LncRNA-LET inhibits cell growth of clear cell renal cell carcinoma by regulating miR-373-3p

Background Clear cell renal cell carcinoma (ccRCC) is the most common renal cell carcinoma subtype with a poor prognosis. LncRNA-LET is a long non-coding RNA (lncRNA) that is down-regulated in ccRCC tissues. However, its role in ccRCC development and progress is unclear. Methods LncRNA-LET expression was detected in ccRCC tissues and ccRCC cells using quantitative real-time PCR. The overexpression and knockdown experiments were performed in ccRCC cells and xenograft mouse model to evaluate role of lncRNA-LET. Cell cycle, apoptosis and JC-1 assays were conducted via flow cytometer. The protein levels were measured through western blot analysis and the interaction between lncRNA-LET and miR-373-3p was identified via luciferase reporter assay. Results LncRNA-LET expression was lower in ccRCC tissues than that in the matched adjacent non-tumor tissues (n = 16). In vitro, lncRNA-LET overexpression induced cell cycle arrest, promoted apoptosis and impaired mitochondrial membrane potential, whereas its knockdown exerted opposite effects. Moreover, we noted that lncRNA-LET may act as a target for oncomiR miR-373-3p. In contrast to lncRNA-LET, miR-373-3p expression was higher in ccRCC tissues. The binding between lncRNA-LET and miR-373-3p was validated. Two downstream targets of miR-373-3p, Dickkopf-1 (DKK1) and tissue inhibitor of metalloproteinase-2 (TIMP2), were positively regulated by lncRNA-LET in ccRCC cells. MiR-373-3p mimics reduced lncRNA-LET-induced up-regulation of DKK1 and TIMP2 levels, and attenuated lncRNA-LET-mediated anti-tumor effects in ccRCC cells. In vivo, lncRNA-LET suppressed the growth of ccRCC xenograft tumors. Conclusion These findings indicate that lncRNA-LET plays a tumor suppressive role in ccRCC by regulating miR-373-3p.


Background
Renal cell carcinoma (RCC), as one of most frequent cancers worldwide, is a common lethal malignancy [1,2]. RCC incidence and death rates are high, with 63,000 new cases and 14,000 deaths per year in United States [3]. Surgical resection and immunotherapy are currently being applied to treat patients with RCC [4][5][6]. RCC includes more than 10 histological and molecular subtypes [7], with clear cell RCC (ccRCC) as the most common subtype [8]. The detailed molecular mechanisms underlying ccRCC development remain elusive.

Open Access
Cancer Cell International *Correspondence: neoneyz@126.com † Zhuo Ye and Jiachen Duan contributed equally to this study 1 Department of Urology, The First Affiliated Hospital of Zhengzhou University, 1 East Jianshe Road, Zhengzhou 450052, People's Republic of China Full list of author information is available at the end of the article role that lncRNA-LET plays is unknown. Interestingly, lncRNA-LET expression is low in serum samples from patients with ccRCC [17], suggesting its involvement in the carcinogenesis of this cancer.
Previous studies have shown that lncRNA-LET has the potential to target miRNAs, thereby regulating the expression of miRNA targets to affect the process of human cancers [18,19]. Although varied miRNAs may be regulated by lncRNA-LET, we here focused on miR-373-3p. Both tumor-promoting and anti-tumor effects of miR-373-3p have been reported before [20,21]. An earlier study has revealed that miR-373-3p acts as an oncomiR in RCC [22]. By analyzing the sequence information of lncRNA-LET and miR-373-3p, we noted that lncRNA-LET contained a potential binding area for miR-373-3p. This study is thus performed to explore whether lncRNA-LET regulates the malignant behaviors of ccRCC cells by regulating miR-373-3p.
Herein, we explored the specific role of lncRNA-LET in regulating ccRCC growth in vitro and in vivo. LncRNA-LET overexpression or knockdown was performed in ccRCC cells. Meanwhile, the xenograft mouse model was constructed. We demonstrated that lncRNA-LET suppressed the growth of ccRCC cells. LncRNA-LETinduced cell cycle arrest and apoptosis in ccRCC cells were attenuated by miR-373-3p mimics.

