LncRNA PVT1 is increased in renal cell carcinoma and affects viability and migration in vitro

Abstract Background Renal cell carcinoma is difficult to diagnose and unpredictable in disease course and severity. There are no specific biomarkers for diagnosis and prognosis estimation feasible in clinical practice. Long non‐coding RNAs (lncRNAs) have emerged as potent regulators of gene expression in recent years. Aside from their cellular role, their expression patterns could be used as a biomarker of ongoing pathology. Methods In this work, we used next‐generation sequencing for global lncRNA expression profiling in tumor and non‐tumor tissue of RCC patients. The four candidate lncRNAs have been further validated on an independent cohort. PVT1, as the most promising lncRNA, has also been studied using functional in vitro tests. Results Next‐generation sequencing showed significant dysregulation of 1163 lncRNAs; among them top 20 dysregulated lncRNAs were AC061975.7, AC124017.1, AP000696.1, AC148477.4, LINC02437, GATA3‐AS, LINC01762, LINC01230, LINC01271, LINC01187, LINC00472, AC007849.1, LINC00982, LINC01543, AL031710.1, and AC019197.1 as down‐regulated lncRNAs; and SLC16A1‐AS1, PVT1, LINC0887, and LUCAT1 as up‐regulated lncRNAs. We observed statistically significant dysregulation of PVT1, LUCAT1, and LINC00982. Moreover, we studied the effect of artificial PVT1 decrease in renal cell line 786–0 and observed an effect on cell viability and migration. Conclusion Our results show not only the diagnostic but also the therapeutic potential of PVT1 in renal cell carcinoma.

specificity, either at an early or advanced stage or which would help predict the development of the disease without or with therapy. 2 In recent decades, long non-coding RNAs (lncRNAs) emerged as potential biomarkers of cancer and other diseases. Specifically in cancer, the role of lncRNAs is currently being addressed by many research groups. LncRNAs act as oncogenes and tumor suppressors and are involved in various signaling pathways. 3 Deregulation of ln-cRNA expression was also detected in RCC and correlated with clinicopathological data, including tumor stage, degree of differentiation, or the presence of metastases. [4][5][6] Many lncRNAs are considered not only diagnostic markers, but their expression is also essential in determining prognosis or monitoring response to treatment. 4 The present study aimed to investigate the lncRNA expression profile using a next-generation sequencing approach with subsequent validation of the results. Moreover, we provided some functional in vitro tests of PVT1 as one of the most significantly dysregulated lncRNAs in our patient cohort.  Table 1.

| RNA extraction and quality measurement
Total RNA enriched for small RNAs was extracted using mirVana™ miRNA Isolation kit (Invitrogen) according to the manufacturer's protocol from all collected samples. RNA concentration was measured using Qubit™ 2.0 (Invitrogen, Thermo Fisher Scientific) fluorometer. According to the manufacturer protocol, RNA integrity has been measured in all samples using Agilent 2200 TapeStation system and RNA ScreenTape (Agilent). For the sequencing analysis, 22 samples with the best RNA integrity (RIN >6, average 7.8) from 16 RCC patients (16 tumor tissue and 6 non-tumor tissue samples) were selected due to the high demands of RNA library preparation for the quality of the material.

| Library preparation and RNA sequencing
According to the results from TapeStation, some samples had a significant amount of genomic DNA, which would be problematic during the transcriptomic library preparation. Thus, the genomic DNA had to be removed using DNA-free™ DNA Removal kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's protocol. RNA concentration was again measured using Qubit™ 2.0 (Invitrogen, Thermo Fisher Scientific) fluorometer. After that, RNA was diluted in 26 µl of nuclease-free water (Qiagen) in the required range 1-1000 ng of total RNA. As the entry amount concentration, 500 ng of RNA was chosen in our case. Using RiboCop rRNA Depletion Kit V1.2 (Lexogen), according to the manufacturer's protocol, ribosomal depletion has been carried out to eliminate ribosomal RNA, which represents the majority of the RNA content and thus would compromise the sequencing capacity after the library preparation. After the ribosomal depletion, the RNA concentration was measured again using Qubit™ 2.0 (Invitrogen, Thermo Fisher Scientific) fluorometer.

| Data analysis
Raw data from the Illumina NextSeq 500 were converted to fastq using bcl2fastq2 Con-version software (version 2.20.0), and read quality was checked using FastQC (version 0.11.7). 7 Adapter sequences were identified using the Kraken system (version 15-065), 8 and poor read ends were removed using Cutadapt (version 1.18). 9 The 3′ ends with a threshold value less than five and reads shorter

| Validation and statistical analysis
Validation of the results from the exploratory phase has been carried out using a High-Capacity cDNA Reverse Transcription

| Cell culture and transfection
The

| Cell proliferation assay
Cell proliferation was determined using the cell counting method.
The 786-0 cells were seeded in a 24-well plate at the density of

| Scratch wound migration assay
The 786-0 cells were plated in a 24-well plate at the density of 25

| Expression profiling in the tissue of RCC patients
Using next-generation sequencing and the DESeq2 tool, we ana-  Table 2. and LUCAT1 (p = 0.0015 [ Figure 2B]), which were down-regulated, and in the expression of LINC00982 (p < 0.0001, Figure 2C). No significant difference in expression has been observed in SLC16A1-AS1 (p = 0.1307, Figure 2D).
In four validated lncRNAs, we also did ROC analysis ( Figure 3A-D).
All lncRNAs except SLC16A1-AS1 showed an ability to distinguish tumor and non-tumor tissue with high sensitivity and specificity and AUC higher than 0.75.
We compared the expression of our four candidate lncRNAs in patients' stage I and in patients with stages II and IV and patients with lower grades (1 and 2) and with higher grades (3 and 4) and found no correlation either for stage or grade (data not shown).

| Functional characterization of PVT1 in vitro
Using qPCR, we measured the expression of PVT1 in three renal Results are shown in Figure 5.
Migration ability has been assessed using the scratch-wound assay. We observed the regrowth of the cells in the wound created by the pipette tip in T0 and T + 12 h ( Figure 8A). Cells transfected with siRNA migrated less than cells transfected with control oligo.
Statistical significance has been tested using the Mann-Whitney U test (p = 0.0332, Figure 8B).

| DISCUSS ION
In the present study, our primary goal was to determine the expression profiles of lncRNAs in RCC using next-generation sequencing Our results were similar in the case of LUCAT1. Increased expression of this lncRNA has been observed in tumor tissue compared to non-tumor tissue, not only in RCC [26][27][28][29] but also in other tumors such as non-small cell carcinoma, lung, 30 glioma, 31 clear cell esophageal carcinoma, 32 or hepatocellular carcinoma. 33 Following the previous works, we observed significantly increased expression of LUCAT1.
No correlation with clinicopathological characteristics has been found. This result contrasts previous results, 26 where a relationship was observed with both the stage and the degree of differentiation, or only at the stage and other characteristics. 27,28 The explanation is likely to be similar to PVT1.
The role of LINC00982 was first mentioned in a study by Fei et al., 34 where the chip identified reduced levels of this lncRNA in gastric cancer. Its relationship with clinicopathological characteristics and influence on the prognosis of the disease was also described. LINC00982 also has reduced expression levels in lung adenocarcinoma compared to non-tumor tissue 35 and also in RCC, 36   obtaining scientific data presented in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.