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SCIENCE CHINA Life Sciences, https://doi.org/10.1007/s11427-019-9580-5

Ribosome profiling analysis identified a KRAS-interacting microprotein that represses oncogenic signaling in hepatocellular carcinoma cells

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  • ReceivedMay 16, 2019
  • AcceptedMay 28, 2019
  • PublishedJun 24, 2019

Abstract

The roles of concealed microproteins encoded by long noncoding RNAs (lncRNAs) are gradually being exposed, but their functions in tumorigenesis are still largely unclear. Here, we identify and characterize a conserved 99-amino acid microprotein named KRASIM that is encoded by the putative lncRNA NCBP2-AS2. KRASIM is differentially expressed in normal hepatocytes and hepatocellular carcinoma (HCC) cells and can suppress HCC cell growth and proliferation. Mechanistically, KRASIM interacts and colocalizes with the KRAS protein in the cytoplasm of human HuH-7 hepatoma cells. More importantly, the overexpression of KRASIM decreases the KRAS protein level, leading to the inhibition of ERK signaling activity in HCC cells. These results demonstrate a novel microprotein repressor of the KRAS pathway for the first time and provide new insights into the regulatory mechanisms of oncogenic signaling and HCC therapy.


Acknowledgment

We thank Shujuan Xie for providing the normal liver tissues and the corresponding paracancer tissue samples. This work was supported by the National Key Research and Development Program of China (2017YFA0504400), the National Natural Science Foundation of China (31370791, 31671349, 31770879), Fundamental Research Funds for the Central Universities (14lgjc18). This research was supported in part by the Guangdong Province Key Laboratory of Computational Science (13lgjc05) and the Guangdong Province Computational Science Innovative Research Team (14lgjc18).


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Table S1 All lncRNAs containing translated ORFs in human HuH-7 cells identified by Ribo-seq analysis

Table S2 Sequences of the primers and oligos used in this study

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Genome-wide analysis of translated ORFs with Ribo-seq data from HuH-7 cells. A, Schematic description of the ribosome profiling (Ribo-seq) and RNA-seq experiments in HuH-7 cells. B, The 29-nt and 30-nt RPF read distributions around the ribosomal P site. The P site positions are colored according to the frame. C, Biotype distribution of all the translated ORFs identified in HuH-7 cells using the RiboCode method (left, ORF ratios; right, ORF numbers; P<0.05). The corresponding positions of ORFs in the genes were shown as the right diagram. D, The fraction of lncRNA-encoded peptides within the indicated length range. E, The 3-nt distribution of the RPF reads in two lncRNA ORFs reported to encode peptides.

  • Figure 2

    Conservation analysis of the predicted microprotein KRASIM. A, Scatter diagram showing the conservation and translation efficiency (TE) of translated lncORFs less than 300 nt in length. Blue, uncharacterized lncORFs; red, the predicted ORF of lncRNA NCBP2-AS2 and two reported lncORFs. B, Conservation analysis of the KRASIM-ORF sequence in vertebrates using the UCSC phastCons and phyloP tracks. C, Distribution of the RPF reads in the KRASIM-ORF. D, Ka and Ks ratios of KRASIM in the indicated pairs of vertebrate species. E, Multiple-species alignment of the amino acid sequences of the predicted NCBP2-AS2-encoded microprotein (KRASIM).

