lncRNA PVT1 and its splicing variant function as competing endogenous RNA to regulate clear cell renal cell carcinoma progression

Long non-coding RNAs (lncRNAs) exert critical regulatory roles in the development and progression of several cancers. Plasmacytoma variant translocation 1 (PVT1), an lncRNA, was shown to be upregulated in clear cell renal cell carcinoma (ccRCC) in our study, while Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival (OS) and disease free survival (DFS) in ccRCC patients. In vitro experiments revealed that PVT1 promoted renal cancer cell proliferation, migration, and invasion, while in vivo studies confirmed its oncogenic roles in ccRCC. Further bioinformatic analysis and RNA immunoprecipitation revealed that PVT1 could function as an oncogenic transcript partly through sponging miR-200s to regulate BMI1, ZEB1 and ZEB2 expression. Besides, a novel splicing variant of PVT1 lacking exon 4 (PVT1ΔE4) was found to have a higher expression in ccRCC and could also promote cell proliferation and invasion as the full-length transcript did. Besides, SRSF1 decreased the inclusion of exon 4 of full-length transcript and increased the relative expression of PVT1ΔE4 in ccRCC. Mechanistic investigations indicated that PVT1ΔE4 could also upregulate the expression of BMI1, ZEB1 and ZEB2 through interacting with miR-200s. Our study helps reveal new molecular events in ccRCC and provides promising diagnostic and therapeutic targets for this disease.


Plasmids transfection
For stable transfection, 1600ng of target lentiviral transfer vector is cotransfected with 800ng each of the three helper plasmids (Gag-Pol, Rev and VSV-G) into 239T cell with Polyethylenimine MAX 40,000(PolyScience) according to the protocol. After 48h transfection, the Supernatant was filtered with 0.22μm sterile Nihon Millipore filters and added to 786-O and ACHN cells plated in 6 well plates. And the lentivirus Supernatant was exchange with fresh culture medium. Three days later, the transfected cells were subject to 2μg/ ml puromycin selection for two weeks and the interference or overexpress efficiency was detected by RT-PCR.
For transient transfection, indicated plasmids or miRNA mimics were transfected into 786-O and ACHN cells with GenMute™ siRNA & DNA Transfection Reagent (signagen) according to manufacturer's instructions Colony formation and EdU assay 786-O cells and ACHN cells were plated at approximately 1,000 cells per well in 6-well plates for Colony formation and 7*10 4 in 48 well plates for Edu assay after stable infection with indicated lentivirus. Culture medium was changed every 3 days. Colony formation was analyzed 10 days after infection by staining cells with 0.05% crystal violet solution for 20 min. EdU assay was performed after the cells reached 70%-80% confluence according to the manufacturer's instructions.

Scratch wound healing assay
The cells were inoculated onto six-well plates and cultured at 37°C in a 5% CO2 cell incubator. After the cells reached 70%-80% confluence, cross lines were made using a 200-μL sterile pipette tip. The cells were washed three times with sterile PBS to remove the scratched cells. The cells were continuously cultured in serumfree culture medium. After 0 h and 48 h, the cells were photographed. Cell migration distance = distance at 0 h-distance at 48 h.

Transwell migratory and invasion assays
The 24-well transwell plate with 8 μm pore polycarbonate membrane inserts (Corning, New York, USA) was used to analyze the migration and invasive potential of cells according to manufacturer's protocol with three replications. For invasion assay, the membrane was coated with the matrigel (200 ng/ml) (BD Biosciences, Bedford, MA). 10 5 of ACHN and 3*10 4 of 786-O cells were seeded, and 12 hours later cells invading into the lower surface of the membrane insert were fixed in 100% methanol, stained with 0.05% crystal violet, and quantified by counting in 10 random fields.

Immunohistochemistory (IHC)
Paraffin-embedded, formalin-fixed tissues were immunostained for PCNA proteins. Tissues were deparaffinized, rehydrated, and incubated at room temperature in 0.3% H 2 O 2 to block endogenous peroxidase and in blocking solution for nonspecific binding. Primary antibody were applied to sections overnight at 4°C. Afterwards, tissues were incubated with anti-mouse HRP conjugated (Abcam, USA) secondary antibody for 1h at room temperature. Then enzyme development was performed with DAB/H2O2 complex for 10 min at room temperature and in the absence of light which provides a brownish precipitation. The primary Ki-67 antibodies (CST, USA), E-cadherin (CST, USA), N-cadherin (CST, USA), BMI1 (Gentex, USA), ZEB1 (CST, USA) and ZEB2 (CST, USA) was used at working dilution 1:100. Stained (brown) cells were quantified as number of positive cells. To evaluate the intensity of antigen immunoreactivity we examined the percent of positive staining cells in 10 fields at random per rat under 200×magnification analysied with image-proplus 6.0.

Subcellular RNA isolation and RT-PCR assay
Total cellular RNA of 786-O cells was extracted using Trizol extraction, and the The Cytoplasmic and nuclear RNA of cells was extracted using a PARIS™ kit (Ambion) according to the manufacturer's instructions. For mRNA detection, the cDNA were Synthesis with PrimeScript RT reagent Kit (TaKaRa) and detected with RT-PCR using SYBR Premix Ex TaqTM (TaKaRa). For the detection of U6, cDNA were Synthesis and detected