KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells

    KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells Xiaoping Yi, Yixiong Li, Hongyan Zai, Xueying Long, Wenzheng Li PII: DOI: Reference:

S0378-1119(16)30195-0 doi: 10.1016/j.gene.2016.03.025 GENE 41231

To appear in:

Gene

Received date: Revised date: Accepted date:

17 November 2015 8 March 2016 12 March 2016

Please cite this article as: Yi, Xiaoping, Li, Yixiong, Zai, Hongyan, Long, Xueying, Li, Wenzheng, KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells, Gene (2016), doi: 10.1016/j.gene.2016.03.025

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT KLF8 knockdown triggered growth inhibition and induced cell phase arrest in human pancreatic cancer cells

PT

Xiaoping Yi 1, Yixiong Li 2, Hongyan Zai2, Xueying Long1, Wenzheng Li1

RI

Departments of 1Radiology and 2General Surgery, Xiangya Hospital, Central

SC

South University, Hunan 410008, P.R. China

NU

Correspondence to: Dr Wenzheng Li, E-mail: [email protected], Department of Radiology, XiangYa Hospital, Central South University, 87# Road,

Changsha

410008,

MA

XiangYa

Hunan,

P.R.China.

Phone:

D

+86-731-84327448; Fax: +86-731-84327448;

TE

Key words: KLF8, pancreatic cancer, lentivirus, RNAi, cell proliferation

AC CE P

Running title: Yi et al: Role of KLF8 in pancreatic cancer

1

ACCEPTED MANUSCRIPT Abstract

PT

Background: The transcription factor Krüppel-like factor 8 (KLF8) plays important role in tumor development and growth, but its role in pancreatic

SC

RI

cancer (PC) is not clear.

Methods: KLF8 expression in human PC cell lines and tumor tissues was

NU

measured by quantitative real-time polymerase chain reaction and Western

MA

blot analyses. The effects of lentivirus mediated knockdown of KLF8 on

D

proliferation and growth in Panc-1 pancreatic cancer cells were examined.

TE

Results: KLF8 was overexpressed in 5 pancreatic cancer cell lines and in

AC CE P

samples from patients with PC. In Panc-1 cells, KLF8 knockdown inhibited cell proliferation, tumorigenicity, and induced G2/M phase arrest. KLF8 knockdown suppressed PC tumor growth in nude mice model. Western blot analysis showed that KLF8 knockdown in Panc-1 cells down-regulated the expression of CDK1/CDC2, cyclin B1, cyclin D1 and up-regulated the expression of p21, p27.

Conclusions: Overexpression of KLF8 may contribute to the progression of pancreatic cancer, and downregulation of KLF8 expression by lentivirus-delivered shRNA is a novel therapeutic approach for PC.

2

ACCEPTED MANUSCRIPT Key words: KLF8, pancreatic cancer, lentivirus, RNAi, cell proliferation

PT

Running title: Yi et al: Role of KLF8 in pancreatic cancer

RI

Introduction

SC

Pancreatic cancer (PC) is a common gastrointestinal malignancy. In recent [1-3]

.

NU

years, PC burden is getting serious around the world including China

While the survival of most cancer patients is steadily increasing, advances

effective treatment

[3, 4]

MA

have been slow for PC because of no reliable tests for early diagnosis and no . PC remains the most aggressive cancer worldwide

TE

D

with an overall 5-year survival of less than 5%. Therefore, it is urgent to

AC CE P

better understand molecular mechanisms underlying PC progression and metastasis in order to develop more effective treatments and new strategies to improve patient survival. Krüppel-like transcription factor (KLF) family consists of 17 distinct members involved in the regulation of diverse cellular processes [5]. KLF8 is a new member of this family and emerges as a crucial regulator of cancer initiation and progression. As a GT-box (CACCC) binding dual-transcription factor, KLF8 is overexpressed in several types of human cancers and regulates various cancer-related cellular processes such as cell cycle progression[6-13], transformation[9,

14]

and invasion[15-19]. Thus KLF8 is a

3

ACCEPTED MANUSCRIPT potential target for cancer therapy. The role of KLF8 in PC is yet to be elucidated although it was reported

PT

recently that KLF8 was highly expressed in paraffin fixed PC specimens [20].

tumorigenesis.