  • Figure 3

    Identification of the microprotein KRASIM in human and mouse cells. A, Schematic diagram of the C-terminally 3×FLAG-tagged vectors fused with the wild-type (WT) and frameshifted (FS) ORFs of KRASIM. B, Western blotting assays to validate the coding ability of hKRASIM using the FLAG antibody in four human cell lines (HuH-7, SK-HEP-1, Hep3B and 293T) transfected with the hKRASIM-3×FLAG and hKRASIM-FS-3×FLAG vectors. The pCFH vector was used as a negative control. C, Western blotting assays to validate the coding ability of mKRASIM using the FLAG antibody in Hepa1–6 and NIH/3T3 cells transfected with the mKRASIM-3×FLAG and mKRASIM-FS-3×FLAG vectors. D, The KRASIM mRNA levels in HuH-7 and SK-HEP-1 cells were detected by qPCR. Cells were transfected with the indicated vectors or siRNAs. GAPDH is shown as an internal reference. E, The protein levels of KRASIM in HuH-7 and SK-HEP-1 cells overexpressing KRASIM or following KRASIM knockdown using the anti-KRASIM antibody. The red arrow shows the specific band corresponding to KRASIM. F, The protein levels of KRASIM were detected in the indicated HCC tissues (T) and their corresponding adjacent nontumorous tissues (NT) using the anti-KRASIM antibody. G, The relative expression of KRASIM mRNA detected by qRT-PCR in a normal hepatocyte cell line (LO2) and five HCC cell lines. H, The protein levels of KRASIM in a normal hepatocyte cell line (LO2) and five HCC cell lines using the anti-KRASIM antibody. I, Western blotting assays showing the subcellular localization of KRASIM in HuH-7 cells that did or did not overexpress KRASIM. J, Immunofluorescence assays showing the expression and distribution of KRASIM in HuH-7 cells transfected with the indicated vectors. Scale bar, 10 µm.

  • Figure 4

    The microprotein KRASIM, but not its lncRNA, suppressed proliferation and regulated the cell cycle in human HCC cells. A and B, CCK-8 (A) and colony formation (B) assays showing the proliferation of HuH-7 and SK-HEP-1 cells transfected with the indicated siRNAs. C and D, CCK-8 (C) and colony formation (D) assays showing the growth of HuH-7 and SK-HEP-1 cells transfected with the indicated vectors. Representative pictures of colonies are shown in the upper panel, and the number of colonies is indicated in the bottom panel. E and F, The proportions of cells transfected with the indicated siRNAs (E) and vectors (F) in each stage of the cell cycle.

  • Figure 5

    The microprotein KRASIM interacted with and colocalized with the KRAS protein. A, Schematic diagram showing the identification of microprotein-interacting proteins using MS following IP. B, The results of silver staining using the immunoprecipitate in HuH-7 cells. Red box, the specific band sent for MS analysis; red arrow, the band corresponding to KRASIM itself. C, Western blotting assays showing the IP efficiency using the FLAG antibody in HuH-7 cells overexpressing KRASIM-3×FLAG. D, List of the candidate KRASIM-interacting proteins identified through MS analysis. Three GTPase- or GTPase-related proteins (KRAS, HRAS and RAB37) are highlighted in red, and the polyubiquitin-B (UBB) protein is highlighted in green. E, Distribution of the best unique peptides from KRAS in the MS analysis. F, Co-IP assays showing the interaction between KRASIM and the KRAS protein in 293T cells (left, treated without RNase A; right, treated with 30 µg mL–1 RNase A). G, The colocalization of KRASIM and the KRAS protein in HuH-7 cells in immunofluorescence assays using the FLAG and KRAS antibodies. Scale bar, 10 µm.

  • Figure 6

    KRASIM suppressed the protein level of KRAS and the activity of the ERK pathway. A and B, The protein level of KRAS in HuH-7 and SK-HEP-1 cells after transfection with the indicated siRNAs or vectors. (A) showing the representative pictures of western blot, and (B) showing the calculation of three biological replicates of the western blot assays. GAPDH was the internal control. C, qPCR assays showing the mRNA level of KRAS in HuH-7 and SK-HEP-1 cells after transfection with the indicated siRNAs or vectors. D, Luciferase reporter assays showing the activity of ERK pathway in HuH-7 and SK-HEP-1 cells transfected with the indicated siRNAs or vectors. E, The working model of the microprotein KRASIM in HCC cells. The microprotein KRASIM, which is encoded by a putative lncRNA, interacts with the KRAS protein. Then, KRASIM suppresses the protein level of KRAS and the ability of the downstream ERK pathway to inhibit human HCC cell proliferation.

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