A novel polypeptide encoded by the circular RNA ZKSCAN1 suppresses HCC via degradation of mTOR

Abstract

Background: hsa_circ_0001727 (circZKSCAN1) has been reported to be a tumor-associated circRNA by sponging microRNAs. Intriguingly, we found that circZKSCAN1 encoded a secretory peptide (circZKSaa) in the liver. The present study aims to elucidate the potential role and molecular mechanism of circZKSaa in the regulation of hepatocellular carcinoma (HCC) progression.

Methods: The circRNA profiling datasets (RNA-seq data GSE143233 and GSE140202) were reanalyzed and circZKSCAN1 was selected for further study. Mass spectrometry, polysome fractionation assay, dual-luciferase reporter, and a series of experiments showed that circZKSCAN1 encodes circZKSaa. Cell proliferation, apoptosis, and tumorigenesis in nude mice were examined to investigate the functions of circZKSaa. Mechanistically, the relationship between the circZKSaa and mTOR in HCC was verified by immunoprecipitation analyses, mass spectrometry, and immunofluorescence staining analyses.

Results: Receiver operating characteristic (ROC) analysis demonstrated that the secretory peptide circZKSaa encoded by circZKSCAN1 might be the potential biomarker for HCC tissues. Through a series of experiments, we found that circZKSaa inhibited HCC progression and sensitize HCC cells to sorafenib. Mechanistically, we found that the sponge function of circZKSCAN1 to microRNA is weak in HCC, while overexpression of circZKSaa promoted the interaction of FBXW7 with the mammalian target of rapamycin (mTOR) to promote the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway. Furthermore, we found that the high expression of cicZKSCAN1 in sorafenib-treated HCC cells was regulated by QKI-5.

Conclusions: These results reveal that a novel circZKSCAN1-encoded peptide acts as a tumor suppressor on PI3K/AKT/mTOR pathway, and sensitizes HCC cells to sorafenib via ubiquitination of mTOR. These findings demonstrated that circZKSaa has the potential to serve as a therapeutic target and biomarker for HCC treatment.

Keywords: Biomarker; HCC; Sorafenib; circZKSaa; mTOR.

Fig. 1

circZKSCAN1 is down-regulated in liver cancer tissues and is sensitive to sorafenib treatment. A Human adult normal liver datasets were analyses for differentially expressed circRNAs. The top 5 up-regulated circRNAs were at list. B Relative expression level of the top 5 up-regulated circRNAs in 30 pairs of HCC and normal tissues. C Relative expression level of the top 5 circRNAs in normal hepatocyte lines (THL-2) and HCC cell lines (SNU-387, SNU-398, HCC-LM3, Hep3B, Huh-7) by RT-qPCR analysis. D Schematic illustration exhibited the significantly different expressions circRNAs by overlapping GSE143233 and GSE140202. E The relative expression of circZKSCAN1, hsa_circ_0006886 and hsa_circ_0071106 in HCC cells with and without sorafenib treatment. F RT-qPCR analysis showed QKI-5 expression level in HCC-LM3 and Hep3B after different concentrations of sorafenib treatment. G RT-qPCR analysis showed expression levels of circZKSCAN1 and ZKSCAN1 in overexpressed QKI-5 HCC-LM3 and Hep3B cells. H RIP analysis showed expression levels of circZKSCAN1 in overexpressed QKI-5 HCC-LM3 cells. H Analysis of apoptosis in HCC-LM3 and Hep3B with or without overexpressed circZKSCAN1 transfection by flow cytometry assays. T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 2

circZKSCAN1 encodes a 206aa peptides. A Vectors schematic diagram showed two IRSE sequences in circZKSCAN1 were cloned into Psi-check2 vector. B The relative luciferase activity of Luc/Rluc was used to tested circZKSCAN1 translation ability in HCC-LM3 cells, Encephalomyocarditis Virus (EMCV) IRES was used as a positive control, empty vector was served as a negative control. C Polysome fractionation and RT-PCR analysis after lysate of HCC-LM3 cells, GAPDH serve as a positive control. D Vectors schematic diagram showed that His/HA-labeled circZKSCAN1-mut, circZKSCAN1, Vector and circZKSaa plasmid. E Upper panel, RT-qPCR results of circZKSCAN1 on vector (circZKSCAN1-mut, circZKSCAN1, Vector and circZKSaa) transfected 293 T cells. Lower panel showed Western bolt assay of circZKSaa expression level on vector (circZKSCAN1-mut, circZKSCAN1, Vector and circZKSaa) transfected 293 T cells. F Western blot analyses showed circZKSaa expression of HCC-LM3, Hep3B and THL-2. G After Co-IP by HA antibody and separation by SDS-PAGE, the protein sample which stained by silver staining was analyzed by mass spectrometry. Data are presented as the mean ± SEM of three independent experiments

