Clinical and Translational MedicineVolume 12, Issue 1 e709 LETTER TO EDITOROpen Access Dysregulation of the miR-16-WWP1 signalling pathway leads to colorectal tumorigenesis Xiaorui Chen, Xiaorui Chen Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYi Zhao, Yi Zhao Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorQing Zhu, Qing Zhu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYanqing Liu, Yanqing Liu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYang Luo, Yang Luo Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorWei Cheng, Wei Cheng Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorBohan Zhang, Bohan Zhang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorKai Wang, Kai Wang Department of Gastrointestinal Surgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, ChinaSearch for more papers by this authorXiaohong Jiang, Xiaohong Jiang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorRui Liu, Rui Liu National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, ChinaSearch for more papers by this authorYanbo Wang, Corresponding Author Yanbo Wang ybwang@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorZhen Zhou, Corresponding Author Zhen Zhou zhenzhou@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorXi Chen, Corresponding Author Xi Chen xichen@nju.edu.cn orcid.org/0000-0002-5807-4219 Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this author Xiaorui Chen, Xiaorui Chen Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYi Zhao, Yi Zhao Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorQing Zhu, Qing Zhu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYanqing Liu, Yanqing Liu Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorYang Luo, Yang Luo Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorWei Cheng, Wei Cheng Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorBohan Zhang, Bohan Zhang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorKai Wang, Kai Wang Department of Gastrointestinal Surgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, ChinaSearch for more papers by this authorXiaohong Jiang, Xiaohong Jiang Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, ChinaSearch for more papers by this authorRui Liu, Rui Liu National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, ChinaSearch for more papers by this authorYanbo Wang, Corresponding Author Yanbo Wang ybwang@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorZhen Zhou, Corresponding Author Zhen Zhou zhenzhou@nju.edu.cn Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this authorXi Chen, Corresponding Author Xi Chen xichen@nju.edu.cn orcid.org/0000-0002-5807-4219 Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China State Key Laboratory of Reproductive Medicine, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Research Unit of Extracellular RNA, Chinese Academy of Medical Sciences, Nanjing, China Correspondence Xi Chen, Zhen Zhou and Yanbo Wang, Nanjing Drum Tower Hospital Center of Molecular Diagnostic and Therapy, State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute of Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China. Email: xichen@nju.edu.cn, zhenzhou@nju.edu.cn and ybwang@nju.edu.cnSearch for more papers by this author First published: 26 January 2022 https://doi.org/10.1002/ctm2.709 Xiaorui Chen, Yi Zhao, and Qing Zhu contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Dear Editor, Colorectal cancer (CRC) is one of the leading malignant tumour-related causes of death worldwide.1 WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a ubiquitin protein ligase2-4 and a potential oncogene in many cancer types.5-8 There is little work to explain the mechanisms by which WWP1 is regulated during tumorigenesis, particularly in CRC. We first determined the expression pattern of WWP1 by performing immunohistochemistry (IHC) in a commercial tissue microarray containing 90 pairs of CRC and adjacent normal tissue (ANT) specimens (Figure 1A; Table S1). The percentage of WWP1-positive cells was dramatically higher in CRC specimens compared with ANT specimens (Figure 1B,C). In addition, WWP1 protein levels were positively associated with clinical grades of CRC (Figure 1D). Kaplan–Meier survival analysis showed an increased risk of CRC-related deaths in patients with higher WWP1 protein