RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo

Liu,Yong‑Chao; Ma,Wen‑Hui; Ge,Yin‑Lin; Xue,Mei‑Lan; Zhang,Zheng; Zhang,Jin‑Yu; Hou,Lin; Mu,Run‑Hong

doi:10.3892/ol.2016.5158

RNAi-mediated gene silencing of vascular endothelial growth factor C suppresses growth and induces apoptosis in mouse breast cancer in vitro and in vivo

Authors:
- Yong‑Chao Liu
- Wen‑Hui Ma
- Yin‑Lin Ge
- Mei‑Lan Xue
- Zheng Zhang
- Jin‑Yu Zhang
- Lin Hou
- Run‑Hong Mu
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Affiliations: Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao, Shandong 266021, P.R. China, Department of Immunology, Medical College, Beihua University, Jilin, Jilin 132013, P.R. China
Published online on: September 21, 2016 https://doi.org/10.3892/ol.2016.5158
Pages: 3896-3904
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Vascular endothelial cell growth factor (VEGF)-C promotes tumorigenesis by allowing lymph node metastasis and lymphangiogenesis, among other actions. RNA interference (RNAi) is a novel technique for suppressing target gene expression and may increase the effectiveness of cancer treatments. The present study assessed the influence of VEGF‑C RNAi on the apoptosis and proliferation of mouse breast cancer cells in vitro and in vivo. A total of three pairs of small interfering RNA (siRNA) targeting mouse VEGF‑C were designed and synthesized prior to transfection into 4T1 cells via a liposomal approach. Reverse transcription polymerase chain reaction, western blot analysis, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining and flow cytometry were performed in vitro to analyze VEGF‑C expression, cleaved caspase‑3 protein expression and 4T1 cell proliferation and apoptosis. Experiments were also conducted in vivo on BALB/c mice with breast cancer. Tumor weight and volume were measured and the number of apoptotic cells in tumor tissues was assessed by a TUNEL assay. Immunohistochemical assays and an enzyme‑linked immunosorbent assay were used to measure the expression of VEGF‑C in tumor tissues. The results demonstrated that the three pairs of siRNA, particularly siV2, significantly reduced VEGF‑C mRNA and protein levels in 4T1 cells. siV2 was deemed to be the most efficient siRNA and therefore was selected to be used in subsequent experiments. Furthermore, in vitro studies indicated that VEGF‑C RNAi significantly decreased cell growth, induced apoptosis and upregulated the expression of cleaved caspase‑3 protein. Tumor weight and volume in breast cancer in vivo models was reduced by the intratumoral injection of siV2. Antitumor efficacy was associated with decreased VEGF‑C expression and increased induction of apoptosis. The present study therefore indicated that VEGF‑C RNAi inhibited mouse breast cancer growth in vitro and in vivo and that it may be a novel targeted therapy for breast cancer.

