A novel NF-κB regulator encoded by circPLCE1 inhibits colorectal carcinoma progression by promoting RPS3 ubiquitin-dependent degradation

Background Constitutive activation of nuclear factor-κB (NF-κB) signaling plays a key role in the development and progression of colorectal carcinoma (CRC). However, the underlying mechanisms of excessive activation of NF-κB signaling remain largely unknown. Methods We used high throughput RNA sequencing to identify differentially expressed circular RNAs (circRNAs) between normal human intestinal epithelial cell lines and CRC cell lines. The identification of protein encoded by circPLCE1 was performed using LC–MS. The function of novel protein was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses. Results A novel protein circPLCE1-411 encoded by circular RNA circPLCE1 was identified as a crucial player in the NF-κB activation of CRC. Mechanistically, circPLCE1-411 promoted the ubiquitin-dependent degradation of the critical NF-κB regulator RPS3 via directly binding the HSP90α/RPS3 complex to facilitate the dissociation of RPS3 from the complex, thereby reducing NF-κB nuclear translocation in CRC cells. Functionally, circPLCE1 inhibited tumor proliferation and metastasis in CRC cells, as well as patient-derived xenograft and orthotopic xenograft tumor models. Clinically, circPLCE1 was downregulated in CRC tissues and correlated with advanced clinical stages and poor survival. Conclusions circPLCE1 presents an epigenetic mechanism which disrupts NF-κB nuclear translocation and serves as a novel and promising therapeutic target and prognostic marker. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-021-01404-9.


RNA extraction and real-time PCR
Total RNA was isolated from cells by TRIzol Reagent (Thermo Fisher Scientific).
ReverTra Ace qPCR RT Kit (Toyobo) was used to perform reverse transcription according to the manufacturer's instructions. The nuclear and cytoplasmic fractions were isolated by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).The Applied Biosystems 7500 Sequence Detection system was used to carry out quantitative real-time reverse transcription PCR (qRT-PCR) with the SYBR Green PCR Master Mix (Applied Biosystems). We generated standard curves and applied the 2 -△△CT method with normalized to 18S rRNA. We next used the gene of human hypoxanthine-guanine-phosphoribosyltransferase (hHPRT) to quantify cancer metastasis in mouse livers. All the gene-specific primers were obtained from Invitrogen and the oligonucleotide sequences are listed in Table S4.

RNase R treatment
RNase R (Epicentre Technologies, Madison, WI, USA) was used to assess the stability of circRNA. Total RNA (2 μg) was mixed with 0.6ul 10 × RNase R Reaction Buffer and 0.2 μl RNase R or DEPC-treated water (control group). The samples were then incubated at 37 °C for 15 min1. The expression levels of circPLCE1 and linear PLCE1 were detected by qRT-PCR.

Actinomycin D assay
For the half-life of circRNA assessment, the gene transcription was blocked by adding 2mg/mL Actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) to the cell culture medium. DMSO was used as a negative control. Cells were harvested at 0, 4, 8, 12, 24h and the stability of circPLCE1 and linear PLCE1 was analyzed by qRT-PCR.

RNA fluorescence in situ hybridization (FISH) assay
According to the manufacturer's instructions, the FISH kit (Ribo Bio, Guangzhou, China) was utilized to perform FISH in cells and the results are visualized by confocal  Table S6.

RNA in situ hybridization (ISH) assay
According to the manufacturer's instructions, the ISH Detector kit (BSTER, Wuhan, China) was used to perform ISH in paraffin-embedded CRC tissues. CRC tissues were fixed with 4% paraformaldehyde at room temperature for 10 min; then they were digested with proteinase K at 37°C for 2 min and pre-hybridized at 37°C for 3 h.

Plasmids construction, transfection and lentivirus infection
For RPS3, HSP70 and HSP90α-expressing plasmids, the full-length ORF sequences with tag of these three genes were respectively subcloned into the pcDNA3.1 vector.  Table S5.