We

demonstrated

that

SC

PC

RI

In the present study, we aimed to investigate the functional role of KLF8 in KLF8

was

aberrantly

NU

overexpressed in fresh frozen pancreatic cancer specimens and cell lines.

MA

Furthermore, KLF8 knockdown in Panc-1 pancreatic cancer cells inhibited

TE

Materials and methods

D

cell proliferation significantly in vitro and in vivo.

AC CE P

Tissue samples, cell lines and cell culture Tissue samples from 30 primary pancreatic cancers and their non-cancerous counterparts were obtained from patients who underwent surgery at the Department of General Surgery in Xiangya Hospital (Changsha, China). The study was approved by Ethics Committee of Xiangya Hospital (No. 201406373). Panc-1, Capan-1, BXPC-3, Miapaca-2 and Sw1990 pancreatic cancer cell lines were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The non-transformed pancreatic epithelial cell line HPDE6c7 (non-cancerous line) was purchased from Rui-Lu Biotech (Shanghai, China). Cells were routinely maintained in DMEM (Gibco)

4

ACCEPTED MANUSCRIPT supplemented with 10% fetal bovine serum (FBS; Gibco), 100 μg/ml penicillin, and 100 μg/ml streptomycin in a humidified 37 °C incubator with

PT

a constant air flow of 5% CO2 and 95% O2.

RI

KLF8-shRNA lentivirus generation and infection. shRNA targeting the

SC

KLF8 gene (CAGCACTGTTTAATGACAT) was inserted into lentivirus

NU

expression plasmid, a pGCSIL-green fluorescent protein (GFP) vector

MA

(GeneChem, Shanghai, China). Scramble sequence (5’-TTCTCCGAACGTGTCACGT-3’) was used as negative control. The expression plasmid

D

carrying siRNA or mock control were transfected into 293T cells, together

TE

with two lentiviral packaging plasmids (pHelper1.0 and pHelper2.0;

AC CE P

GeneChem) according to the manufacturer’s instructions to generate KLF8-shRNA

lentivirus

(lenti/KLF8-shRNA)

or

control

lentivirus

(lenti/N-control). After three days of incubation, the recombinant virus was collected

from

the

culture

medium

and

concentrated

using

Centricon®-plus-20 (Millipore, Billerica, CA, USA). For infection, Panc-1 cells were grown to 30% confluence and incubated with lenti/KLF8-shRNA or lenti/N-control at multiplicity of infection (MOI) =50 for 24 h. The culture medium was then replaced. Cells expressing GFP were observed using fluorescence microscopy (CKX41, Olympus) 72 h after infection to determine the infection efficiency. The knockdown efficiency 5

ACCEPTED MANUSCRIPT was validated with Quantitative real-time PCR (Q-PCR) at 72 h and Western

PT

blot at day 5 post transduction. Quantitative Real-time RT-PCR. Total RNA was extracted using Trizol

RI

reagent (Invitrogen, USA) and reverse transcribed using M-MLV-RTase

SC

(Promega, USA) according to the manufacturer's instructions. cDNA was

NU

used for qPCR using SYBR-Green PCR Master mix (Applied Biosystems,

MA

USA) with specific primers as follows: KLF8 sense 5'-TTCAGAAGGTGGCTCAATGC-3' , antisense

D

5'GGAGTGTTGGAGAAGTCATATTAC-3'; GAPDH sense: 5’-

TE

TGACTTCAACAGCGACACCCA-3’, antisense: 5’-

AC CE P

CACCCTGTTGCTGTAGCCAAA-3’. For each sample, triplicate determinations were made, and mean values were adopted for further calculations. Target gene expression was normalized to that of the endogenous control GAPDH. The relative quantitative expression of the target gene compared with GAPDH was expressed as 2‑ (Ct‑Cc) (Ct and Cc represent the mean threshold cycle differences following normalization to GAPDH). Western blotting. Western blotting was performed according to the standard procedures. Briefly, the protein concentrations were determined according to the Bicinchonic acid (BCA) assay, and then total protein (30 μg) was 6

ACCEPTED MANUSCRIPT subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% skim

PT

milk, and incubated with primary antibodies against KLF8, GAPDH

RI

(AV31533, Sigma-Aldrich, St. Louis, MO, USA), cyclin B1, cyclin D1, p21

SC

p27, human cyclin-dependent kinase (CDK) 1/CDC2, CDK2 (Cell Signaling,

NU

Beverly, MA, USA), and corresponding horseradish peroxidase-conjugated

MA

secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were detected by enhanced chemiluminescence (Amersham, Arlington

D

Heights, IL, USA), and visualized using a LAS3000 luminescent image

TE

analyzer (Fujifilm Life Sciences, Tokyo, Japan).

