Effect of EGFR gene inhibition by lentivirus-mediated small interfering RNA on the proliferation and apoptosis of human pancreatic cancer cells

doi:10.3877/cma.j.issn.2095-3232.2013.02.010

Abstract

Abstract:

Objective

To explore the feasibility of lentivirus-mediated small interfering RNA (siRNA) silencing the epidermal growth factor receptor (EGFR) and its influence on the proliferation and apoptosis of human pancreatic cancer cell.

Methods

The expression vector LV-shEGFR was established and packaged and the lentivirus particles containing EGFR-shRNA was collected for use. The experiment was divided into Panc-1 group (blank group), green fluorescent protein (GFP)-Panc-1 group (control group) and siEGFR-Panc-1 group (interference group). The pancreatic cancer cell line Panc-1 cells at logarithmic growth phase in the blank group received no treatment and the cells in the control group were infected by lentivirus particles without siRNA fragment. For the interfering group, the Panc-1 cells were infected with the prepared lentiviral particles containing EGFR-shRNA. The expression of EGFR gene (described as relative ratio with β-action) and protein (described as gray value) were examined by real-time polymerase chain reaction(real-time PCR)and Western-blot respectively. The cell proliferation, as well as cell cycle and apoptosis were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry respectively. The EGFR expression, absorbance (A) value, apoptosis rate and cell cycle in three groups were compared using one-way analysis of variance (ANOVA) and t-test.

Results

The relative expression level of EGFR gene in the interference group was 0.20±0.04, which was significantly lower compared with the control group (1.00±0.15) and the blank group (1.13±0.13). There were significant differences (LSD-t=7.865, 7.668; P<0.05). The EGFR protein expression level in the interference group was 0.185±0.009, which was down-regulated compared with the control group (0.801±0.087) and the blank group (0.825±0.092) (LSD-t=4.216, 3.975; P<0.05). The cell proliferation of the interference group was obviously suppressed, which was slower compared with the control group. The G₂/M phase cell ratio in the interference group (8.87±0.29)% was significantly decreased compared with that in the blank group (20.00±1.88)% and the control group (21.48±2.13)% and the difference was statistically significant (LSD-t=2.015,2.323; P<0.05). There was significant difference in the S phase cell ratio among three groups (LSD-t=1.987, 2.251; P<0.05), with (50.97±3.04)% in interference group, (36.67±6.18)% in the blank group and (39.91±2.09)% in the control group. The cell apoptotic rate in the interference group (18.4±2.3)% was significantly higher than that in the control group (7.4±1.4)% and the blank control group (7.7±1.2)% and the difference was statistically significant (LSD-t=2.585, 2.667; P<0.05).

Conclusions

The lentivirus-mediated siRNA expression vector can effectively suppress the expression of the EGFR gene in pancreatic cancer cells and lead to cell growth inhibition and apoptosis in vitro.

Key words: Carcinoma, pancreatic ductal, Receptor, epidermal growth factor, Lentiviral, RNA, Small interfering, Cell proliferation, Apoptosis, Cell cycle

Zhen-zhong FA, Yong JIANG, Yu-Lin TAN, Bao-qiang WU, Liang-rong SHI. Effect of EGFR gene inhibition by lentivirus-mediated small interfering RNA on the proliferation and apoptosis of human pancreatic cancer cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2013, 02(02): 113-118.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhgzwkssxdzzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-3232.2013.02.010

https://zhgzwkssxdzzz.cma-cmc.com.cn/EN/Y2013/V02/I02/113

Figures/Tables 5

References 15

[1]	Strimpakos A, Saif MW, Syrigos KN. Pancreatic cancer: from molecular pathogenesis to targeted therapy. Cancer Metastasis Rev, 2008, 27(3):495-522.
[2]	Boeck S, Ankerst DP, Heinemann V. The role of adjuvant chemotherapy for patients with resected pancreatic cancer: systematic review of randomized controlled trials and meta-analysis. Oncology, 2007, 72(5/6):314-321.
[3]	Wheeler DL, Iida M, Kruser TJ, et al. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab. Cancer Biol Ther, 2009, 8(8):696-703.
[4]	Akinc A, Querbes W, De S, et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther, 2010, 18(7):1357-1364.
[5]	Overdijk MB, Verploegen S, van den Brakel JH, et al. Epidermal growth factor receptor (EGFR) antibody-induced antibody-dependent cellular cytotoxicity plays a prominent role in inhibiting tumorigenesis, even of tumor cells insensitive to EGFR signaling inhibition. J Immunol, 2011, 187(6):3383-3390.
[6]	Baselga J, Arteaga CL. Critical update and emerging trends in epidermal growth factor receptor targeting in cancer. J Clin Oncol, 2005, 23(11):2445-2459.
[7]	Chen G, Kronenberger P, Teugels E, et al. Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells. Biol Proced Online, 2010, 13:1.
[8]	Matsubara H, Sakakibara K, Kunimitsu T, et al. Non-small cell lung carcinoma therapy using mTOR-siRNA. Int J Clin Exp Pathol, 2012, 5(2):119-125.
[9]	Hanke S, Valkova C, Stirnweiss J, et al. Activated EGF receptor may balance ERK-inhibitory network signalling pathways. Cell Signal, 2006, 18(7):1031-1040.
[10]	Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer, 2002, 2(7):489-501.
[11]	Bloomston M, Frankel WL, Petrocca F, et al. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA, 2007, 297(17):1901-1908.
[12]	Merlin J, Stechly L, de Beaucé S, et al. Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells. Oncogene, 2011, 30(22):2514-2525.
[13]	Fujita H, Ohuchida K, Mizumoto K, et al. High EGFR mRNA expression is a prognostic factor for reduced survival in pancreatic cancer after gemcitabine-based adjuvant chemotherapy. Int J Oncol, 2011, 38(3):629-641.
[14]	Stein DA, Perry ST, Buck MD, et al. Inhibition of dengue virus infections in cell cultures and in AG129 mice by a small interfering RNA targeting a highly conserved sequence. J Virol, 2011, 85(19):10154-10166.
[15]	Scaltriti M, Baselga J. The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res, 2006, 12(18):5268-5272.

组别	份数	感染后时间(d)
组别	份数	1	2	3	4	5	6	7	8
对照组	6	0.317±0.019	0.516±0.042	0.611±0.043	0.615±0.037	0.950±0.041	1.290±0.071	1.392±0.097	1.398±0.087
干扰组	6	0.290±0.020	0.425±0.037	0.521±0.038	0.612±0.029	0.812±0.037	1.125±0.083	1.168±0.080	1.215±0.079
t值	?	2.189	3.655	3.051	0.143	5.588	3.378	3.984	3.482
P值	?	0.060	0.007	0.008	0.890	<0.05	0.009	0.004	0.008

组别	份数	感染后时间(d)
组别	份数	1	2	3	4	5	6	7	8
对照组	6	0.317±0.019	0.516±0.042	0.611±0.043	0.615±0.037	0.950±0.041	1.290±0.071	1.392±0.097	1.398±0.087
干扰组	6	0.290±0.020	0.425±0.037	0.521±0.038	0.612±0.029	0.812±0.037	1.125±0.083	1.168±0.080	1.215±0.079
t值	?	2.189	3.655	3.051	0.143	5.588	3.378	3.984	3.482
P值	?	0.060	0.007	0.008	0.890	<0.05	0.009	0.004	0.008

Please choose a citation manager

Content to export