Med Oncol (2015) 32:218 DOI 10.1007/s12032-015-0658-z

ORIGINAL PAPER

Lentivirus-mediated silencing of USO1 inhibits cell proliferation and migration of human colon cancer cells Jinke Sui1 • Xu Li1 • Junjie Xing1 • Fuao Cao1 • Hao Wang1 • Haifeng Gong1 Wei Zhang1



Received: 18 May 2015 / Accepted: 15 June 2015 Ó Springer Science+Business Media New York 2015

Abstract Belonging to the tether factors family, USO1 (vesicle transport factor) plays a critical role in endoplasmic reticulum–Golgi trafficking and vesicular transport which is important to tumorigenesis. However, the mechanism of USO1 in colon cancer was still unknown. In our research, the expression of USO1 was knockdown in colon cancer cells (HCT116 and HT-29) by using a special lentivirus shRNA approach. A series of experiments were carried out to evaluate the effect of deregulation of USO1, including cell proliferation, apoptosis, cell migration and cell cycle. Knockdown of USO1 inhibits the ability of cell proliferation and migration. Furthermore, the deregulation of USO1 induces early apoptosis and decreased cells in G2-M phase. We demonstrate for the first time that USO1 gene has a critical role in human colon cancer. Our finding represents that USO1 gene may be a promising target for therapy and diagnosis in treatment of human colon cancer. Keywords USO1  Colon cancer  Proliferation  Migration  Apoptosis

Jinke Sui and Xu Li have contributed equally to this work. & Wei Zhang [email protected] 1

Department of Colorectal Surgery, Shanghai Changhai Hospital, 168 Changhai Road, Shanghai 200433, People’s Republic of China

Introduction The tumorigenesis is a highly complex process involving sequential changes in various genes and cell signaling pathways. The changes in genetics and epigenetics may cause uncontrolled cell proliferation, cell invasion and migration [1, 2]. Vesicular trafficking is related to all these physiological processes and then mediated in cancer formation. Previous reports have demonstrated that the deregulation of vesicle trafficking factors, such as Rab family of small GTPase, may affect the development of colon carcinoma [2, 3]. Therefore, research on the role of vesicle trafficking factors in colon carcinoma is of vital significance to the selection of specific target genes for prevention and therapy of colon carcinoma. USO1 (vesicle transport factor), also known as P115/ TAP, is a member of tether protein family, including a central coiled-coil domain, N-terminal globular domain and C-terminal acidic region in its structure [4, 5]. USO1 has a crucial role not only in vesicle trafficking, especially in endoplasmic reticulum–Golgi transport [6, 7] promoting the homotype vesicles fusion by recruitment of Rab1a [8], but also enhances other proteins to target Golgi membrane by interacting with soluble NSF attachment protein receptors (SNARE) [7]. Previous studies have showed that USO1 could be cleaved by caspase3 and caspase8 in cell nucleus to induce cell apoptosis through interacting with GM130 and Giantin [9, 10]. In mitosis, USO1 and c-tubulin formed a complex to maintain spindles stability [11]. Meanwhile, knockdown of USO1 suppressed gastric cancer cells proliferation by arresting cell cycle on G0–G1 phase and decreased the expression of related genes, such as cyclin D1, Mcm2, PCNA, pERK1/2 [12, 13]. In gastric cancer, USO1 also cooperating with macrophage migration inhibitory factor

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(MIF) regulated cell proliferation. Moreover, Iman Osman et al. [14] reported that the copy number of USO1 was also dramatically changed in superficial invasive melanoma (SSM) and nodular melanoma (NM). Therefore, we hypothesize that USO1 may play an important role in tumorigenesis. However, there is no report about the function of USO1 in colon cancer. To further explore the roles of USO1 in colon cancer, we constructed specific USO1 knockdown cell line in colon cancer cells to examine cell proliferation, migration, apoptosis and the changes in cell cycle before and after USO1 knockdown. This provided a theoretical foundation for the further study of the biological functions and molecular mechanism of USO1.

