Specific interference of urokinase-type plasminogen activator receptor and matrix metalloproteinase-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth, and angiogenesis in gliomas
Authors: Jasti S Rao
Velidi H Rao
Christopher S Gondi
Sajani S Lakka
Chrissa Sioka
Meena Gujrati
Dzung H Dinh
William C Olivero
Journal: Journal of Biological Chemistry (2005)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: adenovirus; antisense; uPAR; MMP-9; RNA interference
Affiliations: Departments of Biomedical and Therapeutic Sciences (Program of Cancer Biology), College of Medicine, University of Illinois, Peoria, IL 61656, USA.

Abstract:  We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy. 

Retracted: Yes

PubPeer comments:
 I have some questions about Figure 1. * Figure 1A, marked with red arrows: Lanes 1 through 5 all look unexpectedly similar * Figure 1A, marked with teal arrows: Lanes 6 through 9 all look unexpectedly similar * Figure 1B, marked with orange, pink, and green boxes: several lanes within the same gel appear to look unexpectedly similar. Could the authors provide some explanation on what might have happened here, please? ![file] , 
 Fig 5.  SNB19 cells spheroids (red fluorescence seen as dark gray) of 100u2013200 u03bcm in diameter were selected, transfected with EV SV, puPAR, puMMP-9 and pUM and co-cultured with fetal rat brain aggregates (green fluorescence seen as light gray). C, progressive destruction of fetal rat brain aggregates (FRBA) and invasion of SNB19 cells was observed for 72 h using confocal laser scanning microscopy. Original at left and black white reversed at right. ![file] I am marvelling how the changes in the aggregate spheroid combinations are limited to the region of contact between them, with no change at all in the morphology, illumination or focus of other regions over a period of 48 hours under the microscope. Shown here for the pMMP-9 and puPAR encounters, by superimposing the 24-hour reversed versions on the 72-hour versions. Invariant sections of each spheroid aggregate cancel out. ![file] , 
 Figure 6: The top right fluorescence H E combo representing vector pUM injected  intracerebrally into mouse brains. It is strikingly similar to a panel in Figure 4 of Lakka et al., Oncogene (2004) 23, 4681u20134689, doi: 10.1038 sj.onc.1207616. Also see: https: pubpeer.com publications 9D2B76E9A952E08E1DAE006B86A449 In Figure 4 of Lakka et al., Oncogene (2004) it represented vector pCM injected intraperitoneally. Here, in Figure 6 of Lakka et al. J Biol Chem (2005) it represents vector pUM injected  intracerebrally into mouse brains. ![file] , 
 J. Biol. Chem. 2020 295: 13136. doi:10.1074 jbc.RX120.015588 This article has been retracted by the publisher. Dzung H. Dinh, Velidi H. Rao, and Jasti S. Rao could not be reached for comment. Lanes 1u20135 of Fig. 1A are the same. Lanes 6u20139 of the same figure are the same. In Fig. 1B, lanes 2 and 4 of the uPAR panel are the same. Lane 1 of the uPAR panel was reused in lanes 1u20133 of the MMP-9 panel. Lane 3 of the uPAR panel was reused in lane 4 of the MMP-9 panel. The GAPDH panel in Fig. 1B was reused as GAPDH in the RNA pol II CMV panels in Fig. 2A. The fluorescent pUM image and the H E Mock EV and pUM images were reused in Lakka, S. S., et al. (2004) Oncogene 23, 4681u20134689. The pUM images in Fig. 6 (A and B) were reused in Lakka, S. S., et al. (2004) Oncogene 23, 4681u20134689. u00a9 2020 by The American Society for Biochemistry and Molecular Biology, Inc. ,