Introduction
Renal cell carcinoma (RCC) accounts for approximately 3% of all adult cancers, and poor survival is manifested in RCC patients, especially for those with metastasis [
1]. RCC exerts socio-economic burden on society and the families of patients. However, the pathogenesis of RCC remains unclarified.
Various literatures reported that cancers are closely related to the abnormal expression of LNCRNA, miRNA and proteins etc., and even co-functions each other. With the rapid development of sequencing technologies, a series of dysregulated long non-coding RNAs (lncRNAs) have been found in many human diseases, especially in cancers [
2‐
8]. Accumulating evidences suggested that long non-coding RNAs (lncRNAs) perform important or vital functions in the malignant tumors [
9‐
15]. LncRNA urothelial cancer associated 1 (UCA1) is abnormally expressed in esophageal squamous cell carcinoma, colorectal cancer, gastric cancer melanoma cells, pancreatic cancer, thyroid cancer, lung cancer and so on [
16‐
30]. In vitro and in vivo assays were conducted to further explore its underlying roles in tumor progression. In this study, we illustrated that UCA1 promoted tumor cell proliferation or migration and suppressed apoptosis of renal cancer and the mechanism of action.
MicroRNAs (miRNAs) are non-coding RNAs with 18–25 nucleotides, which participate in various processes of tumorigenesis. The expression of miR-182-5p is not consistent across tumors. MiR-182-5p is down-regulated in ovarian cancer, colorectal cancer and renal cancer etc. However, miR-182-5p is up-regulated in Oral squamous cell carcinoma, breast cancer and hepatocellular carcinoma etc. [
31‐
36]. However, the underlying mechanisms of miR-182-5p in the malignant behaviors of RCC are unclarified. In this study, we demonstrated that miR-182-5p as negative regulatory factor were involved in the progression of renal cancer as well.
Accumulated evidences reported lncRNAs function as miRNAs sponges. This is firstly reported the relation between UCA1 and miR-182-5p as miRNAs sponges in the renal cancers in the world. This study helps broaden our knowledge of the expression pattern.
Additionally, proteins are the necessary substance to participate in lives’ activities and disease progression as well, which were closely related with lncRNA and miRNA etc. Delta-like ligand 4 (DLL4), one of the ligands of Notch receptors, is predominantly expressed in the endothelial cells and has been shown to play a pivotal role in regulating tumor angiogenesis. During angiogenesis, activation of the Notch/DLL4 pathway selects the stalk cell, and promotes endothelial basement membrane formation and cell adhesion. Some studies reveal a role for DLL4 in tumorigenesis in several cancers, including T acute lymphoblastic leukemia (T-ALL), and glioblastoma etc. [
37‐
47]. DLL4 is one of the ligands of Notch 1. Activation of DLL4-NOTCH signaling is important for the RCC to maintain its malignant properties. DLL4 overexpression significantly reversed cell proliferation inhibition and migration of renal cancer cells induced by silencing UCA1, and remarkably reversed cell apoptosis promotion of renal cancer cells induced by silencing UCA1. DLL4-NOTCH signaling is critical for the progression of renal cancer cells.
In the present study, we showed that UCA1 was involved in the progression of renal cancer. Mechanistically, we found that UCA1 functioned as a miRNA sponge to positively regulate Delta-like ligand 4(DLL4) expression by sponging miR-182-5p in a ceRNA-dependent manner. Together, our results suggest that UCA1 is a powerful tumor biomarker, and UCA1-miR-182-5p-DLL4 axis is involved in proliferation, migration, apoptosis and progression of renal cancer, which highlight its potential clinical utility as a promising diagnostic and therapeutic target of renal cancer. Our findings may provide a new horizon for exploring therapeutic target of renal cancer.
Materials and methods
Patient samples
Eighty eight renal cancer patients (Renal clear cell carcinoma) and 30 renal cancer patients (Papillary renal carcinoma) who received radical nephrectomy were included in this research. Renal cancer tissues and matched normal peritumoral tissues were snap-frozen in liquid nitrogen quickly after resection. Written informed consent was also obtained from all the patients. The study was approved by the institutional research ethics committee of The Second Hospital of Lanzhou University (Granted 2017A-021).
Cell lines and cell culture
Human renal cancer 786-O, Caki-1, human normal renal epithelial cells (293 T) and RPTEC (Renal Tubular Epithelial Cell) were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The 786-O cells were cultured in RPMI-1640 (1640) (HyClone, USA). The Caki-1 cell lines were cultured in McCoy’s 5A (HyClone, USA). The 293 T cells were cultured in DMEM (HyClone, USA). The RPTEC cells were cultured in F12 (HyClone, USA).1640, DMEM, F12 and McCoy’s 5A were mixed with 1% antibiotics (100 U/ml penicillin and 100 μg /ml streptomycin sulfates) and 10% fetal bovine serum (FBS). Then plates were placed in incubator at 37 °C with an atmosphere of 5% CO2.