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the Takara PrimeScript RT Reagent Kit (Takara) following the manufacturer’s protocol. The cDNA was used for quantitative real-time PCR using the one-step SYBRÒ PremixScriptTM RT-PCR Kit (Takara) and was performed on a Bio-Rad CFX96 real-time PCR detection system (BioRad). All expression data were normalized to b-actin expression levels. The primers used are as follows USO1-F: 50 -TCTGTTATCTTTATTGGAGGAGTTTG30 USO1-R: 50 -TTGAAACACCCATAGGACTGACT-30 b-actin F:50 -CGAGCGCGGCTACAGCT-30 b-actin R: 50 -TCCTTAATGTCACGCAC GATTT-30 Cell lysis and Western blot analysis

Materials and methods Cell line and culture The human colon cancer HCT116 and HT-29 cell lines, and HEK-293T cell line were from American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) containing 10 % fetal bovine serum (FBS; Hyclone) and 100 units/mL of penicillin and streptomycin (Gibco), at 37 °C in a humidified incubator containing 5 % CO2. Vector construction and lentivirus infection The sequence coding human USO1 gene was from GenBank. Based on the conserved cDNA fragments within the coding region of the USO1 gene, we designed the shRNA as the following sequence 50 -GCAGCTTTGTACTATCCTAAT-30 . The sequences were cloned into the pLKO.1 lentiviral vector (from Addgene). The constructed plasmid was further co-transfected into HEK-293T cells with lentiviral packaging plasmids to generate an USO1 shRNAexpressing lentivirus or control vector shRNA-expressing lentivirus. Cells were cultured in six-well plates at 50,000 cells/ well and incubated for 24 h. shRNA-containing lentivirus and control vector lentivirus were applied to HCT116 and HT-29 cells, respectively. After selection by puromycin, cells were collected and evaluated for the efficiency of infection. Quantitative real-time PCR assay After infection by lentiviral target USO1 or a control vector, HCT116 and HT-29 cells were collected. Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was obtained using

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Cells were cultured in six-well plates. After fusion rate above 70 %, cells were rinsed with ice-cold PBS and lysed in 1 9 RIPA buffer (Cell Signaling Technology) containing complete protease inhibitors (Roche) and phosphatase inhibitors (Roche), 5 mM DTT (Sigma) and 1 mM PMSF(Sigma) for 30 min, and then centrifuged at 4 °C for 10 min at 15,000g. Collection of the supernatant and the concentration of total protein were determined by the Lowry’s assay (Bio-Rad). The proteins were separated by 10 % SDS-PAGE and blotted into a PVDF membrane (Millipore), and incubated for 1 h at room temperature in 2 % OVA, followed by overnight incubation at 4 °C with anti-USO1 (Abgent) or anti-b-actin (Cell Signaling Technology). Membranes were washed with TBST and incubated for 2 h with a secondary antibody. Immunoreactivity was revealed using an ECL kit (Pierce Chemicla). MTT assay Cells were counted to 1 9 103 and seeded in 96-well plates. The cell viability was assessed on 24, 48, 72 and 96 h. Briefly, at the time point each well was added 20 lL/ well of MTT (thiazolyl blue tetrazolium bromide, 5 mg/ mL, Sigma). The plate was then incubated for an additional 4 h. Then, culture medium was removed from the wells, and 150 lL dimethyl sulfoxide (DMSO) was added to each well to dissolve the crystals for 20 min at 37 °C. Finally, The absorbance was recorded at 570 nm. The migration index was defined as the number of migrated cells by cell counting in at least three random fields (magnification, 9 200) per filter. Each experiment was repeated three separate times. Analysis of cell cycle by flow cytometry Cells were washed twice with ice-cold PBS and fixed with 70 % ethanol overnight at 4 °C. The cells were then

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digested with 50 lg/mL RNase A in 100 lL of PBS and stained with 20 lg/mL propidium (PI) for 30 min at 37 °C. Cell cycle was analyzed using Beckman Moflo XDP (Beckman). Cell migration assay To carry out cell migration assay, we used a modified Boyden chamber (Corning) plate with 8-lm pore size polycarbonate membrane filters [15]. 1 9 104 of serumdeprived human colon HCT116 and HT 29 cells were added to the upper chamber, and the bottom chamber was filled with DMEM containing 20 % serum and incubated for 48 h. Then, 4 % paraformaldehyde was used to fix the cells on the membrane filter. Following staining with 0.05 % Giema (Sigma), the number of migrated cells on the membrane was quantified by cell counting in at least three random fields (magnification, 2009) per filter. Apoptosis detection by FACS Cells were washed twice with PBS, digested with trypsin and then centrifuged. Then, cells were washed with binding buffer after centrifugation and resuspended again. Almost 1 9 106 cells were double stained by adding 1:100 diluted annexin-FITC and PI (Biovion) and then analyzed by Beckman Moflo XDP (Beckman).

