LncRNA RP11-436H11.5 expression is upregulated in RCC tissues
Recently, many studies have indicated that lncRNAs can regulate gene expression by complementary binding with miRNAs [9, 20]. Our previous study demonstrated that miR-335 suppressed RCC cell proliferation and invasion [19]. We hypothesized that there were some lncRNAs that could regulate RCC cell progression by binding with miR-335. Therefore, we used a human lncRNA target prediction tool (DIANA TOOLS) to predict the potential lncRNAs that can interact with miR-335-5p. We selected the top 14 potential lncRNAs for the next screening strategy (Additional file 1: Figure S1).
First, qRT-PCR was performed to detect the expression level of these 14 lncRNAs in 2 matched RCC tissues and adjacent normal renal tissues. The results showed that lncRNA RP11-436H11.5 was the most appropriate lncRNA as it was more highly expressed in RCC tissues than in adjacent normal renal tissues (Fig. 1a). Furthermore, we detected lncRNA RP11-436H11.5 expression in 20 matched primary RCC tissues and adjacent normal renal tissues. The results confirmed that the level of lncRNA RP11-436H11.5 was higher in RCC tissues than in adjacent normal renal tissues (Fig. 1b). Meanwhile, a 4-year OS was analyzed by Kaplan-Meier survival curves. Our data showed that RCC patients in the high lncRNA RP11-436H11.5 group presented significantly worse outcomes compared with those in the low lncRNA RP11-436H11.5 group (Fig. 1c).
LncRNA RP11-436H11.5 promotes RCC cell proliferation and invasion.
Next, we explored the functional effects of lncRNA RP11-436H11.5 on RCC cells. A498 and 786-O cells were transfected with lncRNA RP11-436H11.5-shRNA and functional lncRNA RP11-436H11.5-cDNA. The qRT-PCR results confirmed that the expression of lncRNA RP11-436H11.5 was effectively modulated in A498 and 786-O cells (Fig. 1d and e).
Functionally, the cell proliferation assay demonstrated that knockdown of lncRNA RP11-436H11.5 significantly decreased cell proliferation, whereas overexpression of lncRNA RP11-436H11.5 significantly increased cell proliferation in A498 and 786-O cells (Fig. 1f and g).
As expected, using a transwell invasion assay, we obtained similar results in both A498 (Fig. 1h) and 786-O cells (Fig. 1i).
Together, these results demonstrated that lncRNA RP11-436H11.5 was more highly expressed in RCC tissues than in adjacent normal renal tissues and significantly promoted RCC cell proliferation and invasion.
LncRNA RP11-436H11.5 enhances RCC cell progression by upregulating BCL-W expression
Our previous study demonstrated that miR-335-5p could inhibit RCC cell proliferation and invasion through direct suppression of BCL-W [19]. We know that lncRNA RP11-436H11.5 can regulate many downstream genes to influence RCC cell biological behaviors. We attempted to explore whether BCL-W is one of the downstream genes of lncRNA RP11-436H11.5.
As our expected, western blot assay revealed that knocking down lncRNA RP11-436H11.5 decreased the expression of BCL-W compared to the control in both A498 and 786-O cells (Fig. 2a). In contrast, overexpression of lncRNA RP11-436H11.5 increased BCL-W expression (Fig. 2b).
Furthermore, we investigated the effect of ABT-737, a small molecule inhibitor of BCL-W, on lncRNA RP11-436H11.5-mediated RCC cell invasion. The results showed that ABT-737 dramatically reversed lncRNA RP11-436H11.5-increased RCC cell invasion in A498 and OSRC-2 cells in a concentration-dependent way (Fig. 2c and d).
Together, the results from Fig. 2a-d showed that lncRNA RP11-436H11.5 upregulated BCL-W expression to promote RCC cell progression.
LncRNA RP11-436H11.5 regulates BCL-W expression via miR-335-5p
Since lncRNA RP11-436H11.5 was positively associated with RCC cell progression, we next investigated how lncRNA RP11-436H11.5 regulated BCL-W expression in RCC cell proliferation and invasion.
