Effects Of RNAi Silencing Targeting MMP-2Gene On Growth Of Esophageal Carcinoma Cell In Vivo

AimEsophageal carcinoma is a malignant tumor with Chinese characteristics. In Taihang Mountain area of China, the incidence of esophageal carcinoma ranks first in the world. Although surgery, radiotherapy, chemotherapy and other treatment methods have achieved greater development in recent years, the overall long-term survival in patients with esophageal carcinoma hasn’t made significant improvement. Cancer progression is a complex and multi-step process involving multiple genes changes. MMP-2is believed to play an important role in the development process in a variety of malignant tumors. In this study, by constructing the lentiviral expression vector-mediated siRNA silencing of MMP-2gene, we observed tumor cell growth suppressive effects in human KYSE150esophageal cancer cells in vivo, and provided experimental basis for gene therapy of esophageal cancer.Method1. Constructing the lentiviral expression vector of MMP-2gene:Three specific siRNA interference sequences targeting MMP-2were designed. The siRNA sequences were verified for specificity by BLAST searches against the human genome. The chosen sense sequence is then linked to the antisense sequence with an intervening loop sequence. These three sequences and a negative contral sequence were cloned in lentiviral vectors. DNA sequence analysis was done to confirm the sequences of the recombinant plasmids. To selecte effective interference sequence targeted to MMP-2gene in KYSE150cell, recombinant plasmids were transfected into KTSE150. MMP-2mRNA and protein were detected, and the best inhibited interference sequence was selected for following experiments.2. Cell viability assay:Cell viability was assessed by MTT assay.3. Nude mice pre-experiment:1mice underwent subcutaneous injection of0.1ml cell suspension of KYSE150cell (5X106) in the right flank area of the mice4. The effect of MMP-2expressive inhibition on human esophageal carcinoma cell line KYSE150in vivo:24Athymic mice were randomly divided into three groups with8mices per group:blank control, negative control and siRNA-lentivirus group. KYSE150cells also divided three groups as mentioned above. KYSE150cells were transfected and then were injected s.c. into the right flank area of the mice. Tumor volumes were determined by direct measurement with calipers and calculated using the formula:V=1/2×(long diameter)×(short diameter). Carcinoma development was observed. Athymic mice were exeeuted after30days, and carcinoma tissues were separated subcutaneously. The size and weight of carcinoma were compared.Results1. Three lentiviral vectors of shRNA targeted MMP-2was construced and proved by DNA sequencing.2. For MMP-2-shRNA-1, MMP-2-shRNA-2and MMP-2-shRNA-3, the inhibitory rates of MMP-2Mrna expression were40.5%,41.4%and78.9%(p<0.05). Western blotting detected a similar inhibition of MMP-2protcin levels in cells transfected with shRNA-1, shRNA-2and shRNA-3vectors (p<0.05). Both RT-PCT and western blotting analyses showed the3rd siRNA target sequence was the most effective on MMP-2silencing.3. The titer of this lentiviral stock was determined by serial dilution methods, and the result was4.26×108TU/ml。4. After MMP-2-RNAi-Lentivirus transfection, the viability of KYSE150cells was evidently decreased compared with the blank control group. However, the viability of negative group did not show significant alteration compared with the blank control group (p<0.001)5. The model animals of human esophagus tumor-bearing mice were established suecessfully. As compared with negative control and blank control group, the tumor growth of treatment group was slow and the tumor volume and weight of treatment group decreased significantly (p<0.05).Conclusion1. RNAi could efficiently downregulate the MMP-2gene expression in esophageal carcinoma cell line KYSE150. RNAi could be a potential method for gene therapy.2. The mRNA and protein expression of MMP-2in human esophageal carcinoma celll KYSE150were specifically and efficiently inhibited by lentivirus-mediated siRNA expression vectors targeted to MMP-2gene, which proved the delivery of shRNA using lentiviral vectors was efficient.3. The viability assay indicated that MMP-2could inhibit the viability of KYSE150cells in vitro.4. Knockdown of MMP-2causes a statistically significant inhibition of the growth of the esophageal carcinoma cell line KYSE150in vivo.