LINC00152 is upregulated in glioma tissues and its expression is associated with glioma patient outcomes
LINC00152 is an oncogene that promotes cell proliferation, invasion, and migration in various tumors(Yu et al. 2017). To investigate the expression of LINC00152 in gliomas and normal brain tissues, the GSE16011 database was used. The result showed that the expression of LINC00152 was significantly upregulated in gliomas compared to levels in normal tissue (p < 0.05, Fig. 1a). Furthermore, we analyzed the expression of LINC00152 in different grades of glioma and found that its level increases with WHO grades based on the GSE16011 database (Fig. 1a), which was well validated in TCGA and CGGA datasets (Fig. 1b and supplementary Fig. 1a). Moreover, LINC00152 expression was significantly correlated with MGMT promoter status and IDH status of glioma. LINC00152 expression in IDH-Wt gliomas was markedly higher than that in IDH-Mut gliomas based on TCGA database (Fig. 1c), consistent with result obtained from the CGGA database (Supplementary Fig. 1b). LINC00152 expression in gliomas with MGMT promoter unmethylation was significantly higher than that in gliomas with MGMT promoter methylation based on TCGA database (Fig. 1d). In addition, LINC00152 expression was significantly upregulated in the mesenchymal and classical subtype compared with other two respective molecular subtypes in the TCGA dataset (Fig. 1e).
Since LINC00152 expression significantly associated with WHO grade and molecular subtype in gliomas, we further investigated its prognostic value and the Kaplan–Meier survival analysis was performed. According the median of LINC00152, the glioma patients were grouped into high group and low group. Based on the TCGA database, the result indicated that high expression of LINC00152 is significantly associated with poor prognosis in glioma patients (Fig. 1f), which was well validated in CGGA dataset (supplementary Fig. 1c). To investigate whether LINC00152 could be an independent prognostic marker for glioma, we simultaneously performed univariate and multivariate Cox regression analysis based on the TCGA dataset. Univariate Cox analysis showed that LINC00152 expression, patient age at diagnosis, WHO grade, MGMT promoter status, IDH status and transcriptome subtype were significantly associated with overall survival of glioma patients. According to multivariate Cox analysis, the LINC00152 expression was still a significant predictive factor after adjusting for the aforementioned clinical factors (Table 1).
Table 1
Univariate and multivariable Cox regression analysis of overall survival based on TCGA dataset
|
Univariate analysis
|
Multivariate analysis
|
|
HR
(95% CI for HR)
|
P
|
HR
(95% CI for HR)
|
P
|
LINC00152
|
1.645 (1.551–1.745)
|
***
|
1.206
(1.081–1.345)
|
***
|
gender
male VS Female
|
1.225 (0.955–1.572)
|
0.110
|
-
|
-
|
Age
≤ 40 VS > 40
|
0.231 (0.165–0.324)
|
***
|
0.3947 (0.2565–0.6074)
|
***
|
grade
WHO II VS WHO III + IV
|
0.178 (0.121–0.262)
|
***
|
0.4497 (0.2768–0.7306)
|
**
|
IDH status
WT VS MT
|
10.050
(7.524–13.43)
|
***
|
3.205 (1.863–5.514)
|
***
|
MGMT promoter status
Methylation VS Unmethylation
|
3.228 (2.447–4.259)
|
***
|
-
|
-
|
Transcriptome subtype
NE + PN VS CL + ME
|
0.152 (0.113–0.203)
|
***
|
1.012 (0.6335–1.618)
|
0.959
|
Abbreviations: HR, hazard ratio, CI, Confidence interval. |
Taken together, these results indicated that the expression of LINC00152 was significantly upregulated in GBM and significantly correlated with WHO grade, IDH status, MGMT promoter status and transcriptome subtype of gliomas. Importantly, LINC00152 is an independent prognostic marker for glioma patients and high expression indicates poor clinical prognosis.
The role of LINC00152 in glioma cell proliferation, cell migration, and cell apoptosis
Cell proliferation and invasion are significantly processed in cancer progression(Fang et al. 2015;Huarte 2015;Lathia et al. 2015). To explore the functions of LINC00152 in glioma, we used a short hairpin RNA (shRNA) to decrease the expression levels of LINC00152 in U251 and U87 cells. qRT-PCR analysis showed that this shRNA can downregulate the expression of LINC00152 in both the cell lines with high efficiency (Fig. 2a). As a functional assay, CCK-8 assay was performed to monitor the effects of LINC00152 on cell proliferation and the results showed that knockdown of LINC00152 expression in U251 and U87 cells caused significant inhibition on cell proliferation (Fig. 2b). On the other hand, LINC00152 knockdown can significantly diminish colony formation and invasion abilities of U251 and U87 cells (Fig. 2c and 2d). Furthermore, flow cytometric analysis demonstrated that LINC00152 silencing in U251 and U87 cells induced cell apoptosis and the percentage of apoptotic cells was significantly increased in the LINC00152 knockdown group when compared to that in the control group which were transfected with control shRNA (Fig. 2e). Taken together, these results suggest that LINC00152 plays an important role in the regulation of cell proliferation, invasion, and apoptosis in glioma cells.
LINC00152 regulates tumor growth in vivo
To confirm the functional effects of LINC00152 on glioma cells in vivo, we subcutaneously injected U251 cells with stable LINC00152 knockdowns, or U251 cells were transfected with sh-ctr into the right flanks of 4–6 week-old nude mice. After 35 d, tumors were visible and all the mice were euthanized to harvest xenografts. There was a significant reduction in tumor growth in xenografts with LINC00152 knockdown compared to negative controls and the relative tumor weight in the LINC00152 knockdown group was significantly lighter than that in the control group (Fig. 3a and 3b). H&E staining showed that the tumor tissues were isolated from xenograft mice model (Fig. 3c). In addition, immunohistochemistry (IHC) analysis revealed that the expression of Ki-67 in LINC00152 knockdown group was lower than that in the control group (Fig. 3d). Taken together, these data verify that LINC00152 promotes the growth of GBM cells in vivo.