AC CE P

Cell proliferation assay. Cells were grown in the exponential phase and collected after trypsin treatment. For cellomics assay, viable cells were seeded in 96-well plates at a density of 2x104 cells per well and cultured at 37˚C in 5% CO2 atmosphere. Corresponding data, such as the average cell number per field, fluorescence spot number, area and intensity per cell were collected daily until day five at fixed time points using Cellomics instrumentation and software (Thermo Scientific). BrdU incorporation assay. A 5-bromodeoxyuridine (BrdU) incorporation assay was performed using the BrdU cell proliferation assay kit (Chemicon, Temecula, CA, USA). Briefly, cells (2x103 cells/ml) were plated onto 7

ACCEPTED MANUSCRIPT 96-well plates, and 10 μl of 1/100 diluted BrdU was added per well and incubated for 12 h. Next, the cells were incubated with 100 μl of 1/100

PT

diluted anti-BrdU and peroxidase-conjugated goat anti-mouse IgG

RI

antibodies. Then the plate was washed and 100 μl TMB Peroxidase

For the plate clone forming experiment, 103 cells

NU

Colony formation assay

SC

Substrate was added per well. Plates were read at 450 nm.

MA

was mixed in culture medium, and seeded in culture dish and incubated at 37˚C in air with 5% CO2 and the medium were renewed every three days. 14

TE

was counted.

D

days later, the colonies were stained with Giemsa and the colony number

AC CE P

Cell cycle analysis. For cell cycle analysis, approximately 5×104 cells were seeded in six-well culture plates and incubated in complete medium to 90% confluence. Then the cells were washed with ice-cold PBS twice and fixed with 70 % cold ethanol at 4˚C for 1 h. After washing for three times, the cells were treated with 50 μl/mL PI solution (Sigma, USA) and 100 μl/ml RNase in phosphate-buffered saline (PBS) for 15 min at room temperature in the dark. Propidium iodide (PI) staining of nuclei was used to monitor the phases of the cell cycle. The fluorescence of DNA-bound PI in cells was measured with a FACSCalibur flow cytometer (BD Biosciences). Tumorigenicity in nude mice Cells were resuspended at 5×108 cells/ml, and 8

ACCEPTED MANUSCRIPT a 0.1-mL aliquot of cell suspension was injected subcutaneously into two groups of 4-week-old athymic nude mice (n=12). Subcutaneous tumor

PT

development was monitored by palpation every 4 days, and tumor sizes were

RI

recorded. The tumor volume was calculated according to the equation

SC

V=0.5×L×W2 (V=volume, L=length and W=width). Animals were sacrificed

NU

32 days after injection. Animal care and use was approved by the

MA

Institutional Animal Care and Use Committee. Statistical analysis. Data were expressed as the mean ± standard deviation.

D

All statistical analyses were performed using SPSS v15.0 software (SPSS,

AC CE P

significant.

TE

Chicago, IL, USA). A value of P<0.05 was considered to be statistically

Results and discussion

KLF8 is overexpressed in pancreatic cancer samples and pancreatic cancer cell lines The expression of KLF8 in 30 resected samples from PC patients was examined. KLF8 expression in PC tissues was significantly higher than that in the adjacent nontumor pancreatic tissues, as determined by qPCR (Figure. 1A) and Western blot analysis (Figure. 1B). Furthermore, KLF8 mRNA and protein expression in PC cell lines (Panc-1, Capan-1, BXPC-3, Sw1990, and Miapaca-2) were significantly higher than that in the non-transformed pancreatic epithelial cell line HPDE6c7 (Figure. 1C and D). 9

ACCEPTED MANUSCRIPT These results indicated an association between KLF8 overexpression and

