Circular RNA circPPP6R3 upregulates CD44 to promote the progression of clear cell renal cell carcinoma via sponging miR-1238-3p

Circular RNAs (circRNAs) are a type of covalently closed circular-formed RNAs and play crucial roles in the oncogenesis and progression of various human cancers. Here we identified a novel circRNA, circPPP6R3, to be highly expressed both in clear cell renal cell carcinoma (ccRCC) tissues and cell lines based on analyzing high-throughput sequencing data and qRT-PCR analysis. Highly expressed circPPP6R3 was positively correlated with higher histological grade, T stage, and M stage as well as advanced clinical stage of ccRCC patients. Functionally, knockdown of circPPP6R3 attenuated the proliferation, migration, and invasion of ccRCC cells whereas overexpression had the reverse effects. Mechanistically, the biotin-labeled pull-down assay and dual-luciferase reporter assay revealed that circPPP6R3 directly interacted with miR-1238-3p. miR-1238-3p inhibitors had a rescue effect on the proliferative and metastatic capacities by knockdown of circPPP6R3. Moreover, RNA-sequencing analysis and dual-luciferase reporter assay indicated that circPPP6R3 upregulated CD44, a cell-surface glycoprotein contributed to the cell adhesion and metastasis, via sponging to miR-1238-3p. Further investigation revealed that MMP9 and Vimentin were regulated by CD44 in ccRCC. Our study thus provided evidence that the regulatory network involving circPPP6R3/miR-1238-3p/CD44 axis might provide promising biomarkers as well as a therapeutic approach for ccRCC.


RNA extraction, RNase R treatment, and quantitative real-time PCR assays
The isolation of total RNA from tissues and cell lines was extracted with Trizol reagent (TaKaRa, Japan).
For the RNase R treatment, an amount of 2 mg total RNA was incubated with or without 3 U/mg RNase R (Epicentre Technologies, USA) at 37°C for 15 min. To investigate the expression of mRNA and circRNA, 500 μg of total RNA was subjected to synthesize complementary DNA using the reverse transcription kit PrimeScript RT Master Mix (TaKaRa, Japan). For miRNA analysis, Mir-X TM miRNA First-Strand Synthesis kit (TaKaRa, Japan) was used. The qRT-PCR was conducted with the SYBR Premix Ex Taq TM kit (TaKaRa, Japan) using LightCycler 480 instrument (Roche Diagnostics). GAPDH acted as an internal standard control for mRNA and circRNA analysis while U6 for miRNA analysis using the 2 −ΔΔCT method.

Nucleic acid electrophoresis
To confirm the products amplified by the primers of circPPP6R3 were circular form, gDNA and cDNA PCR products were separated in 2% agarose gel with TAE running buffer using electrophoresis system at 120V for 30 min, followed by UV irradiation. Super DNA Marker (CWBIO, China) was applied as a DNA marker and GAPDH acted as a control.

Small interference RNAs (siRNAs), plasmids construction and stable transfection
siRNAs targeting circPPP6R3 were obtained from RiboBio (China) and were transfected into ccRCC cell lines with Lipofectamine iMax (Invitrogen, USA). The miRNA mimics, inhibitors, and respective control were synthesized by GenePharma (China). The circPPP6R3-overexpressing plasmids were synthesized by IGE Biotech Co (China). Plasmids were co-transfected with lentiviral packaging plasmids into 293T cells to package lentivirus. And then, Caki-1 cells were infected by lentivirus to stably overexpressing circPPP6R3 followed by 7-day selecting with 2ug/mL puromycin.

Cell proliferation assay
For the MTS assay to detect the cell viability, 1000 transfected cells were seeded and cell viability was detected with MTS reagents (Promega, USA) for 6 days. An Absorbance Microplate Reader (CMAX PLUS, China) was utilized to read the absorbance at a wavelength of 490 nm.
For colony formation assay, 1000 transfected cells were respectively seeded into 6-well plates and were cultured for 7-10 days. Afterward, the colonies were fixed with 4% paraformaldehyde and were stained with 0.1% crystal violet, counted and photographed.

Transwell assay and invasion assay
For transwell assays, 200 ul 10 5 transfected ccRCC cells were plated on the upper chamber of transwell chamber inserts (Corning, NY) with or without precoated Matrigel (Corning, NY) as instructed by the manufacturer's protocol. Then, the lower chambers were filled with 600 μl complete medium. After a period of incubating, the migratory or invasive cells were fixed with 4% paraformaldehyde, dyed with crystal violet and were counted in three randomly selected fields with a Nikon Eclipse 80i system (Nikon, Japan).

Wound Healing Assay
The transfected ccRCC cells were seeded in a 12-well culture plate and cultured in a humidified atmosphere overnight. The next day, the monolayer cells were scratched with a 200 ul pipette tip when reached 90% confluence and were photographed with a Nikon Eclipse 80i system. Then, the cells were washed with PBS gently and cultured with FBS-free medium at 37 °C and 5% CO2. The cell migration was photographed at the same sight 12 h or 24 h later.

5-Ethynyl-20-deoxyuridine (EdU) incorporation assay
The EdU incorporation assay was performed to validate the cellular proliferative status with a EdUrelated Kit (RiboBio, China). 10 4 ccRCC cells were plated in 96-wells in advance and then cultured with EdU for 2 h. These cells were fixed using 4% paraformaldehyde and were incubated with Apollo Dye Solution and Hoechst 33342 in order according to the manufacturer's instructions. The ratio of EdU positive cells to Hoechst-stained cells was calculated to evaluate the cell proliferation after photographing and counting under an Olympus IX73 microscope(Olympus, Japan).

Immunohistochemistry was performed according to the protocol of Biotin-Streptavidin HRP Detection
Systems (ASGB-BIO, China). Briefly, tissues were first fixed in 4% formaldehyde, embedded in paraffin, and then cut into 5-μm sections. Next, primary antibody anti-CD44 (1:250, 15675-1, Proteintech Group, China) or anti-Ki-67 (ZM-0165, ASGB-BIO, China) were used to incubate the sections for overnight at 4°C. A Nikon Eclipse 80i system (Nikon, Japan) was adopted to capture the images and three random sites of each slide were detected. The expression of target proteins was detected by the assessment of the proportions and intensities of positive cells.

Western Blotting analysis
ccRCC cells or tissues were lysed in RIPA (CWBIO, China) combined with phosphatase and protease inhibitors (Roche, Switzerland) to extract protein. The quantities of the protein were detected with the BCA Protein assay kit (CWBIO, China). Then, an equal quantity of protein was electrophoresed by SDS-PAGE gels at 120V, transferred onto PVDF membranes which would be blocked by 5% BSA and 3 incubated with specific primary antibodies at 4°C overnight. The next day, membranes were washed by TBST, incubated with HRP-conjugated secondary antibodies and washed three times with TBST before forming an image by G:BOX Chemi XT4 (SYNGENE, USA) with Immobilon ECL substrate (Millipore, Germany). antisense：5'-GGCAGACAGACGAGGAAGUU -3' miR-1238-3p inhibitors 5'-GGGGCAGACAGACGAGGAAG -3'