Effect of lentivirus-mediated integrin αVβ3-shRNA on tumor growth of mice with lung cancer xenografts

Tao Liang, Yong-Fu Ma, Jian Chu, Dao-Xi Wang, Yang Liu*

1Department of Thoracic Surgery, Second Artillery General Hospital of Chinese PLA, Beijing 100088, China

2Department of Thoracic Surgery, Chinese PLA General Hospital, Beijing 100853, China

Effect of lentivirus-mediated integrin αVβ3-shRNA on tumor growth of mice with lung cancer xenografts

Tao Liang1, Yong-Fu Ma2, Jian Chu1, Dao-Xi Wang1, Yang Liu2*

1Department of Thoracic Surgery, Second Artillery General Hospital of Chinese PLA, Beijing 100088, China

2Department of Thoracic Surgery, Chinese PLA General Hospital, Beijing 100853, China

ARTICLE INFO

Article history:

Received 15 November 2015

Received in revised form 20 December 2015

Accepted 15 January 2016

Available online 20 February 2016

Lung cancer

Integrin αVβ3

Xenografts

Angiogenesis

Apoptosis

Invasion

Objective: To study the effect of lentivirus-mediated integrin αVβ3-shRNA on tumor growth of mice with lung cancer xenograft. Methods: Lung cancer tissue, paracancer tissue and normal tissue were collected and integrin αVβ3 expression was detected; BALB/c nude mice were selected, divided into integrin αVβ3 knockdown group (KD group) and negative control group (NC group), and inoculated with cells stably infected by integrin αVβ3-shRNA lentivirus and cells stably infected by negative control-shRNA lentivirus respectively, the growth of tumor tissue was continuously observed, and the number of apoptosis cells as well as the expression of angiogenesis, apoptosis and invasion genes in tumor tissue were detected. Results: mRNA content and protein content of integrin αVβ3 in lung cancer tissue were significantly higher than those in paracancer tissue and normal tissue; increasing trend of tumor tissue volume of KD group was weaker than that of NC group, and tumor volume at various points in time of KD group was lower than that of NC group; mRNA contents and protein contents of VEGF, FGF, EGF, Bcl-2, MMP-9, MMP-12 and MMP-13 in tumor tissue of KD group were lower than those of NC group, and apoptosis index as well as mRNA content and protein content of Bax were higher than those of NC group. Conclusions: The expression of integrin αVβ3 increases in lung cancer tissue, and lentivirus-mediated integrin αVβ3-shRNA can inhibit tumor growth of mice with lung cancer xenografts.

1. Introduction

Lung cancer is one of the most common malignant tumors worldwide and death cases caused by it rank first in cancer-related deaths in China. Non-small cell lung cancer is the most common pathological type of lung cancer, and the cancer cells have strong ability of proliferation and invasion, which directly causes high recurrence rate and distant metastasis rate in non-small cell lung cancer patients after surgical resection or chemotherapy, thus leading to poor long-term prognosis of the disease[1,2]. At present, regulatory mechanisms of lung cancer cell proliferation and invasion haven’t been fully elucidated. Integrins are a family of molecules discoveredin recent years that are closely related to cell adhesion, proliferation, invasion and angiogenesis, are located in cell membrane and play a role in the form of membrane receptors. Integrin αVβ3 has been confirmed to participate in the clinical and pathological process of osteosarcoma, colon cancer and other malignant tumors[3,4], but whether the molecule is involved in the development of lung cancer has not been reported. In the following research, the effect of lentivirus-mediated integrin αVβ3-shRNA on tumor growth of mice with lung cancer xenograft was analyzed.

2. Materials and methods

2.1. Materials

2.1.1. Clinical and experimental subjects

Clinical specimens were lung cancer tissue, paracancer tissueand normal tissue, and tissue was from 100 cases of patients who received radical resection of lung cancer in our hospital from May 2012 to April 2015. Lung cancer cell lines A549 were purchased from the cell bank of Chinese Academy of Sciences, and BALB/c nude mice were purchased from Shanghai Slac Laboratory Animal Company.

2.1.2. Reagents and consumables

Serum and media for cell culture were from Gibco Company and consumables for culture were from Nest Company; RNA extraction and PCR amplification kits were from Beijing Tiangen Company, immunoblot kits and related antibodies were from US Sigma Company, and TUNEL kits were from US Roche Company.