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1	Montazeri A: Health-related quality of life in breast cancer patients: A bibliographic review of the literature from 1974 to 2007. J Exp Clin Cancer Res. 27:322008. View Article : Google Scholar : PubMed/NCBI
2	Jemal A, Siegel R, Xu J and Ward E: Cancer statistics, 2010. CA Cancer J Clin. 60:277–300. 2010. View Article : Google Scholar : PubMed/NCBI
3	Nathanson SD: Insights into the mechanisms of lymph node metastasis. Cancer. 98:413–423. 2003. View Article : Google Scholar : PubMed/NCBI
4	Ran S, Volk L, Hall K and Flister MJ: Lymphangiogenesis and lymphatic metastasis in breast cancer. Pathophysiology. 17:229–251. 2010. View Article : Google Scholar : PubMed/NCBI
5	Joukov V, Kaipainen A, Jeltsch M, Pajusola K, Olofsson B, Kumar V, Eriksson U and Alitalo K: Vascular endothelial growth factors vegf-b and vegf-c. J Cell Physiol. 173:211–215. 1997. View Article : Google Scholar : PubMed/NCBI
6	Fournier E, Birnbaum D and Borg JP: Receptors for factors of the vegf (Vascular Endothelial Growth Family). Bull Cancer. 84:397–405. 1997.PubMed/NCBI
7	Deckers MM, Karperien M, van der Bent C, Yamashita T, Papapoulos SE and Löwik CW: Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology. 141:1667–1674. 2000. View Article : Google Scholar : PubMed/NCBI
8	Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, Chen HY, Hung MC and Kuo ML: The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer. 96:541–545. 2007. View Article : Google Scholar : PubMed/NCBI
9	Ge YL, Zhang X, Zhang JY, Hou L and Tian RH: The mechanisms on apoptosis by inhibiting VEGF expression in human breast cancer cells. Int Immunopharmacol. 9:389–395. 2009. View Article : Google Scholar : PubMed/NCBI
10	Kodama M, Kitadai Y, Tanaka M, Kuwai T, Tanaka S, Oue N, Yasui W and Chayama K: Vascular endothelial growth factor c stimulates progression of human gastric cancer via both autocrine and paracrine mechanisms. Clin Cancer Res. 14:7205–7214. 2008. View Article : Google Scholar : PubMed/NCBI
11	Kerr JF, Wyllie AH and Currie AR: Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26:239–257. 1972. View Article : Google Scholar : PubMed/NCBI
12	Hassan M, Watari H, AbuAlmaaty A, Ohba Y and Sakuragi N: Apoptosis and molecular targeting therapy in cancer. Biomed Res Int. 2014:1508452014. View Article : Google Scholar : PubMed/NCBI
13	Sun P, Gao J, Liu YL, Wei LW, Wu LP and Liu ZY: RNA interference (RNAi)-mediated vascular endothelial growth factor-C (VEGF-C) reduction interferes with lymphangiogenesis and enhances epirubicin sensitivity of breast cancer cells. Mol Cell Biochem. 308:161–168. 2008. View Article : Google Scholar : PubMed/NCBI
14	U.S. National Institutes of Health, . Laboratory animal welfare: Public Health Service policy on humane care and use of laboratory animals by awardee institutions; notice. Fed Regist. 50:19584–19585. 1985.PubMed/NCBI
15	Xue M, Ge Y, Zhang J, Liu Y, Wang Q, Hou L and Zheng Z: Fucoidan inhibited 4T1 mouse breast cancer cell growth in vivo and in vitro via downregulation of Wnt/β-catenin signaling. Nutr Cancer. 65:460–468. 2013. View Article : Google Scholar : PubMed/NCBI
16	Yan Y, Jia L, Zhang J, Liu Y and Bu X: Effect of recombinant Newcastle disease virus transfection on lung adenocarcinoma A549 cells in vitro. Oncol Lett. 8:2569–2576. 2014.PubMed/NCBI
17	Elbashir SM, Lendeckel W and Tuschl T: RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15:188–200. 2001. View Article : Google Scholar : PubMed/NCBI
18	Sioud M: RNA interference: Mechanisms, technical challenges, and therapeutic opportunities. Methods Mol Biol. 1218:1–15. 2015. View Article : Google Scholar : PubMed/NCBI
19	Mansoori B, Shotorbani S Sandoghchian and Baradaran B: RNA interference and its role in cancer therapy. Adv Pharm Bull. 4:313–321. 2014.PubMed/NCBI
20	Li JP, Cao NX, Jiang RT, He SJ, Huang TM, Wu B, Chen DF, Ma P, Chen L, Zhou SF, et al: Knockdown of gcf2/lrrfip1 by rnai causes cell growth inhibition and increased apoptosis in human hepatoma HepG2 cells. Asian Pac J Cancer Prev. 15:2753–2758. 2014. View Article : Google Scholar : PubMed/NCBI
21	Pan XD, Yang ZP, Tang QL, Peng T, Zhang ZB, Zhou SG, Wang GX, He B and Zang LQ: Expression and function of GSTA1 in lung cancer cells. Asian Pac J Cancer Prev. 15:8631–8635. 2014. View Article : Google Scholar : PubMed/NCBI
