RNA interference suppressing PLCE1 gene expression decreases invasive power of human bladder cancer T24 cell line

Cancer Genetics and Cytogenetics 200 (2010) 110e119

RNA interference suppressing PLCE1 gene expression decreases invasive power of human bladder cancer T24 cell line Liping Oua,b, Yongcan Guoa,b, Chunli Luoa,*, Xiaohou Wuc, Yi Zhaoa, Xiaozhong Caia b

a Department of Laboratory Diagnosis, Chongqing Medical University, Chongqing, CQ 400016, China Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing, CQ 400016, China c Urinary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, CQ 400016, China

Received 22 July 2009; received in revised form 28 January 2010; accepted 29 January 2010

Abstract

Mutational activation of the ras proto-oncogenes is frequently found in cancers. The phospholipase C epsilon gene (PLCE1) encodes a novel ras-related protein (R-Ras) effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras is coprecipitated with the PLCE1 protein and can increase its activity. The nature of downstream signaling pathways from Ras involved in bladder cancer remains poorly understood. We aimed to construct a small hairpin RNA (shRNA) expression plasmid against the PLCE1 gene and to observe the inhibition of human bladder carcinoma cell T24 migration by RNA interference suppressing the expression of PLCE1. Two PLCE1 plasmids (P1 and P2) were constructed and inserted into T24 cells. Reverse transcriptaseepolymerase chain reaction and Western blot analyses were performed to investigate inhibition of PLCE1 expression after plasmid transfection. Invasive power of the T24 cell line was measured before and after transfection by a membrane invasion culture system (transwell chamber), gelatin enzymography, and immunocytochemistry of cells. The RT-PCR analysis of BCL2 mRNA levels among different groups of T24 cell line indicated that expression of BCL2 mRNA was lower in the two positive plasmid-transfected cell groups than in the blank control or HK-A groups. Silencing of PLCE1 might downregulate the level of MMP and BCL2 gene expression, decreasing the invasive power of bladder cancer T24 cells and thus inhibiting tumor development. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Urothelial cell carcinoma of the urinary bladder is the fifth most common cancer in industrialized countries [1]. In fact, a large proportion of the patients treated (67%) are deemed poor operative candidates because of medical comorbidities. For this group of patients, combined chemoradiation after transurethral resection of bladder tumor is an attractive alternative, offering the potential of cure. Invasive bladder cancer is an aggressive malignancy. If left untreated, the course is usually fatal, with O85% of patients dying of the disease [2]. A substantial percentage of patients treated with surgery will experience local or metastatic recurrence and ultimately die of the disease despite this treatment. The 5-year overall survival rate for patients with muscle-invasive bladder cancer treated by radical cystectomy ranges between 48% and 64% [3e5]. * Corresponding author. Tel.: þ86-(0)23-68485223. E-mail address: [email protected] (C. Luo). 0165-4608/$ e see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.01.021

Thus, establishing how to inhibit the invasion of bladder cancer is very important. The association between mutation in the RAS gene family (HRAS, KRAS, and NRAS) and 30% of all human cancers, including bladder cancer, suggests that aberrant RAS function is an important contributor to cancer development. Frequently mutated hot spots are Gly (mutating to Val) in codon 12, Gly (mutating to Cys) at codon 13, and Glu (mutating to Arg or Lys or Leu) at codon 61 [6,7]. The findings of Boulalas et al. [8] support the concept that a significant percent of HRAS gene codon 12 mutations are present in bladder cancer cases, and also substantiate the concept that there is a relationship between HRAS codon 12 mutations and low-stage, well-differentiated bladder neoplasms, whereas it seems that advanced transitional cell carcinoma lacks such mutations. Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. Song et al. [9] identified a novel class of human PI-PLC, which they termed