levels (Figures 1E). We then confirmed WWP1 protein levels were consistently upregulated in 22 pairs of CRC specimens compared with ANT specimens (Figure 1F,G). FIGURE 1Open in figure viewerPowerPoint The expression pattern of WWP1 in colorectal cancer (CRC) tissues compared with adjacent normal tissue (ANT) specimens. (A) Image of immunohistochemistry (IHC) staining of WWP1 protein in human CRC tissue microarrays. (B) Representative images of IHC staining of WWP1 protein in CRC tissue microarrays. Scale bar, 100 μm. (C) IHC scores of WWP1 staining in 90 pairs of CRC and ANT specimens (n = 90 per group). (D) IHC scores of WWP1 staining in CRC tissue specimens with different grades (n = 10, 46 and 34, respectively). (E) Kaplan–Meier curves were generated to analyse the association of WWP1 protein levels in CRC tissue specimens with the overall survival of CRC patients. (F) Western blot analysis of the expression levels of WWP1 protein in 22 pairs of CRC and ANT specimens. (G) Densitometry analysis of the immunoblots from panel F (n = 22 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 To investigate whether WWP1 could regulate CRC cell proliferation and migration, we transfected a WWP1 overexpression plasmid into SW480 cells. As expected, a dose-dependent increase in WWP1 protein was observed following transfection with an increasing dose of the WWP1 plasmid; conversely, WWP1 protein expression was inhibited by the transfection of WWP1 siRNA (Figure 2A,B). Both the CCK-8 and EdU assays revealed that WWP1 siRNA could decrease the cell proliferation rate, while the WWP1 overexpression plasmid augmented it (Figure 2C–E). Likewise, transfection of WWP1 siRNA suppressed cell migration in transwell assay, while transfection of the WWP1 overexpression plasmid stimulated it (Figure 2F,G). FIGURE 2Open in figure viewerPowerPoint WWP1 functions as an oncogene and is conversely correlated with miR-16 in colorectal cancer (CRC). (A) Western blot analysis of WWP1 protein levels in SW480 cells transfected with control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid (0.5, 1.0 or 1.5 μg). (B) Densitometry analysis of the immunoblots from panel A (n = 3 per group). (C) The cell proliferation ability was analysed using the CCK-8 assay after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid (n = 3 per group). (D) The EdU proliferation assay was performed 24 h after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (E) Quantitative analysis of EdU-positive cells in panel D (n = 3 per group). (F) The cell migration ability was analysed using a transwell assay after the transfection of SW480 cells with equal doses of control siRNA, WWP1 siRNA, control plasmid or WWP1 overexpression plasmid. (G) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes (n = 3 per group). (H) Quantitative RT-PCR analysis of the relative expression levels of WWP1 mRNA in CRC and ANT specimens (n = 22 per group). (I) Schematic description of the predicted duplexes formed by miR-16 and the 3′-UTR of WWP1 mRNA. The predicted free energy value of the hybrid and the seed recognition are indicated, and all nucleotides in this region are highly conserved across species. (J) Quantitative RT-PCR analysis of the relative expression levels of miR-16 in CRC and ANT specimens (n = 22 per group). (K) Pearson's correlation scatter plot of the fold changes of WWP1 protein and miR-16 in CRC samples. (L) Western blot analysis of WWP1 protein levels in SW480, HT29 and HCT116 cells transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor. (M) The relative luciferase activity in 293T transfected with wild type or mutant WWP1 3′-UTR (n = 3 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 We explored how the WWP1 protein was regulated in CRC. No significant change in WWP1 mRNA levels and low correlation between WWP1 protein and mRNA levels were detected in the above-mentioned 22 pairs of CRC samples (Figure 2H; Figure S1), suggesting a posttranscriptional mechanism for the regulation of WWP1. Because miRNAs serve as vital posttranscriptional regulators in various cancers, we speculated that some miRNAs might target WWP1 in CRC. miR-16 was predicted to be an upstream regulator of WWP1 by two bioinformatic algorithms TargetScan and miRcode. The 3′-UTR of WWP1 possesses a conserved miR-16 binding