| Objective: To make lentivirus based shRNA constructs which target the criticaloncogene Her2/neu. To determine the effect of modulating Her/neu expression on thegrowth and survival of the ovarian cancer cell line SKOV-3in vitro. To confirm in vivo therequirement of Her/neu oncogene in promoting tumor growth using a xenograft modelderived from SKOV-3cells stably transfected with Her2/neu targeting shRNA. To developa novel, non-invasive method using single-photon emission computed tomography(SPECT) to monitor the growth of Her2-neu positive ovarian cancer in mice, and toinvestigate the biologic effect of Her2/neu knockdown on tumor growth.Methods:①Three specific shRNA constructs were designed and clone into alentivirus expression vector. As a negative control, a scramble sequence was also cloned;②Targeting or control shRNA lentiviral constructs were first transfected into293Tpackaging cell using Lipofectamine2000, and the virus supernatant was collected to infectovarian carcinoma cell line SKOV-3. The lentivirus-mediated expression vector with thebest interference effect was screened by RT-PCR;③Stable transfection was selected bypuromycin resistance. Proliferation of SKOV-3derived cells was determined using theMTT assay. Apoptosis of SKOV-3derived cells was determined by flow cytometry (FCM).The expression of HER2/neu was confirmed by Western blot;④SKOV-3mouse xenograftmodels were established by subcutaneous injection of SKOV-3derived cells in the flank ofnude mice. Her2/neu expression in the tumors was determined by immunohistochemistry(IHC), and the size of the tumor in the experimental and control groups was measured as afunction of time to determine the tumor growth rate. Mice in the experimental and controlgroups were injected with131I-Herceptin, which avidly bind to Her2/neu. Radioactivity intumor sites was determined at different intervals following the injection using SPECT scan. Results：①Three shRNA constructs targeting Her2/neu (HER2-shRNA) were clonedsuccessfully, and were confirmed by sequencing.②HER2-shRNA, when transduced intoSKOV-3cells, effectively inhibited the expression of Her2/neu mRNA by82.7%, andHer2/neu protein by (57.9±0.09)%, compared to the non-targeting scramble shRNA(sc-shRNA).③The expression of HER2protein of the experimental group by flowcytometry (FCM) was (48.10±3.19)%, compared to (88.33±2.08)%caused by sc-shRNA(P＜0.05), and the inhibition rate of HER2protein was (53.22±0.04)%. HER2-shRNAcaused an increase in early apoptosis, with the early apoptotic index at (15.42±0.38)%,compared to (5.98±0.16)%caused by sc-shRNA (P＜0.01). HER2-shRNA prevented cellproliferation in the SKOV-3cell line, demonstrated by slower growth rate in theexperimental group compared to the negative control using MTT assay (P＜0.01).④Her2/neu knockdown in SKOV-3xenograft caused a delay in tumorigenic timeand reduced the tumor size, compared to the control group. The immunohistochemicalresults further confirmed that the HER2protein expression was significantly inhibition inthe experimental group. The radioactivity in tumor site appeared1hour after131I-Herceptinwas injected through tail vein, and accumulated gradually. The xenograft displayed a clearimaging. Her2/neu knockdown in SKOV-3xenograft is associated with a lower T/B ratiocompared to sc-shRNA, using SPECT in vivo imaging (P＜0.05).Conclusion: Lentivirus-mediated shRNA knockdown of Her2/neu was demonstratedto change the biologic behavior of the SKOV-3ovarian cancer model, both in vitro and invivo. Molecular imaging of Her2/neu positive tumor using131I-Herceptin acquired clearimage of SKOV-3xenograft by SPECT. Importantly, and the T/B ratio was lower in theHer2/neu knockdown mice than that in the control group, reflecting the differentexpression levels of Her2/neu between these groups. SPECT based molecular imaging isexpected to be an effective, noninvasive method to assess the effect of modulatingHer2/neu expression in vivo. |
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