Cell transfection
Short hairpin RNAs (shRNAs) against UCA1 gene or DLL4 gene were ligated into pGPU6/GFP/Neo vectors (shUCA1, shDLL4) (Gene Pharma, Shanghai, China) and plasmid with non-targeting sequence was used as a negative control (shNC). The microRNA mimics (agomir) and microRNA inhibitor (antagomir) were purchased from Gene Pharma, Shanghai, China. Before transfection, the cells were cultured 24 h. Then, the cells were transiently transfected with corresponding vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with corresponding vector were harvested for quantitative real-time PCR and so on. Experiments were repeated at least three times.
Real-time quantitative PCR
Total RNA was extracted from the tissues or the transfected cells using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The cDNA was synthesized from total RNA(1000 ng) using the Prime Script RT Reagent Kit with gDNA Eraser (Takara, Kyoto, Japan). The expression levels of UCA1 etc. (about 50 ng cDNA, see the relevant specifications for details) were measured by real-time quantitative PCR utilizing SYBR Premix Ex Taq II (Takara) on the CFX96 sequence detection system (Bio-Rad). The primer sequences were as follows in Additional file
1: Table S1. GAPDH and U6 small nuclear RNA were chosen as the internal control. Expressions were normalized to endogenous controls and calculated using relative quantification method (2
-ΔΔCt). Experiments were repeated at least three times.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6was chosen as the endogenous control to normalize the data.
LncRNAs immunoprecipitation
Biotin-labeled lncRNA-UCA1 probe was synthesized by RiboBio. Renal cancer cells were fixed by 1% formaldehyde for 10 min, lysed and sonicated. After centrifugation, 50 μl of the supernatant was retained as input and the remaining part was incubated with lncRNA-UCA1 specific probes-streptavidin dynabeads (M-280, Invitrogen) mixture over night at 30 °C. Next day, M-280 dynabeads-probes-lncRNAs mixture was washed and incubated with 200 μl lysis buffer and proteinase K to reverse the formaldehyde crosslinking. Finally, the mixture was added with TRIzol for RNA extraction and-qPCR detection. Renal cancer cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma).
Cell proliferation assays
Cell Counting Kit-8, CCK-8 (Beyotime Institute of Biotechnology, Shanghai, China) was used for cell proliferation according to the manufacturer’s instructions. For CCK-8 assay, cells were incubated in a 96-well plate for 24 h, and then transiently transfected with siRNAs or plasmids. The absorbance in each well was measured at 0, 24, 48 and 72 h after transfection by a microplate reader (Bio-Rad, Hercules, CA, USA). Experiments were repeated at least three times.
Ethynyl-2-deoxyuridine (EdU) incorporation assay
Cell proliferation was also determined by Ethynyl-2-deoxyuridine incorporation assay using an EdU Apollo DNA in vitro kit (RIBOBIO, Guangzhou, China) following the manufacturer’s instructions. Briefly, after transfected with corresponding vector cells were incubated with 100 μl of 50 μM EdU per well for 2 h at 37 °C, respectively. Finally, the cells were visualized under a fluorescence microscopy. Experiments were repeated at least three times.
Cell migration assay
Cell migration was detected by scratch assay according to the reported methods [
22]. Cells were seeded in 6-well plates and incubated in incubator to get 100% confluence before transfection. The cells were transfected with siRNAs or plasmids. A sterile 200 μl pipette tip was used to generate a clear line in the wells. Pictures were taken from each well quickly using a digital camera system. After one day, pictures were taken again. Migration distance was counted at the time of 0 h and 24 h. Experiments were repeated at least three times.
Flow cytometry assay
Renal cancer 786-O and Caki-1 cells were transiently transfected with siRNAs or plasmid vectors. 48 h after transfection, cells were harvested and resuspended in fixation fluid 5 μl Annexin V-FIFC and 100 μl propidium iodide were added to 500 μl cell suspension. Cell apoptosis was then determined by using flow cytometry (EPICS, XL-4, Beckman, CA, USA). In the graphs, the quadrant respectively stands for dead cells, living cells, early apoptotic cells and late apoptotic cells. Experiments were repeated at least three times.