LINC00152 is a target of miR-107
LncRNAs are known to act as ‘sponges’ to sequester endogenous miRNAs to regulate miRNA targets. Therefore, StarBase v2.0 software (http://starbase.sysu.edu.cn/index.php) was used to identify all potential miRNAs that can bind to LINC00152 gene and the software returned 6 hits, including miR-107, miR-376c-3p, miR-193b-3p, miR-193a-3p, miR-103a-3p, and miR-155-5p. Besides, it has been previously reported that miR-107 is downregulated in glioma and overexpression of miR-107 could inhibit proliferation of glioma(Chen et al. 2016), which compelled us to investigate whether LINC00152 was a real target of miR-107. The site on LINC00152 that miR-107 could potentially bind is shown in Fig. 4a. We also analyzed the expression levels of miR-107 in glioma and normal tissues and further investigated the correction factors between LINC00152 expression and miR-107 expression. The result showed that the expression of miR-107 negatively correlated with the expression of LINC00152 (r =-0.447, p < 0.05, Fig. 4b). Luciferase reporter assays showed that miR-107 mimics significantly suppressed luciferase activity in U251 and U87 cells that carried plasmids with wildtype rather than mutant 3′-UTR of LINC00152 (Fig. 4c), which revealed that LINC00152 was a direct target of miR-107. In addition, LINC00152 silencing could upregulate miR-107 expression in both U251 and U87 cells (Fig. 4d). Taken together, these findings demonstrated that LINC00152 was a direct target of miR-107 and LINC00152 could suppress expression levels of miR-107 in glioma cells.
RAB10 is a target of miR-107
Next, we aimed at identifying the target genes of miR-107 via TargetScan (http://www. targetscan.org/), and observed that RAB10 was predicted as a potential target of miR-107. RAB10, a member of the Ras small GTPase superfamily, is a protein-coding gene with GTP and GDP binding domains. Indeed, recent studies demonstrated that RAB10 is closely associated with the genesis and development of certain cancers(Jiang et al. 2016;Wang et al. 2017). Based on TCGA database, the high expression of RAB10 is significantly associated with short overall survival time in glioma patients (supplementary Fig. 1d). Thus, dual luciferase reporter assays were performed to confirm whether miR-107 binds to the putative miR-107 binding site at the 3′-UTR of RAB10 (Fig. 5a). The results showed that luciferase intensity was significantly attenuated by co-transfected miR-107 mimics and RAB10-WT vectors, but not in the mutant vector lacking the putative miR-107 binding site in both U251 and U87 cells. Also, miR-NC also could not affect the luciferase intensity of RAB10-WT/MT vectors (Fig. 5b). These results suggested that RAB10 is a direct target of miR-107. Then, qRT-PCR and western blotting were performed to assess whether miR-107 could negatively regulate the expression of RAB10 at both the mRNA and protein levels in GBM cell lines, respectively. As expected, miR-107 mimics decreased RAB10 mRNA levels, and conversely, miR-107 inhibitor increased RAB10 mRNA levels (Fig. 5c). Similar results were observed by using western blot analysis (Fig. 5d). These results indicated that RAB10 is a direct binding target of miR-107 and its expression can be regulated by miR-107 levels in glioma samples.
LINC00152 regulates the expression of RAB10 depending on miR-107
Since, LINC00152 harbors an identical miR-107 binding site as RAB10, we wanted to know whether LINC00152 might regulate the expression of RAB10 through miR-107. Firstly, the expression of LINC00152 and RAB10 in glioma tissues were measured and correlation analysis was performed to detect the potential association between them. The results showed that the expression of RAB10 was not only upregulated in glioma tissues, but also had significantly positive correlation with the expression of LINC00152 (Fig. 6a and 6b). Furthermore, knockdown of LINC00152, in U251 and U87 cell lines, decreased the mRNA levels of RAB10 (Fig. 6c) and diminished the expression of RAB10 at the protein level (Fig. 6d). However, overexpression of miR-107 attenuated the effect of LINC00152 silencing on the regulation of RAB10 expression (Fig. 6c and 6d). In summary, these results indicate that LINC00152 can regulate expression of RAB10 depending on miR-107 in GBM cells.
miR-107 reverses the functions of LINC00152 in glioma
To further validate the interactions between LINC00152 and miR-107, we investigated whether miR-107 silencing can rescue the effects caused by LINC00152 knockdown, including cell proliferation, clone formation, invasion abilities in vitro, and tumor growth in vivo. As expected, CCK-8 and colony formation assays demonstrated that miR-107 silencing could weaken the suppressive effects of LINC00152 knockdown on cell proliferation and clone formation in U87 and U251 cells (Fig. 7a-b). Moreover, matrigel transwell assays showed that LINC00152 knockdown and inhibition of miR-107 caused opposite effects on the ability of GBM cell invasion. However, miR-107 inhibitor could partially reverse the effect of LINC00152 knockdown on cell invasion of GBM (Fig. 7c). Furthermore, by in vivo assays, tumor growth in xenografts with co-transfected sh-LINC00152 and miR-107-NC clones was decreased compared with that in negative control group, whereas miR-107 silencing eliminated the suppressor effect induced by LINC00152 knockdown on tumor growth (Fig. 7d-e). Collectively, these results suggest that the suppressive effects of LINC00152 knockdown on GBM could be reversed by miR-107 silencing both in vitro and in vivo.