22	Joukov V, Sorsa T, Kumar V, Jeltsch M, Claesson-Welsh L, Cao Y, Saksela O, Kalkkinen N and Alitalo K: Proteolytic processing regulates receptor specificity and activity of VEGF-C. Embo J. 16:3898–3911. 1997. View Article : Google Scholar : PubMed/NCBI
23	Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E, Saksela O, Kalkkinen N and Alitalo K: A novel vascular endothelial growth factor, VEGF-C, is a ligand for the flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. Embo J. 15:290–298. 1996.PubMed/NCBI
24	Alitalo A and Detmar M: Interaction of tumor cells and lymphatic vessels in cancer progression. Oncogene. 31:4499–4508. 2012. View Article : Google Scholar : PubMed/NCBI
25	Kinoshita J, Kitamura K, Kabashima A, Saeki H, Tanaka S and Sugimachi K: Clinical significance of vascular endothelial growth factor-c (vegf-c) in breast cancer. Breast Cancer Res Treat. 66:159–164. 2001. View Article : Google Scholar : PubMed/NCBI
26	Chambers AF, Groom AC and MacDonald IC: Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2:563–572. 2002. View Article : Google Scholar : PubMed/NCBI
27	Duffy MJ, McGowan PM and Gallagher WM: Cancer invasion and metastasis: Changing views. J Pathol. 214:283–293. 2008. View Article : Google Scholar : PubMed/NCBI
28	Xu C, Gui Q, Chen W, Wu L, Sun W, Zhang N, Xu Q, Wang J and Fu X: Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo. J Exp Clin Cancer Res. 30:632011. View Article : Google Scholar : PubMed/NCBI
29	Decio A, Taraboletti G, Patton V, Alzani R, Perego P, Fruscio R, Jürgensmeier JM, Giavazzi R and Belotti D: Vascular endothelial growth factor c promotes ovarian carcinoma progression through paracrine and autocrine mechanisms. Am J Pathol. 184:1050–1061. 2014. View Article : Google Scholar : PubMed/NCBI
30	Zhang HH, Qi F, Shi YR, Miao JG, Zhou M, He W, Chen MF, Li Y, Zu XB and Qi L: RNA interference-mediated vascular endothelial growth factor-C reduction suppresses malignant progression and enhances mitomycin C sensitivity of bladder cancer T24 cells. Cancer Biother Radiopharm. 27:291–298. 2012. View Article : Google Scholar : PubMed/NCBI
31	Muders MH, Zhang H, Wang E, Tindall DJ and Datta K: Vascular endothelial growth factor-C protects prostate cancer cells from oxidative stress by the activation of mammalian target of rapamycin complex-2 and AKT-1. Cancer Res. 69:6042–6048. 2009. View Article : Google Scholar : PubMed/NCBI
32	Feng Y, Hu J, Ma J, Feng K, Zhang X, Yang S, Wang W, Zhang J and Zhang Y: RNAi-mediated silencing of VEGF-C inhibits non-small cell lung cancer progression by simultaneously down-regulating the CXCR4, CCR7, VEGFR-2 and VEGFR-3-dependent axes-induced ERK, p38 and AKT signalling pathways. Eur J Cancer. 47:2353–2363. 2011. View Article : Google Scholar : PubMed/NCBI
33	Kurenova EV, Hunt DL, He D, Fu AD, Massoll NA, Golubovskaya VM, Garces CA and Cance WG: Vascular endothelial growth factor receptor-3 promotes breast cancer cell proliferation, motility and survival in vitro and tumor formation in vivo. Cell Cycle. 8:2266–2980. 2009. View Article : Google Scholar : PubMed/NCBI
34	Alison MR and Sarraf CE: Apoptosis: A gene-directed programme of cell death. J R Coll Physicians Lond. 26:25–35. 1992.PubMed/NCBI
35	Desoize B and Sen S: Apoptosis or programmed cell death: Concepts, mechanisms and contribution in oncology. Bull Cancer. 79:413–425. 1992.(In French). PubMed/NCBI
36	Snigdha S, Smith ED, Prieto GA and Cotman CW: Caspase-3 activation as a bifurcation point between plasticity and cell death. Neurosci Bull. 28:14–24. 2012. View Article : Google Scholar : PubMed/NCBI
37	Aslakson CJ and Miller FR: Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 52:1399–1405. 1992.PubMed/NCBI
38	Tao K, Fang M, Alroy J and Sahagian GG: Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer. 8:2282008. View Article : Google Scholar : PubMed/NCBI
39	Xu Q, Sun T, Tian H, Wang C and Zhou H: Ultrasound-mediated vascular endothelial growth factor C (VEGF-C) gene microbubble transfection inhibits growth of MCF-7 breast cancer cells. Oncol Res. 20:297–301. 2013. View Article : Google Scholar : PubMed/NCBI
40	Guo B, Zhang Y, Luo G, Li L and Zhang J: Lentivirus-mediated small interfering RNA targeting VEGF-C inhibited tumor lymphangiogenesis and growth in breast carcinoma. Anat Rec (Hoboken). 292:633–639. 2009. View Article : Google Scholar : PubMed/NCBI