the lncRNA expression in the MCF7 breast cancer cell line, we employed siRNAs targeting LOC105371849, listed in Table S3. By targeting the splicing junction of exon 2 and 3 of LOC105371849 with the siRNA, transcript levels were successfully decreased to 10% of the control level. Interestingly, HEATR6 gene ex-pression was also influenced (~60% decreased) by the knockdown of LOC105371849 lncRNA (Fig. 4B).
    3.5. Putative functions of HEATR6 in human breast cancer
    The molecular markers of BCL2, MKI67 and CCND1 genes, which encode the BCL2, Ki-67 and cyclin D1 proteins, have frequently been used to test cancer cell apoptosis and proliferation (Penault-Llorca et al., 2008; von Minckwitz et al., 2008).The cell morphology and the expression of these genes were examined in MCF7 cancer cell line after the treatment of siRNA targeting anti-HEATR6 lncRNA, to cultivate a better understanding of the function of HEATR6 in breast cancer (Fig. 5). Twenty-four hours after transfection, the cells were examined by phase contrast microscopy and analyzed for cell apoptosis and proliferation using real-time PCR. at first, no difference in either mor-phology or cytotoxicity between untransfected cells and those trans-fected with negative control siRNA was noticed. However, some small but consistent morphological changes were then observed in siLOC105371849 lncRNA transfected cells(Fig. 5A). We examined for