Nuclear lncRNA HOXD-AS1 suppresses colorectal carcinoma growth and metastasis via inhibiting HOXD3-induced integrin β3 transcriptional activating and MAPK/AKT signalling
Authors: Mei-Ling Ai
Li Zhao
Wen-Lu Li
Ting-Ting Li
Wen Ou-Yang
Shuang Wang
Sha-Sha Hu
Lan Wang
Yi-Qing Wang
Min-Hui Yang
Yue Han
Yan-Qing Ding
Journal: Molecular Cancer (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Colorectal carcinoma; HOXD-AS1; HOXD3; PRC2; lncRNA.
Affiliations: Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China., Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China., Department of Pathology, The Third Affiliated Hospital of Guangzhou Medical University, Ghuangzhou, 510150, China., The Second Clinical Medical College, Zhujang Hospital, Southern Medical University, Guangzhou, 510515, China., Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China. ., Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. .

Abstract:  Background: Long noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown. Methods: Real-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC. Results: We observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin β3 transcription, thereby activating the MAPK/AKT signalling pathways. Conclusion: Our results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin β3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression. 

Retracted: No

PubPeer comments:
 In Figure 5a, it looks like some of the tumors are not identified as tumor (red arrows). In addtion, many of the marked tumors (blue arrows) look strange, almost artificial, in the way they are formed, placed and colored. ![file] , 
 Dear Dr. Indigofera Tanganyikensis, Thank you for your enquiry. In our study, for in vivo tumorigenicity, a total of 5u00d710^6 tumor cells transfected with HOXD-AS1 vector or mock cells were subcutaneously injected into the right and left bilateral upper limbs of the same mice respectively (Fig.5a upper and middle panel). A total of 5u00d710^6 tumor cells with HOXD-AS1 and HOXD3 stable overexpression were subcutaneously injected into the right upper limbs of the other mice (Fig.5a lower panel). In the middle panel of Fig.5a, the unidentified tumors (red arrow) are actually the tumors formed by the mock cells in the left upper limbs, as indicated by the blue arrow in Fig.5a upper panel. In addition, to fully show the differences between the groups, we put six mice together to take photos. However, the tumor is three-dimensional, influenced by other factors, such as the orientation and skin thickness of the mice, it is difficult to fully show the appearance of the tumor in the two-dementional plane. As a result, we artificially marked the tumor margin using the dotted line. In fact, marking the tumor margin with the dotted line is just to highlight these tumors. The detailed pathological appearances of the tumors were shown in Fig.5b. , 
 Dear Dr. Indigofera Tanganyikensis, Thank you for your enquiry. In our study, for in vivo tumorigenicity, a total of 5u00d710^6 tumor cells transfected with HOXD-AS1 vector or mock cells were subcutaneously injected into the right and left bilateral upper limbs of the same mice respectively (Fig.5a upper and middle panel). A total of 5u00d710^6 tumor cells with HOXD-AS1 and HOXD3 stable overexpression were subcutaneously injected into the right upper limbs of the other mice (Fig.5a lower panel). In the middle panel of Fig.5a, the unidentified tumors (red arrow) are actually the tumors formed by the mock cells in the left upper limbs, as indicated by the blue arrow in Fig.5a upper panel. In addition, to fully show the differences between the groups, we put six mice together to take photos. However, the tumor is three-dimensional, influenced by other factors, such as the orientation and skin thickness of the mice, it is difficult to fully show the appearance of the tumor in the two-dementional plane. As a result, we artificially marked the tumor margin using the dotted line. In fact, marking the tumor margin with the dotted line is just to highlight these tumors. The detailed pathological appearances of the tumors were shown in Fig.5b. ,