We added lncRNA RP11-436H11.5-shRNA in A498 and 786-O cells and detected BCL-W expression. The qRT-PCR results revealed that the mRNA level of BCL-W changed little compared to the control (Fig. 3a), which implied that lncRNA RP11-436H11.5 might not regulate BCL-W expression directly. Therefore, we considered that some non-coding RNAs might play important roles at a post-transcriptional level in this process. To certify our hypothesis, we performed an Argo2 immunoprecipitation assay and detected the mRNA level of BCL-W. The results revealed that the mRNA level of BCL-W increased after knocking down lncRNA RP11-436H11.5 in A498 and 786-O cells (Fig. 3b), which verified our viewpoints that some miRNAs could participate in a RNA-induced silencing complex responsible for repressing BCL-W mRNA degradation and translation.
Using a human miRNA target tool (DIANA TOOLS), we predicted that lncRNA RP11-436H11.5 might be targeted by miR-335-5p. The predicted binding sites of miR-335-5p to lncRNA RP11-436H11.5 were illustrated in Additional file 2: Figure S2. Therefore, we targeted miR-335-5p for further exploration. As shown on Fig. 3c, the expression of miR-335-5p increased after knocking down lncRNA RP11-436H11.5 in A498 cells. In contrast, overexpression of lncRNA RP11-436H11.5 decreased the expression of miR-335-5p in 786-O cells (Fig. 3d). Importantly, there was an inverse correlation (r = −0.5410, p = 0.0138) between lncRNA RP11-436H11.5 level and miR-335-5p level in 20 RCC tissues (Fig. 3e), showing that a lower lncRNA RP11-436H11.5 level was associated with a higher miR-335-5p level (Fig. 3f).
Our previous study demonstrated that miR-335-5p could regulate BCL-W expression by binding to the 3′-untranslated region (3′-UTR) of BCL-W mRNA using a luciferase reporter assay [19].
Together, the results from Fig. 3a-f and Additional file 2: Figure S2 demonstrated that lncRNA RP11-436H11.5 could regulate BCL-W expression via miR-335-5p by binding to the 3′-UTR of BCL-W mRNA.
LncRNA RP11-436H11.5 functions by altering miR-335-5p-BCL-W signals to promote RCC cell proliferation and invasion
Our previous study demonstrated that miR-335-5p inhibited RCC cell progression through the direct suppression of BCL-W. Therefore, we examined whether lncRNA RP11-436H11.5 could affect RCC cell proliferation and invasion by altering miR-335-5p-BCL-W signals. Western blot assay revealed that knocking down lncRNA RP11-436H11.5 decreased the expression of BCL-W in both A498 and 786-O cells, and this shlncRNA RP11-436H11.5-suppressed BCL-W expression was partially reversed by adding a miR-335-5p inhibitor (Fig. 4a). The results also showed that overexpression of miR-335-5p partially reversed oelncRNA RP11-436H11.5-enhanced RCC cell proliferation in A498 cells (Fig. 4b). On the other hand, miR-335-5p inhibitor partially reversed RCC cell proliferation induced by shlncRNA RP11-436H11.5 in 786-O cells (Fig. 4c).
As expected, we obtained similar results in both A498 (Fig. 4d) and OSRC-2 cells (Fig. 4e) using a transwell invasion assay.
Together, the results from Fig. 4a-e illustrated that lncRNA RP11-436H11.5 could promote RCC cell proliferation and invasion by altering miR-335-5p-BCL-W signals.
LncRNA RP11-436H11.5 works as a miR-335-5p decoy to regulate BCL-W expression in RCC cells
Over the years, ceRNA appeared to be a vital mechanism for lncRNA and miRNA interaction. To further elucidate whether lncRNA RP11-436H11.5 can function as a ceRNA, we detected the specific binding sites between miR-335-5p and lncRNA RP11-436H11.5. We cloned full-length lncRNA RP11-436H11.5 containing presumptive miR-335-5p binding sites and conducted a luciferase reporter assay (Fig. 5a). The results showed that oemiR-335-5p inhibited the luciferase activities of wild-type binding sites, but failed to do so with mutation of the miR-335-5p binding seed region in lncRNA RP11-436H11.5 in A498 and 786-O cells (Fig. 5b).