Western blot analysis
The transfected cells were washed with PBS, and total proteins were extracted using RIPA buffer reagent (Thermo Fisher Scientific). The concentrationofthe lysate was detected by a BCA kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Twenty micrograms(ug) of proteins were separated utilizing 10% SDS-PAGE gels and then transferred into PVDF membranes. The lysates of tissues or cells with equal weight were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking in the 5% milk without fat, the membranes were incubated in the primary antibodies for 16 h in 4 °C and then incubated with secondary antibodies at room temperature for 2 h. Immunoblots were visualized using ECL chemiluminescent detection system and analyzed with Chem Imager 5500 V2.03 software. The integrated density values (IDVs) were calculated using Fluor Chem 2.0 software with β-actin as the internal standard. The following antibodies were used as followed as in Additional file
1: Table S2. Goat anti-rabbit antibody and Goat anti-mouse antibody (1:1000, Beyotime, Shanghai, China), Experiments were repeated at least three times.
Luciferase reporter assay
The predicted binding sites of miR-182-5p with UCA1–3’UTR and DLL4–3’UTR were obtained from RNAhybrid. The binding and mutant sequences were respectively cloned into pmirGLO Dual-luciferase vectors (Gene Pharma, Shanghai, China).786-O, Caki-1 and 293 T cells were cultured in 96-well plates in advance and co-transfected with the wild-type pmirGLO-UAC1 reporter plasmid or the mutated type and mimics-182-5p or NC with Lipofectamine 3000. Cells were also co-transfected with the DLL4–3’UTR reporter plasmid or the mutated type and the above listed mimics. Dual-Luciferase Reporter Assay System (Promega, Madison, WI USA) was used to analyze luciferase activity, which was recorded as the ratio of firefly luciferase activity to renilla luciferase activity. Experiments were repeated at least three times.
Animal experiments
Institutional Ethics Review Board approved this experimental procedure (Granted D2017–008). A total of 10 male immune-deficient BALB/c nude mice (5–6 wk. old) were purchased from Shanghai lingchang Laboratory Animals (Shanghai, China), and each group was 5 mice. The shUCA1 and shNC vectors were packed into lentivirus (LV-UCA1 and LV-NC) that were purchased from Gene Pharma (Gene Pharma, Shanghai, China). A total of 5 × 105–6 treated Caki-1 cells were suspended in 100 ml Matrigel (BD Biosciences) and injected subcutaneously into the dorsal flank regions of BALB/c nude mice. Tumor growth of mice was monitored every 5 days, and mice were sacrificed 6 weeks, after inoculation. Tumor volume was calculated using the formula, 0.5ab2, where “a” and “b” meant long diameter and short diameter, respectively. Finally, mice were executed, and the subcutaneous weight of each tumor was observed. All animal experiments were approved by the Committee of Animal Experimental Ethics. Experiments were repeated at least three times.
Immunofluorescence
Immunostaining was performed on the paraffin-embedded tumor tissues from nude mice. The avidin-biotin-peroxidase method was adopted to determine the location and relative expression level of the target proteins. The primary antibodies of DLL4, was used at a dilution of 1:2000. Sections were visualized under a fluorescence microscopy (Olympus, Japan). Experiments were repeated at least three times.
Statistical analysis
All experimental assays were performed in triplicate. All data were presented as mean ± standard deviation (SD) of triplicate biological replicates or samples. All statistical analyses were executed by using SPSS 20.0 software (IBM, Chicago, IL, USA) etc. The UCA1 RNA and miR-182-5p expression differences between renal cancer tissues and matched normal tissues were analyzed using paired samples t-test. CCK-8 assay data were analyzed by ANOVA. The independent samples t-test was used to analyze other data. P value of less than 0.05 was considered to be statistically significant.
Discussion
The lncRNAs are important new members of non-coding RNA family, which are longer than 200 nucleotides. Accumulating evidences suggest that long non-coding RNAs (lncRNAs) perform important functions in regulation of genes which control tumor proliferation, apoptosis, and migration etc., and stretched our understanding of the biological behavior in diseases especially in carcinomas including renal cancer [
2‐
10]. Furthermore, these previous studies indicate that lncRNAs are valuable biomarkers and therapeutic targets [
11‐
15,
50]. The Long non-coding RNA UCA1 was located in chromosome 9p13.12, and has been found to be overexpressed in tumor tissues, such as esophageal squamous cell carcinoma,colorectal cancer, ovarian cancer, bile duct carcinoma and melanoma etc. [
16‐
23].UCA1 are participated in the tumorigenesis and progression, functioning as an oncogene [
24‐
30]. However, the relation between UCA1 and renal cancer is still unknown and mysterious particularly.
This is the first report to illustrate the function of UCA1 and miR-182-5p in renal cancer. In this research, we evaluated the expression patterns and clinical significances of UCA1 in renal cancer, and furthermore explored its possible function in renal cancer cells. Our study validated that UCA1 and miR-182-5p can be considered as a promising biomarker for the diagnosis of renal cancer. The expression of UCA1 was remarkably higher in renal cancer tissues and cell lines, and its up-regulation was positively correlated with differentiation and TNM stage in renal cancer. Furthermore, we explored the function of UCA1 in renal cancer cells and found that inhibition of UCA1 expression through UCA1 shRNA could suppress tumor cell proliferation, inhibited cell migration and induced apoptosis etc. To get a better insight to the role of UCA1, we over-expressed UCA1 in 293 T and PRTEC cell lines and it is found that overexpression of UCA1 could promote cell proliferation, migration and anti-apoptosis. In vivo studies demonstrated that UCA1 knockdown induced the smallest tumor volume and weight and so on in nude mice.