PT

PC. Lentivirirus-mediated knockdown of KLF8 in human pancreatic cells To

RI

examine the biological role of KLF8 in PC, we employed lentivirus-mediad

SC

RNAi to knockdown KLF8 expression in Panc-1 cells. Fluorescence

NU

microscopy for GFP expression showed that the infection efficiency was

MA

over 90% after infection for 72 h (Figure 2A). The expression levels of KLF8 mRNA and protein were significantly decreased in Panc-1 cells

D

infected with lenti/KLF8-shRNA (Figure 2D and 2B). These results

TE

confirmed that lentivirus-mediated KLF8 shRNA depleted endogenous

AC CE P

KLF8 expression in pancreatic cancer cells efficiently. KLF8 knockdown inhibits the proliferation of human pancreatic cells. To investigate the effect of lenti/KLF8-shRNA on PC cell viability in culture, cellomics assay and BrdU incorporation assay were employed. As shown in Figure 2C and E, lenti/KLF8-shRNA significantly inhibited cell growth of Panc-1 cells compared to lenti/N-control infected cells (p<0.05). Moreover, the difference was more pronounced in a time-dependent manner. Colony formation assay showed that the colony-forming ability of Panc-1 was significantly inhibited by lenti/KLF8-shRNA (Figure 2F and G). These results demonstrated that knockdown of KLF8 by lenti/KLF8-shRNA 10

ACCEPTED MANUSCRIPT exerted inhibitory effect on the proliferation of Panc-1 cells.

PT

KLF8 knockdown affects cell cycle progression of human pancreatic cells To detect whether KLF8 has a role in cell cycle regulation, we performed

RI

cell cycle assay. As shown in Figure 3A, the percentage of Panc-1 cells in

SC

G2/M phase in lenti/KLF8-shRNA group (37.8±0.3%) was much higher than

NU

that in lenti/N-control group (26.1±0.9%; p<0.01). Meanwhile, compared

MA

with lenti/N-control group (35.9±0.8%), the percentage of Panc-1 cells in G0/G1 phase was decreased in lenti/KLF8-shRNA group (25.7±0.6%;

D

p<0.01). Taken together, these data indicated that KLF8 knockdown caused

TE

G2/M phase arrest and affected cell cycle progression.

AC CE P

Western blot analysis was then performed to examine the differential expression of cell cycle regulators, including CDK1/CDC2, CDK2, cdk4, cyclin B1, cyclin D1, p21 Waf1/Cip1 and p27 Kip1. The expression of cdk2 and cdk4 showed no significant differences after KLF8 knockdown (data not show). However, CDK1/CDC2, cyclin B1, and cyclin D1 were down-regulated and p21 and p27 were up-regulated (Figure 3B). These results demonstrated that KLF8 knockdown remarkably induces G2/M phase arrest in PANC-1 cells, which may be attributable, at least in part, to the down-regulation of CDK1/CDC2, cyclin B1, cyclin D1 and up-regulation of p21, p27. 11

ACCEPTED MANUSCRIPT KLF8 knockdown inhibits pancreatic tumor growth in vivo To determine whether lenti/KLF8-shRNA could suppress tumor growth in vivo, we

in

mice

inoculated

subcutaneously

RI

volumes

PT

adopted a subcutaneous tumor formation assay, and found that tumor with

SC

lenti/KLF8-shRNA-transfacted cells were dramatically reduced compared to

NU

those in mice receiving lenti/N-control (Figure 4A,B). Western blot analysis confirmed that KLF8 protein expression in lenti/KLF8-shRNA-transfected

MA

cell group was decreased significantly (Figure 4C). Taken together, these

D

data confirmed that lenti/KLF8-shRNA suppressed KLF8 expression and

AC CE P

Discussion

TE

inhibited pancreatic cancer growth in vivo.

PC is an extremely aggressive malignancy with a notably poor prognosis. In recent decades, despite the progress in surgery, chemotherapy, radiotherapy, and immunotherapy, effective treatment of PC remains a major challenge in the clinic [4]. Therefore, there is an urgent need for new therapeutic strategies for PC. However, tumorigenesis and progression of PC are far from being fully elucidated. Recently, increasing studies show that KLF8 plays an important role in the regulation of a variety of cellular processes favoring tumor progression of many cancer types, and it has become a promising therapeutic target for 12

ACCEPTED MANUSCRIPT cancer treatment. KLF8 is aberrantly overexpressed in a variety of human cancers and implicated in the initiation, development, and progression of

[17, 28]

, gastric cancer[24-27],

, ovarian cancer[9, 14], renal cancer[28, 29], and glioma[30, 31].