site with a suitable value of minimum free energy of typical miRNA-target pairs (Figure 2I). Consistently, miR-16 levels were downregulated in the above-mentioned 22 pairs of CRC samples (Figure 2J). Pearson's correlation scatter plots confirmed the negative correlation between WWP1 protein and miR-16 (Figure 2K). We assessed the alteration of WWP1 protein in three CRC cell lines (SW480, HT29, and HCT116) by transfecting miR-16 mimic or inhibitor. As expected, the cellular level of miR-16 altered dramatically after transfection (Figure S2A). Consequently, WWP1 protein expression was markedly reduced by miR-16 transfection, while miR-16 inhibitor promoted WWP1 protein expression in CRC cells (Figure 2L; Figure S2B). However, the WWP1 mRNA level was not altered by miR-16 (Figure S2C). Furthermore, we determined whether miR-16 directly binds to the WWP1 3′-UTR via luciferase reporter assay. Luciferase reporter activity was dramatically altered when miR-16 level was changed while the mutated luciferase reporter was no longer affected by miR-16 (Figure 2M). We investigated the cellular phenotypes mediated by the miR-16-WWP1 axis. Cell proliferation rate was significantly reduced by miR-16 mimic, whereas miR-16 inhibitor markedly boosted proliferation (Figure 3A,C,F). The WWP1 overexpression plasmid was sufficient to rescue the suppression of cell proliferation by miR-16 (Figure 3B,D,F). Furthermore, miR-16 mimic markedly decreased cell migration ability, whereas miR-16 inhibitor increased the number of migrated cells (Figure 3E,G). WWP1 overexpression dramatically attenuated miR-16-mediated suppression on cell migration (Figure 3E,G). Likewise, miR-16 mimic decreased cell invasion; in contrast, miR-16 inhibition had an opposite effect on invasion (Figure S3). Thus, miR-16 may regulate proliferation, migration, and invasion in a WWP1-dependent manner. FIGURE 3Open in figure viewerPowerPoint The effects of miR-16 on WWP1 expression and function in vitro and in vivo. (A) Cell proliferation ability was analysed using the CCK-8 assay after the transfection of SW480 cells with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor (n = 3 per group). (B) Cell proliferation ability was analysed using the CCK-8 assay after the cotransfection of SW480 cells with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid (n = 3 per group). (C) The EdU proliferation assay was performed after the transfection of SW480 cells with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (D) The EdU proliferation assay was performed after the cotransfection with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (E) Cell migration ability was analysed using a transwell assay after the transfection with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or were cotransfected with control mimic + control plasmid, miR-16 mimic + control plasmid, control mimic + WWP1 overexpression plasmid, or miR-16 mimic + WWP1 overexpression plasmid. (F) Quantitative analysis of EdU-positive cells in panels C and D (n = 3 per group). (G) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes (n = 3 per group). (H) Representative images of the excised tumours. SW480 cells were infected with control LV or a lentivirus overexpressing miR-16 (LV-miR-16) or were cotransfected with LV-miR-16 and WWP1 overexpression plasmid. Then, the cells were implanted subcutaneously into four-week-old SCID male mice. Tumour growth was evaluated at day 24 after cell implantation. (I) Representative images of H&E-stained sections of xenografted tumours and representative images of IHC staining for WWP1 and Ki-67 in xenografted tumours. (J) Quantitative analysis of IHC staining for WWP1 and Ki-67 (n = 5 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 We investigated the effects of miR-16-WWP1 axis on CRC tumour growth in vivo. An SW480 cell line with stably knockdown of WWP1 by lentivirus (LV-shWWP1) was implanted into SCID mice. Additionally, SW480 cells infected with control lentivirus or miR-16 overexpression lentivirus (LV-miR-16) or co-infected with LV-miR-16 and the WWP1 overexpression vector were also implanted into SCID mice. Tumour growth was markedly decreased in the LV-shWWP1 group compared to the control lentivirus group (Figure S4A,B). Slower mitosis, reduced malignancy, lower level of WWP1, and the