Patients and tissues
The human ccRCC and matched adjacent non-tumor tissues were obtained from 16 ccRCC patients from the First Affiliated Hospital of Zhengzhou University during September 2018-November 2018. Each patient provided an informed consent prior to specimen collection. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and conferred to Declaration of Helsinki.

Quantitative real-time PCR
Total RNAs were extracted using RL reagent (BioTeke, China). The mRNA was reversely transcribed into complementary DNA with M-MLV Reverse Transcriptase (Takara, China). The expression levels of lncRNA-LET and miR-373-3p were detected via SYBR Green (BioTeke, China). β-actin and U6 were applied as internal controls. The relative expression levels were calculated with the 2 − ΔΔCt method. The primers were shown in Table 2.

Cell apoptosis detection
Cells were collected and centrifuged at 1000g for 5 min. Then, the cells in 195 μl binding buffer were incubated with 5 μl AnnexinV-FITC and 10 μl PI for 15 min at room temperature in the dark according to the manufacturer's instruction (Beyotime, China). Cell apoptosis was analyzed by flow cytometer.

Caspase activity assay
The activities of caspase-3 and caspase-9 were analyzed with corresponding Caspase Assay Kits (Beyotime or Solarbio, China). Briefly, proteins were extracted from cells and then qualified with Bradford Protein Assay Kit (Beyotime, China). Subsequently, samples were incubated with the caspase substrate for 24 h at 37 °C. The absorbance was determined at 405 nm.

JC-1 assay
Cells were obtained and centrifuged at 550g for 5 min. Then, the cells were resuspended in 500 μl JC-1 staining working solution (Beyotime, China). After incubation for 20 min in the incubator at 37 °C, cells were centrifuged at 600g for 5 min and washed twice with 1× JC-1 staining buffer, and resuspended with 500 μl 1× JC-1 staining buffer. JC-1 aggregate was measured via the flow cytometer.

Hematoxylin-eosin (HE) staining
The tumor tissues were fixed with 4% paraformaldehyde, embedded with paraffin and then cut into 5-µm sections. Afterwards, the sections were deparaffinized and rehydrated before being stained with hematoxylin (Solarbio, China) and eosin (Sangon, China). The staining was visualized under a microscope.

TUNEL staining
The tumor tissues were fixed with 4% paraformaldehyde and 5-µm sections were embedded in paraffin, followed by deparaffinization and rehydration. The TUNEL-positive cells were labeled by Label Solution with Enzyme solution for 60 min at 37 °C in the dark, and then these sections were incubated with converter-peroxidase (POD) according to the manufacturer's protocol. Afterwards, hematoxylin (Solarbio, China) was used for the counterstaining of cell nuclei. The analysis of apoptotic cells was conducted and images were taken under a microscope.

Immunofluorescence analysis
Cells were fixed in 4% paraformaldehyde for 15 min and incubated with 0.1% Triton X-100 (Beyotime, China) for 30 min. Additionally, tumor tissues were fixed in 4% paraformaldehyde, embedded with paraffin and cut into 5-µm sections. Then, the sections were incubated with goat serum to block nonspecific binding. The sections were subsequently incubated with anti-Ki67 antibody

Luciferase reporter assay
293 T cells were seeded onto 12-well plates. The partial lncRNA-LET sequences containing wild-type (WT) and mutant (MUT) binding sites for miR-373-3p were synthesized and subcloned into pmirGLO luciferase reporter vectors. The 293 T cells were transfected with the luciferase reporter constructs together with miR-373-3p mimics or mimics NC with Lipofectamine 2000. After a 48-h incubation, the transfected cells were collected and the luciferase activity analysis was conducted.

In vivo xenograft mouse model
Male 5-week-old BALB/c nude mice were obtained from BEIJING HFK BIOSCIENCE Co., LTD (China).
All animal experiments were conducted according to the Guideline for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Mice acclimated for 1 week and then randomly assigned to four groups (n = 6/group): EV group, lncRNA-LET group, shRNA Ctrl group, lncRNA-LET shRNA group. 5 × 10 6 cells stably expressing EV, lncRNA-LET, shRNA Ctrl or lncRNA-LET shRNA vectors were subcutaneously injected in the right fore-flank of each nude mouse. Then, the size of the tumor was recorded every 3 days for 21 days. Finally, the mice were sacrificed and tumor tissues were photographed. The tumor volume was calculated with the equation volume (mm 3 ) = length × width 2 /2.