RI

breast cancer

[21-23]

PT

these cancers, including hepatocellular carcinoma

SC

In addition, a recent study reported that KLF8 may be a potential prognostic

NU

factor for pancreatic cancer[20]. In the present study, our findings conformed that KLF8 expression was significantly increased in pancreatic cancer cell

MA

lines and fresh frozen cancer tissues compared with adjacent non-tumor

D

pancreatic tissue. These results suggest an association between the

TE

overexpression of KLF8 and PC.

AC CE P

In this study, we constructed shRNA lentivirus targeting KLF8 to efficiently inhibit KLF8 expression at mRNA and protein levels. Subsequent findings showed that KLF8 knockdown exhibited potent inhibitory effects on cell proliferation and colony formation in Panc-1 cells in vitro. Furthermore, KLF8 knockdown inhibited pancreatic cancer growth in vivo, as

tumor

volumes

were

significantly

suppressed

when

lenti/KLF8-shRNA-transfected panc-1 cells were injected into nude mice. In agreement with our findings, other studies showed that down-regulation of KLF8 in U251 and U87-MG glioma cells[30, 31], 786-0 renal cells[29], CAL27 human oral cancer cells[13], Saos-2 osteosarcoma cells[12], and SGC-7091

13

ACCEPTED MANUSCRIPT gastric cancer cells[24, 25], HCC hepatocellular carcinoma cells[23] dramati-

PT

cally inhibited cancer cell proliferation. We further explored the potential molecular mechanism of oncogenic role

RI

of KLF8 by identifying differential expression of cell cycle related factors

SC

between lenti/KLF8-shRNA and lenti/N-control treated cells. CDC2 and

NU

cyclin B1 are two key regulators of G2-to-M phase transition[34]. In this study,

MA

our findings showed that KLF8 knockdown remarkably decreased CDC2, cyclin B1, and cyclin D1 in PANC-1 cells, suggesting that KLF8 knockdown

D

may induce cell cycle arrest in G2/M phase. Indeed, we observed a

TE

significantly decreased G0/G1-phase population and increased G2/M phase

AC CE P

population in PANC-1 cells treated with lenti/KLF8-shRNA. These results indicated that KLF8 knockdown significantly downregulated the expression of CDC2, cyclin B1, and cyclin D1 in PANC-1 cells, which contributes, at least in part, to cell cycle arrest in G2/M phase and the proliferation suppression in this cell line. Furthermore, p21 and p27 play an important role in the regulation of cell cycle[35,

36]

. Notably, our findings demonstrated that KLF8 knockdown

significantly increased the expression levels of p21, which is an inhibitor of cell cycle progression via the inhibition of CDK activity and serves as a tumor suppressor protein[37]. Moreover, KLF8 knockdown led to significant 14

ACCEPTED MANUSCRIPT increase in the expression level of p27, a member of the Cip/Kip family of CDK inhibitors and inducing cell cycle arrest via its inhibitory effect on

PT

CDK2/cyclin E and other CDK/cyclin complexes[38]. Taken together, KLF8

RI

knockdown induced cell cycle arrest in G2/M phase could suppress cell

SC

proliferation via the modulation of a number of cell cycle regulators in

NU

PANC-1 cells. Further studies are required to explore the detailed

MA

mechanism. Conclusions

D

In summary, our findings show that KLF8 is overexpressed in PC tissues and

TE

cell lines and plays an oncogenic role in PC progression. The significant

AC CE P

downregulation of KLF8 expression by lentivirus mediated RNAi in PC cells resulted in cell proliferation suppression both in vitro and in vivo. Collectively, inhibition of KLF8 by RNAi may provide a potential therapeutic approach for gene therapy of pancreatic cancer. Informed consent: Informed consent was obtained from all individual participants included in the study. Ethical approval： All applicable international, national, and/or institutional guidelines for the

15

ACCEPTED MANUSCRIPT care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the

RI

PT

institution or practice at which the studies were conducted.

SC

Conflicts of interests

NU

The authors declare no conflicts of interests. References

Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et

MA

[1]

D

al. Cancer incidence and mortality worldwide: sources, methods and

[2]

AC CE P

E359-86.