Accumulated evidences indicate the reciprocal inhibition between lncRNAs and miRNAs [
16,
20,
22,
24,
25,
28‐
30], and our study is also a good example on the relation furthermore. LncRNAs function as miRNAs sponges or decoys that titrate the concentration of miRNAs and therefore prevent miRNAs binding specific mRNAs. The role of UCA1 as a miRNA sponge in neoplasms has been reported in several studies. UCA1 promotes the progression of melanoma by sponging miR-28-5p [
22]. In Prostate cancer, UCA1 works as an oncogene by targeting miR-204 [
28]. The above studies are consistent with our research. As lncRNAs could function as sponges, they also might be saturated with miRNAs. Therefore, distinct expressions of lncRNAs and miRNAs in different tissues may induce different binding actions. Bioinformatics prediction was performed in order to investigate the underlying mechanism of UCA1 in renal cancer in our study, and miR-182-5p were predicted as potential targets. The prediction was confirmed by luciferase assay, and meanwhile miR-182-5p was found to be up-regulated in UCA1 knockdown cell lines. Furthermore, miR-182-5p were found to be down-regulated in renal cancer specimens and cell lines. And its down-regulation was positively correlated with differentiation and TNM stage in renal cancer. Over-expression of miR-182-5p respectively inhibited the malignant behaviors of renal cancer cells, and inhibited cell proliferation, migration and promoted apoptotic while the opposite effects were found in renal cancer cells with down-regulated miR-182-5p. MiR-182-5p plays tumor-suppression and anti-angiogenesis roles through altering the microenvironment and regulating DLL4 expression. Furthermore, we found that UCA1 knockdown combined with miR-182-5p over-expression significantly suppressed the malignant behaviors of renal cancer cells. Down-regulation of miR-182-5p could partially rescue the inhibition induced by UCA1 knockdown. This suggested that the suppression of malignancy caused by UCA1 knockdown might result from up-regulating of miR-182-5p.
As we known, miRNAs regulate lives’ activities by means of the protein indirect role and regulate target genes by binding to the 3’UTR of specific mRNAs and cause degradation or transcriptional inhibition of target genes [
31‐
36]. Biological softwares were used to predict the possible target genes of miR-182-5p to investigate the mechanisms of these miRNAs in renal cancer. DLL4 was a shared target gene of miR-182-5p, which was identified by luciferase reporter assay. Overexpression of miR-182-5p suppressed DLL4 protein expression, while down-regulation of miR-182-5p enhanced DLL4 expression. Thus, the expression of DLL4 was negatively correlated with the expression of miR-182-5p. Moreover, DLL4 had been proved to be involved in the malignant behaviors of renal cancer in previous studies [
37].
Delta-like ligand 4 (DLL4), one of the ligands of Notch receptors, is predominantly expressed in the endothelial cells and has been shown to play a pivotal role in regulating tumor angiogenesis [
38‐
45]. Some studies reveal a role for DLL4 in tumorigenesis in several cancers, including T acute lymphoblastic leukemia (T-ALL) [
46], and glioblastoma [
47] etc. We found that overexpressing of DLL4 reverses malignant phenotypes inhibition of renal cancer cells induced by silencing UCA1. DLL4 overexpression significantly reversed cell proliferation inhibition and migration of renal cancer cells induced by silencing UCA1, and significantly reversed cell apoptosis promotion of renal cancer cells induced by silencing UCA1. DLL4-NOTCH signaling is critical for the progression of renal cancer cells.
In brief, further experiments demonstrated that knockdown of UCA1 increased miR-182-5p expression and subsequently inhibited the expression of DLL4 in a ceRNA-dependent manner. Moreover, knock-down of miR-182-5p reversed DLL4 expression and DLL4 overexpression reversed the malignant phenotype inhibition of renal cancer cells induced by silencingUCA1.This study is giving us a novel point of view for specific molecular targets in human cancers, especially renal cancers, and it deepened our understanding of the relationship between lncRNA,miRNA and proteins in disease progression, and provides us a direction to further discover the diseases’ occurrence mechanism in the future, and to overcome and conquer the fatal disease etc. Maybe, it can be converted into the more freshly and validly curative mean for the renal carcinomas. It may be a bright development prospect to radically cure renal cancer that could alter the current dilemma of treatment for the advanced renal cancer.
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