LINC02418 expression is upregulated and associated with poor prognosis in CRC patients
To identify the LncRNA expression profile in CRC patients, we examined the expressional level of LncRNAs in human CRC samples and normal intestinal tissue. By using the Cancer Genome Atlas (TCGA) database, we found LINC02418 abundance was significantly up-regulated in CRC samples (n = 478) when compared with normal tissue (n = 41) (Fig. 1a).To validate the results, LINC02418 level in 20 pairs of CRC samples and adjacent tissue were examined by RT-qPCR. Similarly, quantification test showed LINC02418 was highly expressed in 19 out of the 20 CRC patients (Fig. 1b). These results implied that expression of LINC02418 in CRC tissues was markedly higher than that in normal tissues. Moreover, the expression of LINC02418 is increased by 2–6 folds in SW620, HCT116, LoVo, Colo205, HT-29 and SW480 cell line when compared with normal colon epithelial cell line NCM460 (Fig. 1c). The HCT116 and LoVo cell lines were chosen for further experiments, because they had the highest level of LINC02418.
Next, to study the clinical significance of LINC02418 in CRC, the correlation between LINC02418 expression and clinicopathologic characteristics was assessed by Kaplan-Meier survival assay. As shown in Fig. 1d and e, in both early (Stage 1–2) and late stages (Stage 3–4), CRC patients with high expression of LINC02418 had poorer overall survival (OS) rate, which indicate LINC02418 is one potential indicator for prognosis prediction and LINC02418 may exert promoter role in CRC progress.
Knock-down of LINC02418 inhibits tumor growth, cell mobility and cell invasion
In order to dissect the effect of LINC02418 on the biologic activity of CRC cell lines, HCT116 and LoVo cells stably transduced with shLINC02418 were established. Based on the endogenous level of LINC02418, HCT116-sh-LINC02418#1 and LoVo-sh-LINC02418#1 were selected for further analysis (Fig. 2a). The CCK-8 assay revealed that compared with sh-NC transduced CRC cells and blank control (without shRNA transducing) group, stable knockdown of LINC02418 statistically inhibited the proliferation of HCT116 and LoVo cells (Fig. 2b). Colony formation assay was carried out to further analyze the effect of LINC02418 on tumor cell growth. As revealed in Fig. 2c, stable down-regulation of LINC02418 significantly reduced the proliferation of HCT116 and LoVo cells.
To evaluate whether LINC02418 affect CRC growth in vivo, subcutaneous tumor formation experiment was set up in nude mice. HCT116 and LoVo cells transduced with sh-LINC02418 or sh-NC were subcutaneously injected into the nude mice for 21 days and tumor size was recorded at indicated days. As shown in Fig. 2d and e, tumor size of xenograft tumor from sh-LINC02418-transduced HCT116 and LoVo cells were obviously less than that in sh-NC-transduced cells, which was consistent with in vitro experiments.
The transwell experiment showed the stable inhibition of LINC02418 significantly repressed mobility and invasiveness of HCT116 and LoVo cells (Fig. 2f). Taken together, these data suggested that LINC02418 contributed to CRC cell growth and metastasis in vivo and in vitro.
LINC02418 acts as a ceRNA for miR-34b-5p in CRC cells
It is documented that LncRNAs could exert their function trough acting as ceRNA of miRNAs . The lncRNA-miRNA-mRNA regulatory network has been proved participate in cancer malignization. To assess whether LINC02418 interacted with other miRNAs in CRC, the potential binding partner for LINC02418 was analyzed by online software StarBase v2.0. As shown in Fig. 3a, LINC02418 contained putative binding sequence for miR-34b-5p.
To confirm the binding between LINC02418 and miR-34b-5p, dual luciferase assays were performed in both HCT116 and LoVo cell lines. The luciferase activity was markedly inhibited when LINC02418-WT and miR-34b-5p mimic were co-transfected into HCT116 and LoVo cells. Co-transfection of LINC02418-WT and miR-NC and co-transfection of LINC02418-MUT and miR-34b-5p mimic had no statistical impact on luciferase activity (Fig. 3b). In addition, knockdown of LINC02418 in HCT116 and LoVo cells greatly enhanced the expression level of miR-34b-5p (Fig. 3c).Taken together, it could be found that LINC02418 negatively regulated miR-34b-5p expression in CRC cells.
Subsequently, we detected the relationship between LINC02418 and miR-34b-5p in clinical samples. In 20 pairs of CRC samples, LINC02418 expressional level negatively correlated with miR-34b-5p level (Fig. 3d, e).
BCL2 is the target of miR-34b-5p in CRC cells
BCL2, the gatekeeper for cell apoptosis, was identified as one possible target for miR-34b-5p by using software TargetScan (Fig. 4a). Interestingly, previous articles reported that miR-34b-5p regulates multiple cellular processes including cell apoptosis and cell proliferation through participating in a several critical signal pathway like VEGF-A, BCL2 and Notch . To determine the interaction between BCL2 and miR-34b-5p, wild type 3’UTR (containing miR-34b–5p recognition site) and the mutant 3’UTR of BCL2 were cloned into luciferase reporter plasmid. Dual-luciferase assay showed that miR-34b-5p mimic transfection reduced luciferase activity in wild type construction but not in mutant type construction (Fig. 4b, c). In parallel, miR-34b-5p transfection reduced endogenous protein level of BCL2, while miR-34b-5p inhibitor addition significantly restored BCL protein expression in both HCT116 and LoVo cells (Fig. 4d). These results proved that BCL2 was one of the target genes of miR-34b-5p. Moreover, quantification of BCL2 in 20 pairs of CRC samples revealed that BCL2 expressional level was positively correlated with the amount LINC02418 (Fig. 4e, f).
LINC02418 promotes colon cancer cell proliferation by upregulating BCL2 via sponging miR-34b-5p
To elucidate the effect of LINC02418 on miR-34b-5p/BCL2 axis, protein level of several apoptotic markers were detected in HCT116 and LoVo cells. Western blot analysis showed that knockdown of LINC02418 greatly enhanced the amount of cleaved-Caspase 9 and cleaved-Caspase 3, and strongly reduced BCL2 expression. Transfection of miR-34b-5p inhibitor into cells with defective expression of LINC02418 not only reduced the level of cleaved forms of Caspase 9 and Caspase 3, but also promoted protein expression of BCL2.Overexpression of BCL2 also reduced protein level of cleaved-Caspase 9 and cleaved-Caspase 3 (Fig. 5a). Subsequently, CCK-8 and colony formation assays showed inhibition of LINC02418 repressed cell growth in HCT116 and LoVo cells. However, transfection of miR-34b-5p inhibitor and overexpression of BCL2 abolished the effect of LINC02418 depletion on cell proliferation (Fig. 5b, c). These findings demonstrated that LINC02418 regulated colon cancer cell proliferation through upregulating BCL2 expression via sponging miR-34b-5p.