| Objective: To explore the influence of biological behavior of tumor cells aftersilencing the epidermal growth factor receptor2(HER2) gene by using specificHER2-shRNA lentiviral vector to infect breast cancer cell line SKBR-3in vitro, and toresearch the gene therapy in vivo by xenograft model derived from SKBR-3cells stablytransfected with Her2targeting shRNA, and to determine feasibility of SPECT imagingmodalities for in vivo detection of RNAi effects in order to explore the prospect of SPECTin gene therapy.Methods:①The breast cancer cell line SKBR-3was infected with effectivelentivirus-mediated HER2-short hairpin (sh) RNA expression vector and scrambled controllentivirus vector respectively. Stable infection was screened by puromycin.②Theexpression of HER2mRNA and protein were detected by Real time PCR, flowcytometry(FCM) and Western blot respectively. Cell apoptosis were detected by FCM.MTT assay, Transwell cell invasion assay and scratch experiment were used respectively todetect the ability of cell proliferation, invasion and migration,③Both stably infectedcells were inoculated into nude mice to establish two types of breast cancer xenograftmodels, which were named KD group and NC group. The uninfected SKBR-3mousexenograft model served as CON group. The growth status of nude mice and the sizes ofxenografts in each group were recorded at different time. The expression of HER2proteinwas detected by immunohistochemistry.④T/B (tumor/background) ratios werecalculated by SPECT radioimmunoimaging with131I-Herceptin for breast cancer nudemice.Results:①Stably infected cells were obtained by puromycin resistance screening.FCM results showed that the expression rate of fluorescence was above80%in each group. ②Real time PCR analysis showed the HER2mRNA relative content (HER2/β-actin) ofCON, NC and KD group were1.000,1.081and0.326, respectively. The difference wasstatistically significant (P <0.01). The Her2mRNA expression of KD group was inhibitedby67.4%. In addition, the expression of HER2protein of KD group by FCM was (55.13±1.65)%, compared to (92.40±2.01)%of CON group and (92.97±2.44)%of NC group(P＜0.01), and the inhibition rate of HER2protein was (40.58±0.96)%; Western blot showedthat the relative expression of the HER2protein were (0.5481±0.0102),(0.5494±0.0135)and (0.2784±0.0099) respectively. The difference was statistically significant (P <0.01).The inhibition of cell proliferation was demonstrated by slower growth rate in theexperimental group compared to the negative control using MTT assay (P＜0.01);HER2-shRNA caused an increase in early apoptosis, with the early apoptotic index at (12.8±0.82)%, compared to (2.67±0.15)%caused by sc-shRNA (P＜0.01); Transwell and cellscratch experiments showed that HER2-shRNA caused significant decrease in cell invasionand migration ability.③Her2knockdown in breast cancer xenograft reduced the tumorsize and weight (P＜0.05), compared to the control group. The immunohistochemicalresults further confirmed that the HER2protein expression was significantly inhibited inthe experimental group; The radioactivity in tumor site accumulated gradually with thetime after131I-Herceptin was injected through tail vein, and the adjusted ratio of T/B in KDgroup at different times was significantly lower than the CON and NC group (P＜0.05)except for that of the first time.Conclusion: The HER2-shRNA lentiviral vector can significantly inhibit theexpression of HER2gene and impair the malignant biological behaviors of breast cancercell SKBR-3.The molecular imaging with131I-Herceptin may reflect HER2proteinexpression in vivo, and provide a feasible experimental data for assessing and monitoringHER2targeted RNA interference. |
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