Statistical analysis
All date were analyzed with GraphPad Prism version 7.0 and presented as mean ± SD. The two-tailed paired and unpaired Student's t-test was used to test for significant differences between two groups. One-way ANOVA analysis followed by Tukey's test was used to analyze the multi-sample analysis. p value less than 0.05 was considered statistically significant.

LncRNA-LET expression is down-regulated in ccRCC tissues
To examine the clinical significances of lncRNA-LET in ccRCC tissues, we conducted quantitative real-time PCR. The lncRNA-LET expression level was significantly decreased in ccRCC tissues compared with matched adjacent non-tumor tissues (Fig. 1a). On the contrary, miR-373-3p expression was higher in ccRCC tissues (Fig. 1b).

LncRNA-LET arrests cell cycle at G1 stage in ccRCC cells
The basal expression levels of lncRNA-LET in three ccRCC cancer cell lines were first determined via quantitative real-time PCR. The lowest and highest lncRNA-LET expression were observed in 786-O cells and 769-P cells, respectively (Fig. 2a). Next, lncRNA-LET overexpression plasmid was transfected into 786-O cells, while two lncRNA-LET siRNAs were transfected into 769-P cells. The overexpression and knockdown efficiencies were confirmed via quantitative real-time PCR in these two cell lines (Fig. 2b). EdU incorporation assays demonstrated that lncRNA-LET inhibited ccRCC cell  (Fig. 2c). Cell cycle progression detection revealed that lncRNA-LET overexpression caused a dramatic accumulation in G1-phase and reduction in S-phase of 786-O cells, whereas lncRNA-LET silencing accelerated cell cycle of 769-P cells to S-phase (Fig. 3a). Moreover, lncRNA-LET overexpression down-regulated  (Fig. 3b). These findings suggest that lncRNA-LET suppresses the proliferation and arrests cell cycle progress of ccRCC cells.

LncRNA-LET promotes cell apoptosis in ccRCC cells
Results from flow cytometry indicated that lncRNA-LET overexpression significantly promoted cell apoptosis while lncRNA-LET silencing suppressed cell apoptosis (Fig. 4a). Meanwhile, lncRNA-LET increased caspase-3 and caspase-9 activities (Fig. 4b), up-regulated Bax expression and reduced Bcl-2 expression (Fig. 4c) in 786-O cells. Data from JC-1 assay illustrated that lncRNA-LET increased the ratio of green/monomeric forms of JC-1 in ccRCC cells (Fig. 5a). Further western blot analysis confirmed that lncRNA-LET led to the release of Cytochrome C from mitochondria (Fig. 5b). Immunofluorescence also showed that lncRNA-LET overexpression significantly facilitated cytosolic translocation of Cytochrome C and lncRNA-LET silencing Fig. 4 LncRNA-LET promotes cell apoptosis in ccRCC cells. a Cell apoptosis was studied by flow cytometry. b The caspase-3 and caspase-9 activities were detected through corresponding caspase assay kits. c Western blot analysis was used to determine the Bax and Bcl-2 protein levels. # p < 0.05, ## p < 0.01, ### p < 0.001. ccRCC clear cell renal cell carcinoma inhibited its translocation in ccRCC cells (Fig. 6a, b). The data reveal that lncRNA-LET promotes cell apoptosis in ccRCC cells possibly.

LncRNA-LET inhibits tumor growth in vivo
In order to evaluate the function of lncRNA-LET in tumor growth in vivo, we stably overexpressed lncRNA-LET in 786-O cells or knocked lncRNA-LET down in 769-P cells. These stably transfected cells were subcutaneously injected into nude mice. LncRNA-LET led to reduction of tumor volume (Fig. 9a, b). Further HE staining, TUNEL staining and Ki67 immunostaining results showed that lncRNA-LET increased apoptosis, and suppressed cell proliferation (Fig. 9c-e). These results indicate that lncRNA-LET functions similarly in vivo and in vitro.