2.2. Experimental methods

2.2.1. Cell culturing and transfecting methods

Lung cancer A549 cell lines were conventionally recovered and cultured, when cell density reached about 70%-80%, 0.125% trypsin was used for digestion and sub-culture, and sub-cultured cells were inoculated in Petri dishes and infected with integrin αVβ3-shRNA lentivirus and negative control-shRNA lentivirus respectively; four days after infection, 2 μg/mL puromycin was added for pressure screening, then media were replaced every 3 d to expand the culture, and cells stably infected by integrin αVβ3-shRNA lentivirus and negative control-shRNA lentivirus were obtained respectively.

2.2.2. Establishment of mouse models with lung cancer xenografts

Sixty BALB/c nude mice were divided into integrin αVβ3 knockdown group (KD group) and negative control group (NC group), each group with 30, and 200 μL tumor cell suspension with density of 1×108/mL was subcutaneously injected in necks of two groups. KD group were inoculated with cells stably infected by integrin αVβ3-shRNA lentivirus, and NC group were inoculated with cells stably infected by negative control-shRNA lentivirus.

2.2.3. Detection of tumor growth

8 d, 12 d, 16 d, 20 d and 24 d after tumor cell inoculation, diameters of xenografts were measured in three directions and marked as a, b and c respectively, and tumor tissue volume V=πabc/6.

2.2.4. Tumor tissue collecting and processing methods

Tweenty-four days after tumor cell inoculation, mice were killed and anatomized, obtained tumor tissue was washed with normal saline and equally divided into two, one was frozen in liquid nitrogen and then preserved at -80 ℃, and the other was fixed in formalin.

2.2.5. Detecting methods of gene expression in tumor tissue

Clinical lung tissue specimens and mouse tumor tissue preserved at -80 ℃ were taken, RNA extraction kits and protein extraction kits were used to extract RNA specimens and protein specimens respectively, then PCR reaction and immunoblot test were performed respectively, and mRNA contents and protein contents of related genes were detected.

2.2.6. Detecting methods of cell apoptosis index

Tumor tissue fixed in formalin was taken and made into paraffin slices, then TUNEL kits were used for staining, all procedures were conducted in accordance with the kit instructions, finally DAB was used for color development, 200 cells were observed under high power lens, cells with tan nuclei were taken as positive cells, the number of positive cells was counted, and the percentage of positive cells was calculated and used as apoptosis index.

2.3. Statistical methods

SPSS21.0 was used for data processing, comparison of measurement data between two groups was by t test, and differences were considered to be statistically significant at a level of P＜ 0.05.

3. Results

3.1. Integrin αVβ3 expression in lung cancer tissue and paracancer normal tissue

mRNA content and protein content of integrin αVβ3 in lung cancer tissue were significantly higher than those in paracancer tissue and normal tissue, and differences of pair wise comparison were statistically significant (P＜0.05) (Table 1).

Table 1Comparison of integrin αVβ3 expression in lung cancer tissue, paracancer tissue and normal tissue.

3.2. Dynamic change of tumor tissue volume

After tumor cell inoculation, tumor tissue volume of both groups showed increasing trend, the increasing trend of tumor tissue volume of KD group was weaker than that of NC group, tumor tissue volume at various points in time of KD group was lower than that of NC group, and differences between two groups were statistically significant (P＜0.05) (Table 2).

Table 2Dynamic change of tumor tissue volume of two groups.

3.3. Expression of angiogenesis molecules in tumor tissue

mRNA contents and protein contents of VEGF, FGF and EGF in tumor tissue of KD group were significantly lower than those of NC group, and differences between two groups were statistically significant (P＜0.05) (Table 3).

3.4. Cell apoptosis in tumor tissue

Apoptosis index in tumor tissue of KD group was higher than that of NC group, mRNA content and protein content of pro-apoptosis gene Bax were higher than those of NC group, and mRNA content and protein content of anti-apoptosis gene Bcl-2 were lower than those of NC group (P＜0.05) (Table 4).

3.5. Expression of invasion-related molecules in tumor tissue

mRNA contents and protein contents of MMP-9, MMP-12 and MMP-13 in tumor tissue of KD group were significantly lower than those of NC group, and differences between two groups were statistically significant (P＜0.05) (Table 5).

Table 3Expression of angiogenesis molecules in tumor tissue of two groups.

Table 4Comparison of cell apoptosis in tumor tissue of two groups.

Table 5Expression of invasion-related molecules in tumor tissue of two groups.