proliferation marker gene Ki-67 were observed in the LV-shWWP1 group by H&E staining and immunohistochemical staining (Figure S4C,D). LV-miR-16 clearly reduced tumour growth in vivo, whereas the WWP1 overexpression plasmid attenuated this effect (Figure 3H; Figure S5). Western blotting and immunohistochemical staining showed decreased WWP1 protein level in the LV-miR-16 group and rescued expression of WWP1 in the co-infection group (Figure 3I,J; Figure S6). H&E staining showed that LV-miR-16 caused slower mitosis and less malignancy, but the WWP1 overexpression plasmid neutralized this effect (Figure 3I). The Ki-67 level was decreased in tumours from the LV-miR-16 group but was restored in the co-infection group (Figure 3I,J). These results demonstrated that CRC tumour growth could be inhibited in vivo by miR-16 via targeting WWP1. Moreover, we investigated the relationship between miR-16 and another important regulatory miRNA of WWP1, miR-452, in CRC cells. miR-452 could target and repress WWP1 translation in two CRC cell lines (Figure 4A–C). However, miR-452 and miR-16 did not display synergism in vitro (Figure 4D–I). FIGURE 4Open in figure viewerPowerPoint The effects of miR-452 on WWP1 expression and function in colorectal cancer (CRC) cells. (A) Quantitative RT-PCR analysis of the relative expression levels of miR-452 in SW480 and HT29 cells transfected with control mimic, miR-452 mimic, control inhibitor or miR-452 inhibitor (n = 3 per group). (B) Western blot analysis of WWP1 protein levels in SW480 and HT29 cells transfected with control mimic, miR-452 mimic, control inhibitor or miR-452 inhibitor. (C) Densitometry analysis of the immunoblots of WWP1 protein from panel B (n = 3 per group). (D) Western blot analysis of WWP1 protein levels in SW480 and HT29 cells transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfected with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. (E) Densitometry analysis of the immunoblots of WWP1 protein from panel D (n = 3 per group). (F) The Edu proliferation assay was performed after transfection with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfection with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all of the cells. (G) Cell migration ability was analysed using a transwell assay after transfected with control mimic, miR-16 mimic, control inhibitor or miR-16 inhibitor or co-transfected with miR-16 mimic + miR-452 mimic or miR-16 inhibitor + miR-452 inhibitor. (H) Quantitative analysis of EdU-positive cells in panel F (n = 3 per group). (I) Quantitative analysis of the cells that migrated to the bottom of the transwell membranes in panel G (n = 3 per group). Data are shown as the means ± SEMs. *p < .05; **p < .01; ***p < .001 Overall, we showed that miR-16 can target WWP1 to suppress CRC tumorigenesis and that downregulation of miR-16 in CRC abolishes this repression of WWP1, resulting in more malignant tumour features. Our findings also have clinical relevance because targeting WWP1 protein with miR-16 has the potential to inhibit cellular proliferation and migration for CRC treatment. Indeed, the ubiquitin-proteasome system has become a popular target for developing therapeutics against tumors.9 Our results strongly support this promising strategy because overexpression of miR-16 or silencing of WWP1 strongly inhibits tumour growth in vivo. Future studies are needed to evaluate the therapeutic efficacy of WWP1 inhibition (e.g., with miR-16 or siRNA) in disrupting target protein ubiquitination and inducing antitumor activity. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 32022015 and 31871295), the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (No. SN-ZJU-SIAS-008), the CAMS Innovation Fund for Medical Sciences (No. CIFMS-2021-I2M-5-015), and the Fundamental Research Funds for the Central Universities (No. 020814380146 and 020814380162). CONFLICT OF INTEREST The authors declare no conflict of interest. Supporting Information Filename Description ctm2709-sup-0001-SuppMat.docx35.7 KB SUPPORTING INFORMATION ctm2709-sup-0002-tablesS1.xlsx28.7 KB SUPPORTING INFORMATION ctm2709-sup-0003-FigureS1-S7.pdf1.1 MB SUPPORTING INFORMATION Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Siegel RL, Miller KD, Jemal A. 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CrossrefCASWeb of Science®Google Scholar Volume12, Issue1January 2022e709 FiguresReferencesRelatedInformation