TE

major patterns in GLOBOCAN 2012. Int J Cancer. 2015. 136(5):

He Y, Zheng R, Li D, Zeng H, Zhang S, Chen W. Pancreatic cancer incidence and mortality patterns in China, 2011. Chin J Cancer Res. 2015. 27(1): 29-37.

[3]

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015. 65(1): 5-29.

[4]

Garrido-Laguna I, Hidalgo M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies. Nat Rev Clin Oncol. 2015. 12(6): 319-334.

[5]

Limame R, de Beeck K O, Lardon F, De Wever O, Pauwels P.

16

ACCEPTED MANUSCRIPT Kruppel-like factors in cancer progression: three fingers on the steering wheel. Oncotarget. 2014. 5(1): 29-48. Lloyd JA. KLF8 sets the pace for the cell cycle through interactions

PT

[6]

Mehta TS, Lu H, Wang X, Urvalek AM, Nguyen KH, Monzur F, et al.

SC

[7]

RI

with p300 and PCAF. Cell Cycle. 2010. 9(4): 650-1.

NU

A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal

Wang X, Urvalek AM, Liu J, Zhao J. Activation of KLF8 transcription

D

[8]

MA

zinc-fingers. Cell Res. 2009. 19(9): 1098-109.

TE

by focal adhesion kinase in human ovarian epithelial and cancer cells.

[9]

AC CE P

J Biol Chem. 2008. 283(20): 13934-42. Wang X, Zhao J. KLF8 transcription factor participates in oncogenic transformation. Oncogene. 2007. 26(3): 456-61. [10]

Zhao J, Bian ZC, Yee K, Chen BP, Chien S, Guan JL. Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol Cell. 2003. 11(6): 1503-15.

[11]

Liang K, Liu T, Chu N, Kang J, Zhang R, Yu Y, et al. KLF8 is required for bladder cancer cell proliferation and migration. Biotechnol Appl Biochem. 2014 Oct 17. doi: 10.1002/bab.1310. [Epub ahead of print].

17

ACCEPTED MANUSCRIPT [12]

Lin F, Shen Z, Tang LN, Zheng SE, Sun YJ, Min DL, et al. KLF8 knockdown suppresses proliferation and invasion in human

Bin Z, Ke-Yi L, Wei-Feng Z, Li-Cheng J, Xian-Bin L, Chun-Peng X,

RI

[13]

PT

osteosarcoma cells. Mol Med Rep. 2014. 9(5): 1613-7.

SC

et al. Downregulation of KLF8 expression by shRNA induces

NU

inhibition of cell proliferation in CAL27 human oral cancer cells. Med Oral Patol Oral Cir Bucal. 2013. 18(4): e591-6. Lu H, Wang X, Urvalek AM, Li T, Xie H, Yu L, et al. Transformation

MA

[14]

D

of human ovarian surface epithelial cells by Kruppel-like factor 8.

Zhang H, Liu L, Wang Y, Zhao G, Xie R, Liu C, et al. KLF8 involves

AC CE P

[15]

TE

Oncogene. 2014. 33(1): 10-8.

in TGF-beta-induced EMT and promotes invasion and migration in gastric cancer cells. J Cancer Res Clin Oncol. 2013. 139(6): 1033-42. [16]

Lahiri SK, Zhao J. Kruppel-like factor 8 emerges as an important regulator of cancer. Am J Transl Res. 2012. 4(3): 357-63.

[17]

Wang X, Zheng M, Liu G, Xia W, McKeown-Longo PJ, Hung MC, et al. Kruppel-like factor 8 induces epithelial to mesenchymal transition and epithelial cell invasion. Cancer Res. 2007. 67(15): 7184-93.

[18]

Liu N, Wang Y, Zhou Y, Pang H, Zhou J, Qian P, et al. Kruppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of

18

ACCEPTED MANUSCRIPT gastric cancer via epithelial to mesenchymal transition. Oncol Rep. 2014. 32(6): 2397-404. Lu H, Hu L, Yu L, Wang X, Urvalek AM, Li T, et al. KLF8 and FAK

PT

[19]

RI

cooperatively enrich the active MMP14 on the cell surface required

SC

for the metastatic progression of breast cancer. Oncogene. 2014.

[20]

NU

33(22): 2909-17.