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Knockdown of USO1 suppressed the proliferation of colon cancer cells Then, we examined cell proliferation in colon cancer cells HCT116 and HT-29 by MTT assay. The result showed that cell proliferation was obviously inhibited after USO1 knockdown, as compared to the control vector cells (Fig. 2a), which was coincident with previous reports [13]. Compared to non-infected cells and vector cells, cell proliferation did not change significantly, suggesting that lentivirus system has no toxicity to cells. Next, we hypothesize that USO1 influence cell proliferation by regulating cell cycle. To verify this hypothesis, we analyzed cell cycle of USO1 knockdown in HCT116 and HT29 cells by flow cytometry. We observed that the percentage of cells in S phase is 2.67 ± 0.09 and 7.4 ± 0.05 % (p \ 0.01), and in G2-M phase is 50.42 ± 2.13 and 44.43 ± 1.36 % (p \ 0.05), in control group and USO1 knockdown in HCT116 cells, respectively (Fig. 2b, c). Our results showed that USO1 knockdown increased cells in S phase and decreased cells in G2-M phase. Therefore, our data revealed that USO1 knockdown arrests cells in S phase, thereby inhibiting colon cancer cells proliferation. USO1 knockdown decreased cell migration in colon cancer cells

Results

Vesicular trafficking plays a critical role in cell migration. It suggested that USO1 may participate in colon cancer cell migration. The results of transwell assay demonstrated that in contrast to control vector cells, USO1 knockdown dramatically reduced cell migration index of HCT116 cells (p \ 0.001) (Fig. 3a, b), and the similar result was also obtained in HT-29 cells (p \ 0.01). These results indicated that cell migration ability was inhibited after USO1 knockdown in colon cancer cells.

USO1 knockdown in colon cancer cells HCT116 and HT-29

USO1 knockdown promotes early apoptosis in colon cancer cells

In order to study the function of USO1 in colon cancer, we used the lentivirus pLKO.1-USO1 shRNA and pLKO.1 control vector to infect colon cancer cells HCT116 and HT29, respectively. Real-time quantitative PCR and Western blot were carried out to identify USO1 knockdown efficiency. The results of real-time quantitative PCR showed that USO1 mRNA level was reduced more than 50 % in USO1 KD HCT116 and HT-29 cells compared to control vector cells (Fig. 1a). Similarly, Western blot also displayed that infected with pLKO.1-USO1 shRNA, cells had a significant down-regulation of the expression of USO1 protein, in contrast to control cells (p \ 0.05, Fig. 1b, c).

To assess the changes in apoptosis, USO1 knockdown in HCT116 and HT-29 cells was examined by flow cytometry (Fig. 4a, b). We found that early apoptosis was significantly increased (15.29 ± 1.41 %, p \ 0.001) in USO1 knockdown in HCT116 cells as compared to the control vector cells (3.13 ± 0.14 %, p \ 0.001), and we obtained a similar result (p \ 0.001) in HT-29 cells. According to these results, down-regulating USO1 expression induced early apoptosis in colon cancer cells. In conclusion, our results indicated that USO1 knockdown in colon cancer cell significantly inhibited cell proliferation and migration and induced early cell apoptosis.

Statistical analysis Each experiment was repeated at least three times. The data were presented as mean ± SD, and statistical comparisons were made using Student’s t test. Significance was presented as a ***p \ 0.001, **p \ 0.01 or *p \ 0.05.