Subsequently, a RNA-pull down assay was applied to confirm that miR-335-5p could interact with lncRNA RP11-436H11.5 and BCL-W. Functional lncRNA RP11-436H11.5-cDNA and lncRNA RP11-436H11.5-shRNA were added in 786-O and A498 cells, respectively. Specific biotinylated antisense oligoes were used to isolate lncRNA RP11-436H11.5 and BCL-W from the above RCC cells. The qRT-PCR results revealed that lncRNA RP11-436H11.5 absorbed more miR-335-5p, whereas BCL-W absorbed less miR-335-5p after overexpression of lncRNA RP11-436H11.5 in A498 cells (Fig. 5c). Similar results were obtained when we knocked down lncRNA RP11-436H11.5 in 786-O cells (Fig. 5c).
Finally, luciferase reporter assay was applied through psiCheck2 vector holding wild-type or mutant 3′-UTR of BCL-W. The results showed that luciferase activities decreased after adding oemiR-335-5p in A498 cells (Fig. 5d, pLVTHM + oemiR-335-5p vs pLVTHM + pWPI). The restraint effect of oemiR-335-5p on BCL-W was reversed with the addition of functional lncRNA RP11-436H11.5-cDNA (Fig. 5d, oeRP11-436H11.5 + oemiR-335-5p vs pLVTHM + oemiR-335-5p). No prominent differences were found for mutant 3′-UTR of BCL-W (Fig. 5d). On the other hand, the luciferase activities increased after adding miR-335-5p inhibitor in 786-O cells (Fig. 5e, pLVTHM + inh-miR-335-5p vs pLVTHM + pWPI). The enhanced effect of miR-335-5p inhibitor on BCL-W was reversed with the addition of lncRNA RP11-436H11.5-shRNA (Fig. 5e, shlncRNA RP11-436H11.5 + inh-miR-335-5p vs pLVTHM + inh-miR-335-5p). No prominent differences were found for mutant 3′-UTR of BCL-W (Fig. 5e).
Together, the results from Fig. 5a-e illustrated that lncRNA RP11-436H11.5 could function as a miRNA decoy to defend BCL-W from degradation by miR-335-5p in RCC cells.
Deregulation of lncRNA RP11-436H11.5 suppresses RCC cell proliferation and invasion in RCC orthotopic xenografts
The above in vitro data indicated that lncRNA RP11-436H11.5 promoted RCC cell proliferation and invasion. Furthermore, orthotopic xenograft mouse models were applied to detect the anti-tumorigenic role of shlncRNA RP11-436H11.5.
A498 cells were labeled with luciferase expression and transfected with lncRNA RP11-436H11.5-shRNA and functional lncRNA RP11-436H11.5-cDNA. Then, the transfected cells were inoculated into the left renal capsule of nude mice. Tumor size and metastasis were monitored through an In Vivo Imaging System (IVIS). After 6 weeks, IVIS results revealed that a reduction of tumor luciferase expression was found in the shlncRNA RP11-436H11.5 group compared to the shRNA-control group (Fig. 6a). Meanwhile, the results also showed that deregulation of lncRNA RP11-436H11.5 effectively suppressed tumor size and metastasis (Fig. 6b and c). Conversely, oelncRNA RP11-436H11.5 increased tumor size and metastasis compared to the control group (Fig. 6d–f). Furthermore, oelncRNA RP11-436H11.5 promoted lung, liver, spleen and diaphragm metastasis (Fig. 6g–j).
Together, the results from Fig. 6a-j demonstrated that shlncRNA RP11-436H11.5 functioned as a tumor suppressor by inhibiting tumorigenesis and metastasis in RCC cells.