Background We previously report that yes-associated protein (YAP), the core downstream effector of Hippo pathway, promotes the malignant progression of glioblastoma (GBM). However, although classical regulatory mechanisms of YAP are well explored, how YAP is modulated by the Hippo-independent manner remains poorly understood. Meanwhile, the nonreceptor tyrosine kinase Fyn-related kinase (FRK), which exhibits low expression and possesses tumor suppressor effects in GBM, is reported to be involved in regulation of protein phosphorylation. Here, we examined whether FRK could impede tumor progression by modulating YAP activities. Methods Human GBM cells and intracranial GBM model were used to assess the effects of FRK and YAP on the malignant biological behaviors of GBM. Immunoblotting and immunohistochemistry were used to detect the expression of core proteins in GBM tissues. Co-immunoprecipitation, proximity ligation assay, luciferase assay and ubiquitination assay were utilized to determine the protein-protein interactions and related molecular mechanisms. Results The expression levels of FRK and YAP were inversely correlated with each other in glioma tissues. In addition, FRK promoted the ubiquitination and degradation of YAP, leading to tumor suppression in vitro and in vivo. Mechanistically, FRK interacted with and phosphorylated YAP on Tyr391/407/444, which recruited the classical E3 ubiquitin ligase Siah1 to catalyze ubiquitination and eventually degradation of YAP. Siah1 is required for YAP destabilization initiated by FRK. Conclusions We identify a novel mechanism by which FRK orchestrates tumor-suppression effect through phosphorylating YAP and inducing its ubiquitination by Siah1. FRK-Siah1-YAP signaling axis may serve as a potential therapeutic target for GBM treatment.

Due to the hypoxia and nutrient deficiency microenvironment, malignant glioma exhibits high autophagy activity and autophagy plays a significant role in the occurrence and development of glioma. However, the potential molecular mechanism of autophagy in glioma remains unknown. In this study, we demonstrated that Golgi phosphorylation protein 3 (GOLPH3), a highly conserved protein basically concentrates in the trans-Golgi network, promoted glioma autophagy. Inhibiting autophagy by using chloroquine suppressed the stimulating effect of GOLPH3 on glioma malignant development both in vitro and in vivo. Mechanistically, GOLPH3 interacted with and recruited prohibitin-2 (PHB2), an autophagy receptor of mitochondrion, and LC3-II. PHB2 promoted cell autophagy and down-regulation of PHB2 abolished the effect of GOLPH3 on autophagy. On the side, the relative mRNA and protein levels of GOLPH3 and PHB2 were positively associated with each other and both also correlated with autophagy in glioma tissues. Together, our results revealed that GOLPH3 promotes glioma progression by enhancing PHB2-mediated autophagy and inhibiting autophagy may benefit glioma patients with GOLPH3 high level. The novel GOLPH3-PHB2-autophagy axis maybe a potential and prospective therapeutic target for gliomas.

Glioma is the most common primary cancer in the central nervous system. Despite advances in surgery, radiotherapy and chemotherapy over the past decades, the prognosis of glioblastoma patients remains poor. We aim to identify robust gene signatures to better understand the complex molecular mechanisms and to discover potential novel molecular biomarkers for glioma. By exploring GSE16011, GSE4290 and GSE50161 data in Gene Expression Omnibus (GEO) database, we screened out 380 differentially expressed genes between non-tumor and glioma tissues, and further selected 30 hub genes through the Molecular Complex Detection (MCODE) plug-in in Cytoscape. In addition, LMNB1 and DLGAP5 were selected for further analyses due to their high expression in gliomas and were verified by using our cohort. Our study confirmed that LMNB1 and DLGAP5 were up-regulated in gliomas, and patients with high expression of LMNB1 or DLGAP5 had poor survival rate. Furthermore, silence of LMNB1 and DLGAP5 inhibited the proliferation of glioma cells. Together, LMNB1 and DLGAP5 were two potentially novel molecular biomarkers for diagnosis and prognosis of glioma.