Fig. 3

circZKSaa has diagnostic value in HCC. A The circZKSaa content was tested via Western blot in 12 pairs of HCC and normal tissues. β-actin was used to indicate the amount of loading proteins. B Diagnostic value of circZKSaa in 29 pairs of HCC and normal tissues by ROC curve. C After transfected circZKSaa plasmid in HCC-LM3 and 293 T cells, the circZKSaa expression level in cells supernatant. D The RNA-Seq databases of HCC patient’s serum databases was analyzed by Linux’s CIRIquant, find_circ and other softwares. E The location of circZKSaa was determined by Nucleo-cytoplasmic separation, GAPDH was used as the positive control of cytoplasmic protein, while PCNA was used as the positive control of nuclear protein. F Western blot assay showed circZKSaa expression level in HCC-LM3 cells after treated sorafenib with different concentrations and different duration. G Analysis of apoptosis in HCC-LM3 and Hep3B with or without circZKSaa transfection by flow cytometry assays. Quantitative data from three independent experiments are presented as mean ± SEM (error bars). T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 4

circZKSCaa inhibits tumor growth. A The mRNA and protein levels of circZKSaa and circZKSCAN1 in stable HCC-LM3 and Hep3B cells were demonstrated by real-time RT-qPCR and Western blot. Cell proliferation assay including B CCK8 analysis, C colony formation assays, D KI67 immunofluorescence assay, E Cell cycle progression was determined via flow cytometry assay for circZKSCAN1-mut, circZKSCAN1, sh-scramble, sh-circZKSCAN1, Vector and circZKSaa stable cell lines (HCC-LM3 and Hep3B). Quantitative data from three independent experiments are presented as mean ± SEM (error bars). Scale bars = 100 μm. T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 5

circZKSaa promotes tumor apoptosis. Cell apoptosis analyses contain A TUNEL staining and B Flow cytometry analysis for circZKSCAN1-mut, circZKSCAN1, sh-scramble, sh-circZKSCAN1, Vector and circZKSaa stable cell lines (HCC-LM3 and Hep3B). C Xenograft BALB/c nude mice (n = 5 or 6 each group) experiment analyses which including the images of tumors in each group and tumor volume to demonstrated the HCC cell growth in vivo. Quantitative data from three independent experiments are presented as mean ± SEM (error bars). Scale bars = 1 cm. T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 6

circZKSaa prevents AKT activation through mTOR. HCC-LM3 and Hep3B cells were transfected corresponding vectors (circZKSCAN1-mut\circZKSCAN1\sh-scramble\sh-circZKSCAN1\Vector\circZKSaa) to detect the proliferation signal pathway. A The expression level of β-catenin, ERK, phosphorylated ERK, AKT, T308 site phosphorylated-AKT and S473 site phosphorylated-AKT were determined via Western blot, and GAPDH expression level served to indicate the amount of loading proteins. B The protein expression level of PDK1, phosphorylated PDK1, mTOR and phosphorylated mTOR were determined via Western blot, and GAPDH expression level served to indicate the amount of loading proteins. C After Co-IP by HA antibody and separation by SDS-PAGE, the protein sample which stained by silver staining was analyzed by mass spectrometry. D Co-IP analyses by HA and mTOR antibody determined the interaction between circZKSaa and mTOR. E Immunofluorescence staining of mTOR and circZKSaa-HA in HCC-LM3 cells, Scale bars = 20 μm. F Western blot assay were used to detect MDM2, phosphorylated-MDM2, S6K1 and phosphorylated-S6K1 in both HCC-LM3 and Hep3B cells transfected with vectors (circZKSCAN1-mut\circZKSCAN1\sh-scramble\sh-circZKSCAN1\Vector\circZKSaa), GAPDH expression level indicated the amount of loading proteins. Quantitative data from three independent experiments are presented as mean ± SEM (error bars). T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 7

circZKSCaa promotes ubiquitination of mTOR. A RT-qPCR demonstrated mTOR and 5’UTR-mTOR expression level in circZKSCAN1-mut\circZKSCAN1\sh-scramble\sh-circZKSCAN1\Vector\circZKSaa transfected cells (HCC-LM3 and Hep3B). B Western blot detected half-life of mTOR by adding CHX for different times. The amount of mTOR protein level was calculated by ImageJ. C Western blot analyses mTOR protein degradation by MG-132 treatment. D Before treated with MG132, HCC-LM3 and Hep3B cells were transfected with HA-ubiquitin and circZKSaa-Flag. Whole cell lysates were IP with mTOR antibody followed by detection with ubiquitin antibody. GAPDH, Flag and mTOR serve as amount of input proteins. Quantitative data from three independent experiments are presented as mean ± SEM (error bars). T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001

Fig. 8

circZKSCaa degrades mTOR by interacting with FBXW7. A Co-IP assay by His antibody measured the interaction between mTOR, circZKSaa-His and FBXW7 in HCC-LM3 and Hep3B cells; Immunofluorescence staining of FBXW7-HA and circZKSaa-His in HCC-LM3 cells, Scale bars = 200 μm. B Western blot analyses mTOR ubiquitination level in FBXW7-overexpresion cells with or without down-regulated circZKSCAN1, both cells were pre-incubated with MG-132. C Western blot analyses mTOR ubiquitination level in circZKSaa-His overexpresion cells with or without down-regulated FBXW7, both cells were pre-incubated with MG-132. D FBXW7-HA, mTOR-Flag, circZKSaa-His vector plasmid was transfected into 293 T cells as indicated. Immunoprecipitation was performed with anti-Flag protein A/G magnetic beads and proteins from the immunoprecipitatte was subjected to Western blot for evaluation of FBXW7-HA and mTOR-Flag. E mTOR-Flag, mTOR-631/635-mut-Flag, circZKSaa-His vector plasmid was transfected into 293 T cells as indicated. Immunoprecipitation was performed with anti-Flag protein A/G magnetic beads and proteins from the immunoprecipitatte was subjected to Western blot for evaluation of circZKSaa-His and mTOR-Flag. F FBXW7-HA, mTOR-Flag, mTOR-631/635-mut-Flag, circZKSaa-His vector plasmid was transfected into 293 T cells as indicated. Immunoprecipitation was performed with anti-Flag protein A/G magnetic beads and proteins from the immunoprecipitatte was subjected to Western blot for evaluation of Ub and mTOR-Flag