Wei Y, Chen G, You L, Zhao Y. Krupel-like factor 8 is a potential

MA

prognostic factor for pancreatic cancer. Chin Med J (Engl). 2014.

Yang T, Cai SY, Zhang J, Lu JH, Lin C, Zhai J, et al. Kruppel-like

TE

[21]

D

127(5): 856-9.

AC CE P

factor 8 is a new Wnt/beta-catenin signaling target gene and regulator in hepatocellular carcinoma. PLoS One. 2012. 7(6): e39668. [22]

Han S, Han L, Sun H, Zan X, Zhou Z, Xu K, et al. Kruppellike factor expression and correlation with FAK, MMP9 and Ecadherin expression in hepatocellular carcinoma. Mol Med Rep. 2013. 8(1): 81-8.

[23]

Li JC, Yang XR, Sun HX, Xu Y, Zhou J, Qiu SJ, et al. Up-regulation of Kruppel-like factor 8 promotes tumor invasion and indicates poor prognosis for hepatocellular carcinoma. Gastroenterology. 2010. 139(6): 2146-2157.e12.

19

ACCEPTED MANUSCRIPT [24]

Chen G, Yang W, Jin W, Wang Y, Tao C, Yu Z. Lentivirus-mediated gene silencing of KLF8 reduced the proliferation and invasion of

Liu L, Liu N, Xu M, Liu Y, Min J, Pang H, et al. Lentivirus-delivered

RI

[25]

PT

gastric cancer cells. Mol Biol Rep. 2012. 39(10): 9809-15.

SC

Kruppel-like factor 8 small interfering RNA inhibits gastric cancer

[26]

NU

cell growth in vitro and in vivo. Tumour Biol. 2012. 33(1): 53-61. Hsu LS, Wu PR, Yeh KT, Yeh CM, Shen KH, Chen CJ et al. Positive

MA

nuclear expression of KLF8 might be correlated with shorter survival

Wang WF, Li J, Du LT, Wang LL, Yang YM, Liu YM, et al.

TE

[27]

D

in gastric adenocarcinoma. Ann Diagn Pathol. 2014. 18(2): 74-7.

AC CE P

Kruppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer. World J Gastroenterol. 2013. 19(27): 4309-15. [28]

Wang X, Lu H, Urvalek AM, Li T, Yu L, Lamar J, et al. KLF8 promotes human breast cancer cell invasion and metastasis by transcriptional activation of MMP9. Oncogene. 2011. 30(16): 1901-11.

[29]

Fu WJ, Li JC, Wu XY, Yang ZB, Mo ZN, Huang JW, et al. Small interference RNA targeting Kruppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo. J Cancer Res Clin

20

ACCEPTED MANUSCRIPT Oncol. 2010. 136(8): 1255-65. [30]

Schnell O, Romagna A, Jaehnert I, Albrecht V, Eigenbrod S, Juerchott

PT

K, et al. Kruppel-like factor 8 (KLF8) is expressed in gliomas of

Wan W, Zhu J, Sun X, Tang W. Small interfering RNA targeting

NU

[31]

SC

PLoS One. 2012. 7(1): e30429.

RI

different WHO grades and is essential for tumor cell proliferation.

Kruppel-like factor 8 inhibits U251 glioblastoma cell growth by

Cox BD, Natarajan M, Stettner MR, Gladson CL. New concepts

D

[32]

MA

inducing apoptosis. Mol Med Rep. 2012. 5(2): 347-50.

TE

regarding focal adhesion kinase promotion of cell migration and

[33]

AC CE P

proliferation. J Cell Biochem. 2006. 99(1): 35-52. He HJ, Gu XF, Xu WH, Yang DJ, Wang XM, Su Y. Kruppel-like factor 8 is a novel androgen receptor co-activator in human prostate cancer. Acta Pharmacol Sin. 2013. 34(2): 282-8. [34]

Hu X, Moscinski LC. Cdc2: a monopotent or pluripotent CDK. Cell Prolif. 2011. 44(3): 205-11.

[35]

Williams GH, Stoeber K. The cell cycle and cancer. J Pathol. 2012. 226(2): 352-64.

[36]

Diaz-Moralli S, Tarrado-Castellarnau M, Miranda A, Cascante M. Targeting cell cycle regulation in cancer therapy. Pharmacol Ther.