Although radiotherapy is a well-known effective non-surgical treatment for malignant gliomas, the therapeutic efficacy is severely limited due to the radioresistance of tumor cells. Previously, we demonstrated that Yes-associated protein (YAP) promotes glioma malignant progression. However, whether YAP plays a role in radioresistance and its potential value in cancer treatment are still unclear. In this study, we found that high YAP expression is associated with poor prognosis in malignant glioma patients undergoing radiotherapy. Research in immortalized cell lines and primary cells from GBM patients revealed that YAP exhibited a radioresistant effect on gliomas via promoting DNA damage repair. Mechanistically, after radiation, YAP was translocated into the nucleus, where it promoted the expression and secretion of FGF2, leading to MAPK-ERK pathway activation. FGF2 is a novel target gene of YAP. Inhibition of YAP-FGF2-MAPK signaling sensitizes gliomas to radiotherapy and prolongs the survival of intracranial cell-derived and patient-derived xenograft models. These results suggest that YAP-FGF2-MAPK is a key mechanism of radioresistance and is an actionable target for improving radiotherapy efficacy.

Patients with malignant glioma often suffered from depression, which leads to an increased risk of detrimental outcomes. Imipramine, an FDA-approved tricyclic antidepressant, has been commonly used to relieve depressive symptoms in the clinic. Recently, imipramine has been reported to participate in the suppression of tumour progression in several human cancers, including prostate cancer, colon cancer and lymphomas. However, the effect of imipramine on malignant glioma is largely unclear. Here, we show that imipramine significantly retarded proliferation of immortalized and primary glioma cells. Mechanistically, imipramine suppressed tumour proliferation by inhibiting yes-associated protein (YAP), a recognized oncogene in glioma, independent of Hippo pathway. In addition to inhibiting YAP transcription, imipramine also promoted the subcellular translocation of YAP from nucleus into cytoplasm. Consistently, imipramine administration significantly reduced orthotopic tumour progression and prolonged survival of tumour-bearing mice. Moreover, exogenous overexpression of YAP partially restored the inhibitory effect of imipramine on glioma progression. Most importantly, compared with imipramine or temozolomide (TMZ) monotherapy, combination therapy with imipramine and TMZ exhibited enhanced inhibitory effect on glioma growth both in vitro and in vivo, suggesting the synergism of both agents. In conclusion, we found that tricyclic antidepressant imipramine impedes glioma progression by inhibiting YAP. In addition, combination therapy with imipramine and TMZ may potentially serve as promising anti-glioma regimens, thus predicting a broad prospect of clinical application.

The roles of lncRNAs in cardiac diseases have received increasing attention. The biological role of taurine upregulated gene 1 (TUG 1) in hypoxia-induced damage of cardiomyocytes is still poorly defined. Our study aimed to investigate the function of TUG 1 in hypoxia-treated cardiomyocytes and the possible underlying mechanism. TUG 1 and miR-133a expression levels in hypoxia-cultured human AC16 cardiomyocytes were examined by RT-qPCR. The role of TUG 1 and miR-133a in cell proliferation was assayed by CCK-8 assay. AC16 cell apoptosis was assessed by flow cytometry and caspase-3/7 activity assay. The expression levels of cleaved poly ADP ribose polymerase (PARP) and cleaved caspase-3 were evaluated by Western blot analysis. We found that TUG 1 expression was elevated, while miR-133a expression was reduced under hypoxic condition in AC16 cells. TUG 1 silencing and miR-133a restoration relieved hypoxia-induced reduction of proliferation as well as repressed hypoxia-induced AC16 cell apoptosis, while the opposite effects were observed after TUG 1 overexpression and miR-133a inhibition. We identified that TUG 1 acted as a competing endogenous RNA to suppress miR-133a expression. Mechanistically, miR-133a overturned TUG 1 overexpression-mediated inhibition of proliferation and promotion on apoptosis in AC16 cells under hypoxic condition. Conversely, inhibition of miR-133a abolished TUG 1 knockdown-mediated promotion of proliferative ability and repression of apoptosis in hypoxia-cultured AC16 cells. In conclusion, TUG 1 knockdown relieved hypoxia-induced reduction of proliferation and repressed hypoxia-induced AC16 cell apoptosis by up-regulating miR-133a expression.