4. Discussion

Integrin αVβ3 is one of the key members of the integrin family, it is a type of receptor on cell membrane and it mainly mediates intercellular adhesion, cell-matrix adhesion, cell invasion, angiogenesis and other processes[5-7]. In recent years, more and more studies have confirmed that integrin αVβ3 is overexpressed in osteosarcoma, colorectal cancer and other malignant tumors, and it is closely related to the clinical and pathological processes of tumors[3,4]. According to studies of foreign scholars, integrin αVβ3 can be combined with fibrinogen, type I collagen, LM, FN and so on, and increase the capacity of heterogeneous adhesion of tumor cells[8,9]. In addition, integrin αVβ3 can crosslink with VEGF receptor, enhance VEGF function and induce VEGF receptor activation, thus promoting angiogenesis[10].

Tumor tissue growth involves many complex links that specifically include large proliferation of cancer cells in tumor lesion, cell separation from primary lesion and invasion into extracellular matrix, formation of local new blood vessels and lymphatic vessels, and cancer cell invasion into vascular system as well as colonization and growth in distal tissue and organs. Lung cancer cells are with strong ability of proliferation and invasion, so the incidence of recurrence and metastasis is high after surgical resection. At present, there is no report about the relationship between integrin αVβ3 and the development of lung cancer. Based on the trend of integrin αVβ3 in osteosarcoma, colorectal cancer and other malignant tumors, it was speculated that there was abnormal expression of integrin αVβ3 in lung cancer tissue. Detection of integrin αVβ3 expression in lung cancer tissue and paracancer normal tissue showed that mRNA content and protein content of integrin αVβ3 in lung cancer tissue were significantly higher than those in paracancer tissue and normal tissue, which indicated that the overexpression of integrin αVβ3 was involved in the occurrence and development of lung cancer.

In order to verify the specific relationship between integrin αVβ3 and lung cancer occurrence and development, lung cancer cell lineswere cultured at first, adenovirus-mediated integrin αVβ3-shRNA was transfected to inhibit the expression of integrin αVβ3, then cells transfected with integrin αVβ3-shRNA and cells transfected with negative control-shRNA were subcutaneously injected into the mice respectively, and observation of tumor tissue growth showed that after tumor cell inoculation, tumor tissue volume of both groups showed increasing trend, the increasing trend of tumor tissue volume of KD group was weaker than that of NC group and tumor tissue volume at various points in time of KD group was lower than that of NC group, which indicated that transfection of integrin αVβ3-shRNA could transfer? the growth of lung cancer xenograft.

Large formation of local angiogenesis is the key link of the occurrence and development of tumors, and links such as tumor growth and metastasis are highly dependent on rich blood supply of tumor tissue. Physiological angiogenesis process specifically means that new blood vessels are formed in the vascular structures that already exist, the formation of new blood vessels in tumor tissue mainly begins with small lesions without vascular structures, and new blood vessels that are formed under the effect of proangiogenesis molecules such as VEGF, FGF and EGF can provide the necessary oxygen and nutrients for tumor tissue and thus be conducive to tumor growth[11,12]. After knocking down integrin αVβ3 expression in tumor cells, it was detected that mRNA contents and protein contents of VEGF, FGF and EGF in tumor tissue of KD group were significantly lower than those of NC group, which indicated that inhibiting integrin αVβ3 could reduce expression of pro-angiogenesis factors in lung cancer xenografts.

Formation of new blood vessels in local xenografts provides the necessary nutrients for the completion of a variety of biological behaviors of tumor cells. In the occurrence and development process of lung cancer, proliferation and invasion are the most important two biological behaviors that cause tumor recurrence and metastasis, and they involve the expression changes of a variety of related genes. Bcl-2 family is a family of apoptosis-regulating molecules located in mitochondrial outer membrane, Bcl-2 has the effect of inhibiting apoptosis and enhancing anti-apoptotic ability of cells, and Bax can antagonize Bcl-2 function and induce apoptosis[13,14]. MMPs are a group of protease molecules involved in the degradation of extracellular matrix, and among them, MMP-9, MMP-12 and MMP-13 are closely related to lung cancer invasion and metastasis[15,16]. Analysis of proliferation and invasion-related gene expression in tumor tissue of mice with xenografts showed that mRNA contents and protein contents of Bcl-2, MMP-9, MMP-12 and MMP-13 in tumor tissue of KD group were significantly lower than those of NC group, and mRNA content and protein content of Bax were significantly higher than those of NC group, which indicated that inhibiting integrin αVβ3 could regulate the expression of proliferation and invasion-related genes, induce apoptosis and inhibit invasion.

Based on above discussion, it can be concluded that the expression of integrin αVβ3 increases in lung cancer tissue, and lentivirusmediated integrin αVβ3-shRNA can inhibit tumor growth of mice with lung cancer xenografts.