Cell proliferation assays
Cell proliferation was examined using plate colony formation, 3D anchorage-free colony formation, soft agar cell colony formation and patient-derived organoids (PDOs) growth. For plate colony formation, 500 cells were plated into 6-well platesand cultured for two weeks. Then the colonies were fixed with methanol and stained with 0.1% crystal violet for 15 minutes at room temperature. Cell colonies were counted and imaged. For 3D anchorage-free colony formation assay, 5,000 cells were seeded in 200 μL DMEM supplemented with 10% FBS in 96-well ultra-low attachment microplate (7007; Corning, USA). Cell culture media was refreshed every three days, and images of 3D colonies were captured using a phase-contrast microscope (DMI4000B, Leica, Wetzlar, Germany). The volume of 3D colonies was calculated using the formula: Volume = 4/3πR3. For soft agar cell colony formation assay, 5,000 cells were suspended in 75 μL 0.4% agar DMEM solution and seeded over 50 μL of bottom agar in 96-well plates. Images of colonies were captured after 10 days using a phase-contrast microscope.

Migration and invasion assays
Cell migration and invasion were examined using cell migration assays and wound healing assay. Cell migration assays were performed with 24-well plates with 8-μm pore size chamber inserts (Corning). In general, 5×10 4 cells resuspend with 200 μl serum-free DMEM were seeded in the upper chamber well and 800 μl of DMEM with 10% FBS was added into the lower chamber. After 24 h, cells migrating through the membrane were fixed with 4% paraformaldehyde for 15 min, and then stained with 0.1% crystal violet for 15 min. The cells were viewed under an inverted microscope (DMI4000B, Leica, Wetzlar, Germany) and quantified using software ImageJ. For wound healing assay, a total of 2×10 6 cells were seeded in six-well plates and 14 / 15 incubated until confluency was reached. A 100ul pipette tip was used to create a rectilinear scratch. After 12 h, cells were fixed with 4% paraformaldehyde for 15 min, and then stained with 0.1% crystal violet for 15 min. An inverted microscope (DMI4000B, Leica, Wetzlar, Germany) was utilized to image the wound closure.

Western blot
Cell and tissue samples were lysed with radio-immunoprecipitation assay buffer GAPDH served as a control for western blot analysis.

Immunoprecipitation assay
For immunoprecipitation, the cells transfected with indicated plasmids were lysed in Pierce IP lysis buffer containing protease inhibitors cocktail (Thermo Scientific, IL, USA) for 30 min at 4°C. Cell lysates were centrifuged at 13,000g for 20 min at 4°C.

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Protein A/G Magnetic Beads (Thermo Scientific) were prewashed with washing buffer two times. Then, indicated antibodies were added to the beads for antibody crosslink 4h at 4°C. The cell lysates were added to the antibody-crosslinked beads overnight at 4°C. After washing with washing buffer for five times, the beads were incubated with 2x sample loading buffer and boiled for 10 minutes. Finally, the lysate samples were resolved on SDS-PAGE for further study.

Mass spectrometry assay
The gel was chopped into small fragments with a razor blade, destained, and subjected to digestion by modified porcine trypsin (50-100 ng/digestion; Promega).
After trypsin digestion, peptides were dissolved in 0.1% FA and 2% ACN, directly loaded onto a reversed-phase analytical column (75 um i.d. x 150mm, packed with Acclaim PepMap RSLC C18, 2 um, 100Å, nanoViper). The gradient was comprised of an increase from 5% to 50% solvent B (0.1% FA in 80% ACN) over 40 min, and climbing to 90% in 5 min, then holding at 90% for the 5 min. All at a constant flow rate of 300 nl/min. The MS analysis was performed on Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 27; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 30.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 1E5 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800 m/z. Fixed first mass was set as 100 m/z.
Protein identification were performed with MASCOT software by searching Uniprot_Aedis Aegypti.