Discussion
In this study, low lncRNA-LET was found in ccRCC tissues compared with matched adjacent non-tumor tissues.
LncRNA-LET induced cell cycle arrest and apoptosis of ccRCC in vitro and inhibited the growth of xenografts in vivo. Further study showed lncRNA-LET performed its role in ccRCC by targeting miR-373-3p.
LncRNAs are reported to regulate various biological processes. They can contribute to the tumor progression or act as tumor-suppressors in ccRCC [12,[23][24][25]. It was reported that the low lncRNA-LET level was correlated to the poor prognosis of patients with lung cancer or gastric cancer [26,27]. Wu et al. [17] showed a low lncRNA-LET level in the serum of ccRCC patients and regarded it as highly indicative of ccRCC diagnosis. In this study, we confirmed a low lncRNA-LET level in ccRCC tissues, which was consistent with the report of Wu et al. However, the role of lncRNA-LET in the growth of ccRCC is still unclear and needs further exploration. In our study, lncRNA-LET inhibited the growth of ccRCC, both in vitro and in vivo. Cell cycle contributes to the growth of cells, and cyclins are key drivers of cell cycle. The growth-inhibitory effect of lncRNA-LET was accompanied with cell cycle arrest, which was further confirmed by declines in cyclins. Besides, lncRNA-LET also induced apoptosis in ccRCC cells, which was further confirmed by increased activities of caspase-3 and caspase-9. Interestingly, we found that lncRNA-LET increased the level of Bax and decreased the level of Bcl-2. As the ratio of Bax/Bcl-2 contributes to the opening of mitochondrial permeability transition pore, it indicated that the induction of apoptosis by lncRNA-LET may be associated with mitochondria-mediated apoptosis. Therefore, the mitochondrial membrane potential and release of cytochrome C were detected. Our results demonstrated that lncRNA-LET modulated mitochondrial membrane potential and enhanced the release of cytochrome C, indicating that apoptosis of ccRCC induced by lncRNA-LET may belong to mitochondriamediated apoptosis. Additionally, we found that lncRNA-LET increased the levels of DKK1 and TIMP2, which are involved in the regulation of cell migration [28][29][30], suggesting that lncRNA-LET may also have an effect on the metastasis of RCC. More researches are needed to verify this speculation.
Furthermore, how lncRNA-LET performed its tumorsuppressor role in ccRCC was explored. Li et al. revealed that miR-373-3p promoted tumorigenesis of RCC in vitro and in vivo [22]. In our study, we identified lncRNA-LET as a direct target of miR-373-3p. Interestingly, we found that the levels of DKK1 and TIMP2, which are two verified targets of miR-373-3p, were increased by lncRNA-LET. These results prompt us that lncRNA-LET may also modulate the expression of miR-373-3p target genes. Thus, rescue experiments were performed in our study. The results showed that miR-373-3p down-regulated the lncRNA-LET-induced increase of DKK1 and TIMP2 levels, and reversed the effects of lncRNA-LET on cell cycle and apoptosis. These results indicate that lncRNA-LET may perform its tumor-suppressor role in ccRCC cells through regulating the expression of miR-373-3p target genes. In 2011, Salmena et al. proposed a concept of competing endogenous RNA (ceRNA) [31] that targets of microRNA, such as mRNAs, lncRNAs and pseudogenes, can inversely target microRNAs using their micro-RNA response elements, thus modulating the expression of target genes and resulting in their various roles. We hypothesize that lncRNA-LET may act as a tumorsuppressor in ccRCC through a ceRNA pattern. As the ceRNA network is a large-scale regulatory network, there must be other microRNAs which lncRNA-LET may target to perform its role in ccRCC. However, our study focused on only miR-373-3p, there remains a large scale of microRNAs which can be targeted by lncRNA-LET. Hence, further explorations are needed to reveal the mechanism underlying lncRNA-LET.

Conclusions
In the present study, lncRNA-LET repressed cell cycle, induced apoptosis and inhibited tumor growth of ccRCC by targeting miR-373-3p. We identified lncRNA-LET as a tumor-suppressor in ccRCC. The results of the present study provide a potential biomarker and therapeutic target for ccRCC treatment.

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