21

ACCEPTED MANUSCRIPT 2013. 138(2): 255-71. Warfel NA, El-Deiry WS. p21WAF1 and tumourigenesis: 20 years after. Curr Opin Oncol. 2013. 25(1): 52-8.

Yoon MK, Mitrea DM, Ou L, Kriwacki RW. Cell cycle regulation by

RI

[38]

PT

[37]

SC

the intrinsically disordered proteins p21 and p27. Biochem Soc Trans.

NU

2012. 40(5): 981-8.

MA

Figure legends

Figure. 1 Quantitative real-time RT-PCR and Western blot analysis of KLF8

D

mRNA and protein expression in pancreatic cancer samples and adjacent

TE

non-tumor pancreatic tissues. A PC tissues had higher KLF8 mRNA levels

AC CE P

than adjacent non-tumor pancreatic tissues (P<0.01). B Western blot analysis of whole-cell protein extracts prepared from seven paired pancreatic cancer tissues (T) and adjacent non-tumor pancreatic (N). C and D KLF8 mRNA and protein expression levels were higher in pancreatic cancer cell lines compared to non-transformed pancreatic epithelial cell line HPDE6c7 (non-cancerous line). GAPDH was used as internal controls. Figure 2. Lenti/KLF8-shRNA mediated KLF8 knockdown in pancreatic cell line Panc-1. A Micrographs were taken 72 h following infection (magnification, x100). B and D The protein and mRNA levels of KLF8 were down-regulated by lenti/KLF8-shRNA in Panc-1 cells (*P<0.05). The 22

ACCEPTED MANUSCRIPT lenti/N-control group was used as negative control. C Cell number was counted using Cellomics Arrayscan following 1, 2, 3, 4 and 5 days of

PT

incubation, and the increased fold in cell number was calculated.

RI

lenti/KLF8-shRNA suppressed the growth curves of Panc-1 cells compared

SC

with lenti/N-control in a time-dependent manner. E The BrdU incorporation

NU

of cells was examined following 1 and 4 days of incubation. F and G Colony formation assay showed that Lenti/KLF8-shRNA inhibited the number of

MA

grown clones compared with lenti/N-control.

D

Figure 3. The effects of lenti/KLF8-shRNA on cell cycle as determined by

TE

flow cytometric analysis. A Flow cytometry analysis showed that KLF8

AC CE P

knockdown induced G2/M phase arrest and decreased the G0/G1 phase population of the cells. B Cell cycle-related molecules were detected by Western blot analysis. KLF8 knockdown resulted in down-regulation of CDK1/CDC2, cyclin B1, cyclin D1 and up-regulation of p21, p27 expression in Panc-1 cells. Figure 4. Local injection of lenti/KLF8-shRNA-transfected Panc-1 cells into mice suppressed tumor growth in vivo. A The tumor size of lenti/KLF8-shRNA group was significantly decreased compared to lenti/N-control group. B Tumor growth curves showed a significant growth tendency in lenti/N-control group, while tumor growth in lenti/KLF8-shRNA 23

ACCEPTED MANUSCRIPT group was suppressed (p<0.001). C KLF8 protein expression in lenti/KLF8-shRNA group decreased compared to the other groups, as

AC CE P

TE

D

MA

NU

SC

RI

PT

determined by Western blot analysis. GAPDH was used as internal control.

24

Figure 1

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

25

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

Figure 2

26

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

Figure 3

27

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

Figure 4

28

ACCEPTED MANUSCRIPT Abbreviations list：

PT

PC : Pancreatic cancer ;

lenti/KLF8-shRNA : KLF8-shRNA lentivirus;

SC

lenti/N-control: control-shRNA lentivirus;

RI

KLF : The Krüppel-like transcription factor ;

NU

Q-PCR : Quantitative real-time PCR;

AC CE P

TE

D

BrdU: 5-bromodeoxyuridine;

MA

WB: Western blotting;

29

ACCEPTED MANUSCRIPT Highlights： 1. KLF8 was overexpressed in pancreatic cancer tissues and cell lines;

PT

2.KLF8- knockdown resulted in cell proliferation suppression both in vitro and in vivo.

AC CE P

TE

D

MA

NU

SC

4. KLF8 may play an important role in PC progression.

RI

3. KLF8-knockdown influence expression of CDC2, p21, p27, resulted in G2/M arrest.

30