Conflict of interest statement

We declare that we have no conflict of interest.

[1] Okayama A, Miyagi Y, Oshita F, Nishi M, Nakamura Y, Nagashima Y, et al. Proteomic analysis of proteins related to prognosis of lung adenocarcinoma. J Proteome Res 2014; 13(11): 4686-4694.

[2] Kadota K, Nitadori J, Woo KM, Sima CS, Finley DJ, Rusch VW, et al. Comprehensive pathological analyses in lung squamous cell carcinoma: single cell invasion, nuclear diameter, and tumor budding are independent prognostic factors for worse outcomes. J Thorac Oncol 2014; 9(8): 1126-1139.

[3] Zhi LQ, Ma W, Wang WH, Wang XP. Study on expression and relationship between adrenomedullin and intergrin αVβ3 in human osteosarcoma. Chongqing Med 2014; 43(19): 2428-2431.

[4] Zhang MK, Wang FJ, Yang MP, Li SL, Wang CJ. ADAM23 and αVβ 3 expression in colorectal cancer and their relations with liver metastases. Chin J Gen S 2013; 22(10): 1297-1301.

[5] Shan D, Li J, Cai P, Prasad P, Liu F, Rauth AM, et al. RGD-conjugated solid lipid nanoparticles inhibit adhesion and invasion of αVβ3 integrin-overexpressing breast cancer cells. Drug Deliv Transl Res 2015; 5(1): 15-26.

[6] Wu FH, Luo LQ, Liu Y, Zhan QX, Luo C, Luo J, et al. Cyclin D1b splice variant promotes αVβ3-mediated adhesion and invasive migration of breast cancer cells. Cancer Lett 2014; 355(1): 159-167.

[7] Zhang Z, Nie F, Kang C, Chen B, Qin Z, Ma J, et al. Increased periostin expression affects the proliferation, collagen synthesis, migration and invasion of keloid fibroblasts under hypoxic conditions. Int J Mol Med 2014; 34(1): 253-261.

[8] Reyhani V, Seddigh P, Guss B, Gustafsson R, Rask L, Rubin K. Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels. Biochem J 2014; 462(1): 113-123.

[9] Wu W, Okamoto O, Kato A, Matsuo N, Kumai J, Nomizu M, et al. Functional peptide of dermatopontin produces fibrinogen fibrils and modifies its biological activity. J Dermatol Sci 2014; 76(1): 34-43.

[10] Zanella S, Mingozzi M, Dal Corso A, Fanelli R, Arosio D, Cosentino M, et al. Synthesis, characterization, and biological evaluation of a dualaction ligand targeting αVβ3 integrin and VEGF receptors. Chemistry Open 2015; 4(5): 633-641.

[11] Lee SH, Lee J, Jung MH, Lee YM. Glyceollins, a novel class of soy phytoalexins, inhibit angiogenesis by blocking the VEGF and bFGF signaling pathways. Mol Nutr Food Res 2013; 57(2): 225-234.

[12] Xiao L, Yang S, Hao J, Yuan X, Luo W, Jiang L, et al. Endostar attenuates melanoma tumor growth via its interruption of b-FGF mediated angiogenesis. Cancer Lett 2015; 359(1): 148-154.

[13] Han EB, Chang BY, Jung YS, Kim SY. Lantana camara induces apoptosis by Bcl-2 family and caspases activation. Pathol Oncol Res 2015; 21(2): 325-331.

[14] Bai L, Chen J, McEachern D, Liu L, Zhou H, Aguilar A, et al. BM-1197: a novel and specific Bcl-2/Bcl-xL inhibitor inducing complete and longlasting tumor regression in vivo. PLoS One 2014; 9(6): e99404.

[15] Wen Y, Cai L. Research progress of matrix metalloproteinase 12 in nonsmall cell lung cancer. Chin J Lung Cancer 2014; 17(1): 30-33.

[16] Xu XY, Pei LT, Li XX. Expression of MMP-9 and MMP-13 in non-small cell lung cancer and their significance. Chin J Clin Exp Patho 2014; 30(12): 1358-1364.

Document heading doi: 10.1016/j.apjtm.2016.01.005

ent heading

10.1016/j.apjtm.2016.01.004

*Corresponding author: Yang Liu, Thoracic Surgery Department, Chinese PLA General Hospital, No. 28, Fuxing Road, Haidian District, Beijing 100853, China.

Tel: 13501009331

Foundation project: It is supported by Surface Project of National Natural Science Foundation of China (No 81573026).