Dysregulated lncRNAs are proposed to be tightly associated with the progression of various tumors including glioblastoma (GBM). LncRNA Survival Associated Mitochondrial Melanoma-Specific Oncogenic Non-Coding RNA (SAMMSON) has been reported to be an oncogenic lncRNA in several tumors. Nevertheless, the specific role and molecular mechanism of SAMMSON in GBM progression remain unknown. Expression of SAMMSON in GBM tissues and cells was detected by qRT-PCR. CCK-8 and LDH release assays were applied to evaluate cellular viability. Invasion effect was assessed by Transwell invasion assay and western blot analysis of E-cadherin and N-cadherin expression. Apoptosis was detected using flow cytometry analysis and caspase-3 activity assay. The protein levels of phosphatidylinositol-3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt) and p-Akt were estimated by western blot. We found that SAMMSON was highly expressed in GBM tissues and cells. SAMMSON knockdown suppressed cell viability and increased LDH release in GBM cells. Moreover, SAMMSON silencing impeded the invasive ability of GBM cells by regulating epithelial-to-mesenchymal transition (EMT). Furthermore, SAMMSON downregulation increased the apoptotic rate and caspase-3 activity in GBM cells. Additionally, it was demonstrated that the PI3K/Akt pathway was inhibited following SAMMSON silencing in GBM cells. Rescue assays revealed that activation of the PI3K/Akt pathway by 740Y-P abolished SAMMSON knockdown-induced viability reduction, invasion suppression and apoptosis in GBM cells. Taken together, lncRNA SAMMSON knockdown inhibited the malignancy of GBM cells by inactivation of the PI3K/Akt pathway.

FBXW7 is a potential tumor suppressor that regulates ubiquitination and proteolysis of multiple targets such as cyclin E, c-Myc, c-Jun and Notch. However, little knows about the correlation between FBXW7 and prognosis of patients with colorectal cancer (CRC). In this study, we detected FBXW7 expression in CRC tissue microarray which includes 568 cases cancer tissue and their paired adjacent non-cancerous tissues. We found that FBXW7 expression was significantly reduced in CRC tissues versus paired normal colon tissues (P < 0.001). Moreover, low FBXW7 expression was significantly associated with increased lymph node metastasis (P < 0.001) and advanced TNM stage (P < 0.001). Besides, the low expression of FBXW7 indicated the poor prognosis in CRC patients for both overall and disease-free cumulative survival (P < 0.001 and P = 0.003, respectively). Multivariate Cox regression analysis showed that low FBXW7 expression was an independent unfavorable prognostic factor of CRC (hazard ratio = 0.45, P = 0.001). In conclusion, we can indicate that FBXW7 may play essential roles in the progression of CRC and function as an independent prognostic marker for clinical diagnosis and therapy treatment of patients with CRC.

Objective To investigate the association between susceptibility to colorectal cancer (CRC) and a 4-bp insertion/deletion polymorphism (rs10680577) in the proximal promoter of the EGLN2 gene. Method The first step in genotyping EGLN2 was PCR, then the PCR products were separated using 7% nondenaturing polyacrylamide gel electrophoresis and visualized by silver staining according to the final product band location and quantity to determine the genotype of the sample. The final count was done by two different pathologists. Result In the codominant model, compared with the ins/ins genotype, subjects with the heterozygous ins/del or homozygous del/del genotype had a significantly increased risk of CRC (adjusted OR = 1.45, p<0.0001 and OR = 2.44, p = 0.0001, respectively). Each additional copy of the 4-bp deletion allele conferred a significantly increased risk of CRC (OR = 1.47, 95% CI 1.28-1.66, p<0.0001). In the stratification analysis, we further proved that the association was more prominent in TNM stage III and IV cancer compared with stage I and II (adjusted OR = 1.43, 95% CI 1.07-1.93, p for heterogeneity = 0.02). Conclusions Our study provided initial evidence that the insertion/deletion polymorphism rs10680577 may play a functional role in the development of CRC in the Chinese population.