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Fig. 1 Expression of USO1 was measured in human colon cancer cell lines HCT116 (up) and HT-29 (down) after infected with USO1 shRNA or control vector lentivirus and non-infected cells. a USO1 knockdown efficiency was verified by using quantitative real-time

PCR b Western blotting examined the expression level of USO1 protein in human colon cancer cell lines HCT116 and HT-29 compared with control vector and c semi-quantification of USO1 protein level from WB

Discussion

and cell migration, especially after knockdown of USO1 in colon cancer cells. We deregulated the expression of USO1 by using lentivirus shRNA approach (Fig. 1a, b), and confirmed that USO1 plays an important role in cell proliferation, growth, apoptosis, and cell migration in colon cancer cells (Figs. 2, 3 and 4). Firstly, cell proliferation was significantly suppressed in USO1 knockdown in colon cancer cells HCT116 and HT-29. Moreover, we found that lower expression of USO1 in HCT116 cells (Fig. 2a) destroyed the cell cycle by arresting cell on S phase and reducing the cell number in G2-M phase. Furthermore, the percentage of early apoptosis was dramatically raised in USO1 knockdown in HCT116 and HT-29 cells (Fig. 4). In addition, knockdown of USO1 also attenuated cell migration ability (Fig. 3). Therefore, our results indicate that the USO1 expression level is closely related to colon cancer progression. Programmed cell death induced by apoptosis was considered as a necessary barrier in tumorigenesis [17, 18]. Nowadays, mechanism studies on the tumor apoptosis inhibition have drawn more and more attention. The studies on USO1 in mitosis and apoptosis revealed that USO1 was cleaved by caspase3 pathway and promoted cell apoptosis

Vesicular trafficking is an essential biological process in both normal cells and tumor cells. The malignant tumor has some specific characters including uncontrolled proliferation, migration and invasion in which vesicular trafficking possessing is highly active [2]. Vesicular transport factor USO1 is a member of the tether protein family, which has unique functions in vesicular trafficking due to its peculiar structure [5]. The mechanism of USO1 in vesicular trafficking, especially in endoplasmic reticulum–Golgi transportation, has been abundantly reported, but the researches on relationship between USO1 and tumor are only reported in gastric cancer and melanoma [13, 14]. Currently, colon cancer has become one of the most highlighted causes of tumor mortality. However, gene targeting therapy in colon cancer is still short of theoretical foundation [16]. Therefore, we want to reveal how USO1 exerts its biological role in colon cancer. According to previous reports, USO1 has a role in cell apoptosis and proliferation in gastric cancer [9, 13], but there is no research on USO1 in colon cancer. Thus, we aim to reveal the changes in cell proliferation, apoptosis

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Fig. 2 Cell proliferation rate was inhibited, and cell cycle was arrested in S phase by infecting USO1 shRNA lentivirus in HCT116 and HT-29 cells. a The viability of HCT116 and HT-29 cells following infection with USO1 shRNA lentivirus was significantly

decreased by MTT assay. b Cell cycle of HCT116 cells infected with control vector or USO1 shRNA lentivirus or non-infected cells was analyzed by flow cytometry. c The percentage of each cell cycle phase was determined

Fig. 3 USO1 knockdown inhibits human colon cancer cell motility. The migration ability was analyzed by a migration assay in HCT116 (a) cell and HT-29 (b) cell following infection with control vector or

USO1 shRNA lentivirus or non-infected cells. All experiments were repeated three times with identical results

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Fig. 4 Knockdown of USO1 in human colon cancer cells induced cell apoptosis. Cell apoptosis of HCT116 (a) and HT-29 (b) cells infected with control vector or USO1 shRNA lentivirus or non-infected cells was analyzed by flow cytometry

which is related to p53 signaling pathway [19, 20]. Our results are consistent with the previous reports, but whether there are other proteins interacting with USO1 involved in cell proliferation and apoptosis in colon cancer still needs further research. In conclusion, our research revealed the important role of USO1 in colon cancer development. Lower expression of USO1 significantly inhibits colon cancer cell proliferation and growth, suggesting that USO1 plays an important role during colon cancer development. Altogether, USO1 may serve as a molecular target for treatment and diagnosis of colon cancer and may be a biomarker for colon cancer diagnosis or prediction. Compliance with Ethical Standards Conflict of interest

None.

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Lentivirus-mediated silencing of USO1 inhibits cell proliferation and migration of human colon cancer cells.

Belonging to the tether factors family, USO1 (vesicle transport factor) plays a critical role in endoplasmic reticulum-Golgi trafficking and vesicular...
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