Volume 68, Issue 1, Pages 171-184.e6 (October 2017) A Peptide Encoded by a Putative lncRNA HOXB-AS3 Suppresses Colon Cancer Growth  Jin-Zhou Huang, Min Chen, De Chen, Xing-Cheng Gao, Song Zhu, Hongyang Huang, Min Hu, Huifang Zhu, Guang-Rong Yan  Molecular Cell  Volume 68, Issue 1, Pages 171-184.e6 (October 2017) DOI: 10.1016/j.molcel.2017.09.015 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 The lncRNA HOXB-AS3 Was Frequently Downregulated in CRC Tissues and Encodes a Small Peptide (A) HOXB-AS3 RNA levels were compared between primary CRC tissues (T) and corresponding adjacent non-tumoral tissues (NT) in the indicated cancers by RT-PCR. (B) Alignment of HOXB-AS3 amino acid sequences of three species of primates. (C) Diagram of the GFP fusion constructs used for transfection. The start codon ATGGTG of the GFP (GFPwt) gene is mutated to ATTGTT (GFPmut). The start codon ATG of the HOXB-AS3 ORF is mutated to ATT. (D–F) The indicated constructs were transfected into HeLa cells for 24 hr, GFP fluorescence (D) and HOXB-AS3 peptide immuno-staining using an anti-HOXB-AS3 antibody (F) were detected, and HOXB-AS3-GFP fusion protein levels were determined by western blotting with anti-GFP and HOXB-AS3 antibodies (E). (G) Diagram of the Flag fusion constructs used for transfection. The start codon ATG of the HOXB-AS3 ORF is mutated to ATT. (H–J) The indicated constructs were stably expressed in HTC-116 cells; Flag (H) and HOXB-AS3 peptide (J) were immuno-stained using anti-Flag and HOXB-AS3 antibodies, respectively; and HOXB-AS3-Flag fusion protein levels were determined by western blotting with anti-Flag and HOXB-AS3 antibodies (I). See also Figures S1–S4. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 HOXB-AS3 Peptide Is Endogenously Produced and Its Low Expression Is Associated with a Poor Prognosis in CRC Patients (A) HOXB-AS3 peptide in SW480 and SW620 cells was immune-stained with anti-HOXB-AS3 antibody. (B) HOXB-AS3 peptide in the indicated cancer cells with different levels of metastasis was detected. (C) SW480 cells were transfected with anti-HOXB-AS3 translation-blocking antisense oligo; HOXB-AS3 peptide was detected. (D) HOXB-AS3 peptide levels were detected in the indicated cancer tissues (T) and their corresponding adjacent non-tumoral tissues (NT). HOXB-AS3 peptide expression in SW480 cells was positive control. (E) Representative IHC images of HOXB-A3 peptide expression in CRC tissues and their corresponding NT tissues. (F) Differences in HOXB-AS3 peptide expression scores between CRC tissues (T) and the corresponding NT tissues are presented as a boxplot (n = 90). (G) Associations between HOXB-AS3 peptide levels and the percentage of patient death were analyzed in CRC samples. (H) A Kaplan-Meier survival analysis of CRC patients according to HOXB-AS3 peptide expression ratios of cancer/corresponding NT tissues. See also Figure S3 and Table S1. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The HOXB-AS3 Peptide, Not Its lncRNA, Suppresses Colony Formation and Tumor Growth In Vitro and In Vivo (A) HTC-116 and SW620 cells were transfected with the indicated constructs, and their colony-forming abilities were measured after 2 weeks (n = 3). (B) HTC-116 and SW620 cells were transfected with the indicated constructs for the indicated times, and the cell numbers were measured (n = 3). MUT indicates the 5′ UTR-ORFmut vector. (C) HTC-116 and SW620 cells were transfected with the indicated constructs for the indicated times, and migration and invasion abilities were determined using Transwell assays. (D) The in vivo growth of the indicated cell lines stably expressing the indicated constructs was examined. Mouse xenograft tumors are shown in the top panel. The images and weights of the xenograft tumors are presented in the left and right sides of the bottom panel, respectively (n = 5). (E) NOD-SCID mice were transplanted with the indicated Luc-labeled HTC-116 cells (2 × 106 cells/mouse) via tail vein injection; luciferase activity was visualized 8 weeks post-transplantation (n = 5). (F) Histological analysis of pulmonary metastases in the mouse model was conducted using hematoxylin and eosin (HE) staining, as shown in (E). The data are represented as the means ± SEM. See also Figure S5. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 HOXB-AS3 Peptide Interacts with the Arginine Residues in RGG Box of hnRNP A1 (A) Proteins that interacted with the HOXB-AS3 peptide were identified by combining co-immunoprecipitation (coIP) and mass spectrometry. (B) Proteins that interacted with the HOXB-AS3 peptide were shown to participate in RNA splicing. (C) hnRNP A1-HA plasmid was transfected into HEK293T cells, cellular lysates were treated with 30 μg/mL RNaseA for 30 min, hnRNP A1-HA complexes were then co-immunoprecipitated by anti-HA antibody, and HOXB-AS3 peptide was detected. (D) HOXB-AS3 ORF-Flag plasmid was transfected into HEK293T cells, cellular lysates were treated with 30 μg/mL RNase A for 30 min, HOXB-AS3-Flag complexes were co-immunoprecipitated by anti-Flag antibody, and hnRNP A1 peptide was detected. (E) Diagram of hnRNP A1 wild-type and mutation constructs with the different domain. (F and G) The indicated hnRNP A1-HA (A1-HA) plasmid together with HOXB-AS3 ORF-Flag (H3-Flag) vector were co-transfected into HEK293T cells, and hnRNP A1-HA (F) and HOXB-AS3-Flag (G) complexes were co-immunoprecipitated by anti-HA and Flag antibody, respectively; HOXB-AS3-Flag and hnRNP A1-HA were then detected using anti-Flag and HA antibodies, respectively. (H) The wild-type HA-hnRNP A1 and its AGG mutant were transfected into HEK293T cells; HA-hnRNP A1 expression was detected. (I and J) The indicated hnRNP A1-HA plasmid with HOXB-AS3 ORF-Flag vector was co-transfected into HEK293T cells; the interactions of HOXB-AS3 with hnRNP A1 AGG mutant were determined as described in (F) and (G). Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Silencing of hnRNP A1 Phenocopied the Functions of HOXB-AS3 Peptide Overexpression, and HOXB-AS3 Peptide, Not HOXB-AS3 lncRNA, Blocked the Enhancement of Cancer Phenotypes Induced by hnRNP A1 Overexpression (A–D) CRC cells were transfected with anti-hnRNP A1 siRNAs; hnRNP A1 expression (A), cell growth (B), colony formation (C), migration, and invasion (D) were determined. (E–H) HTC-116 cells were co-transfected with the indicated HOXB-AS3 plasmid together with hnRNP A1 vector; the indicated proteins (E), cell growth (F), colony formation (G), migration, and invasion (H) were determined. The data are represented as the means ± SEM. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 HOXB-AS3 Peptide Blocks the Binding of the Arginine Residues in RGG Motif of hnRNP A1 to the Intronic Sequences Flanking Exon 9 (A) HTC-116 cells were transfected with the indicated HOXB-AS3 plasmids and the indicated proteins were detected. (B) The HTC-116 nuclear extract (NE) were affinity-purified using the indicated biotin-labeled RNAs, and the eluted proteins were detected using anti-hnRNP A1 and HOXB-AS3 antibodies. (C) HTC-116 cells were transfected with HOXB-AS3 ORF-Flag plasmid and RNA affinity purification was performed as in (B). (D) HTC-116 cells were transfected with HOXB-AS3 ORF-Flag plasmid at the indicated doses and RNA affinity purification was performed as in (B) using biotin-labeled RNA E19 (50–68). (E and F) HTC-116 cells were transfected with the indicated hnRNP A1-truncated constructs (E) or hnRNP A1 AGG mutant (F) and RNA affinity purification was performed as in (B) using biotin-labeled RNA E19 (50–68). (G) HTC-116 cells were co-transfected with the indicated hnRNP A1 constructs together with HOXB-AS3 ORF-Flag vector, and RNA affinity purification was performed as in (B) using biotin-labeled RNA E19 (50–68). See also Figure S6. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 HOXB-AS3 Peptide Blocks the Increase of PKM2, the Decrease of PKM1, and Lactate Production Induced by hnRNP A1 Overexpression (A) HTC-116 cells were transfected with the indicated HOXB-AS3 vectors, followed by the PKM splicing assay outlined in Figure S7A. (B) HOXB-AS3 expression in HTC-116 cells was silenced by two anti-HOXB-AS3 siRNAs, followed by the PKM splicing assay. (C) HTC-116 cells were co-transfected with the indicated hnRNP A1 constructs together with HOXB-AS3 ORF-Flag vector, followed by the PKM splicing assay. (D–F) HTC-116 cells were co-transfected with the indicated HOXB-AS3 plasmid together with PKM2 vector; cell growth (D), colony formation (E), migration, and invasion (F) were determined. (G) The PKM1 and PKM2 mRNA levels were positively and negatively correlated with HOXB-AS3 peptide levels in six pairs of matched fresh CRC tissues and corresponding colon NT tissues, respectively. (H) HTC-116 cells were transfected with the indicated HOXB-AS3 vectors and lactate production was measured (n = 3). (I and J) HOXB-AS3 and hnRNP A1 expression in HTC-116 cells was silenced by two anti-HOXB-AS3 (I) and hnRNP A1 (J) siRNAs, respectively, and lactate production was measured (n = 3). (K and L) HTC-116 cells were co-transfected with the indicated hnRNP A1 (K) and PKM2 (L) constructs together with HOXB-AS3 ORF-Flag vector, and lactate production was measured (n = 3). The data are represented as the means ± SEM. See also Figure S7. Molecular Cell 2017 68, 171-184.e6DOI: (10.1016/j.molcel.2017.09.015) Copyright © 2017 Elsevier Inc. Terms and Conditions