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

PLC3, that is characterized by the presence of a Rasassociating domain at its C terminus and a CDC25-like domain at its N terminus. Ras directly regulates phosphoinositide breakdown through membrane targeting of PLC3. The role of PI-PLC in carcinogenesis remains obscure. This enzyme produces two vital intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which induce activation of protein kinase C and mobilization of Ca2þ from intracellular stores, respectively. At least 13 PLC isoforms have been identified in mammalian species, and are classified into b, g, d, e, z, and h groups [10]. PLC3 is identified as a direct downstream effector of the small GTPases Ras, Rap1, and Rap2. PLC3 is characterized by possession of the Ras-associating domains, which are responsible for PLCe activation through direct association with the GTP-bound active forms of Ras, Rap1, and Rap2. The PLCE1 protein is also reported to be regulated by a12, a13, and b1g2 subunits of heterotrimeric G proteins and the Rho family of small GTPases [11]. Identification of PLC3 as a Ras effector prompted us to examine the role of PLC3 in carcinogenesis. The Rasassociating domain of PLC3 specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIPLC is a key event initiating intracellular signal transduction from various receptor molecules at the plasma membrane. This reaction yields two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which induce activation of protein kinase C (PKC) and mobilization of Ca2þ from intracellular stores, respectively. The PLCE1 protein binds not only to Ras but also to Rap1A. Furthermore, Rap1A may mediate translocation of PLC3 to the perinuclear region in response to EGF. Considering that Rap1A and PKC have been reported to be localized at the Golgi apparatus [12], PLC3 (which is a downstream molecule of Rap1A) may have some role in the regulation of Golgi functions mediated by PKC. Some researchers have demonstrated that activation of PKC leads to upregulation of matrix metalloproteinase (MMP), a finding that makes PKC a target of cancer invasive power research [13]. Protein kinase C is an important signaling molecule in tumor cell transformation, and it is also a key factor in mediating tumor cell adhesion, movement, and invasion. Protein kinase C is a cell membrane receptor of phorbol ester, a carcinogenic promoting agent. Phorbol ester can thoroughly activate PKC and increase the invasion power of tumor cell, and a PKC suppressor has displayed anti-invasion characteristics in vitro and vivo experiments. Matrix metalloproteinases function in tumor metastasis and vasoformation. In many tumor metastasis models, phorbol ester can regulate cell expression of tissue degradation factors such as MMPs. On the other hand, the MMP family of enzymes play an essential role in maintaining the cellular microenvironment. They have a range of biological functions, including the liberation of cytokines and membrane-bound receptors,

111

as well as the promotion of tumor invasion and angiogenesis [14,15]. Matrix metalloproteinases have been implicated in the development of cancer. Previous studies have shown that MMP-1, MMP-2, MMP-3, and MMP-9 are associated with bladder cancer and can predict stage, grade, and even disease outcome [16,17]. MMP-9 likely has an important role in a variety of cancers, including bladder cancer [18]. MMP-9 degrades type IV collagen, a major component of the basement membrane, which is breached in invasive bladder cancer [19]. Cell proliferation, invasive ability, and cell motility are crucial factors for cancer metastasis. Apoptosis is a crucial event in various physiological processes (including embryogenesis, organ development, and cell proliferation), as well as in pathological processes) including autoimmune disease and cancer development). These phenomena are regulated by several genes. One of these genes, BCL2, initially identified as the proto-oncogene translocated to the immunoglobulin heavy chain locus in human follicular B-cell lymphomas, is recognized as one of the most potent inhibitors of apoptosis induced by a wide variety of stimuli, including radiation, chemotherapeutic agents, and growth factor deprivation. Some studies have identified BCL2 protein expression in bladder cancer cells as a possible candidate marker predicting development of bladder cancer [20]. Corrective gene therapy can be attempted, in which absent or defective cellular genes are introduced. The insertion of tumor suppressor genes to correct genetically disabled cell cycle regulatory proteins has been extensively studied in bladder cancer [21]. Tumor suppressor gene therapy represents a favored anticancer strategy [22]. Recent research in a mouse model, using 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter through augmentation of TPA-induced inflammation, has demonstrated that PLC3, an effector of Ras and Rap small GTPases, plays a crucial role in two-stage skin chemical carcinogenesis. The results suggest that the PLCE1 protein plays crucial roles in intestinal tumorigenesis through two distinct mechanisms, augmentation of angiogenesis and inflammation, depending on the tumor stage [10]. No previous study has examined the effect of targeted inhibition or downregulation of PLCE1 in bladder cancer. In the present study with the human bladder cancer cell line T24, we used short hairpin RNA (shRNA) designed according to the PLCE1 gene to implement gene silencing by means of RNA interference. Measures included the regulatory effect on gene expression and effect on the invasive power of T24.

2. Materials and methods 2.1. Cell culture and reagents Human bladder cancer cell line T24 were obtained from the Research Institute of Ultrasound, Chongqing University of Medical Science, and were cultured in RPMI 1640 culture

112

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

medium (Gibco, Rockville, MD) with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/mL penicillin, 100 units/mL streptomycin at 37 C under 5% CO2 and 95% humidified air. Before hypoxia, the medium was replaced with RPMI 1640 free of serum. The cells were then incubated overnight and perfused with 1% O2, 94% N2, and 5% CO2 in a CO2 incubator for 24 hours [23]. Then cells were preserved with RPMI 1640 free of serum or antibiotics and prepared for transfection assays. The shRNA transcription template synthesis was proceeded by Wuhan Genesil Biotechnology company. Reagents for transfection assays were prepared, including kit of pGenesil green fluorescent protein plasmid (Wuhan Genesil Biotechnology, Hubei, China), T4 DNA ligase, SalI incision enzyme (New England Biolabs, Beverly, MA; Beijing, China), BamHI and HindIII restriction enzyme (TaKaRa Biotechnology), lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) and TIANprep mini plasmid kit (Tiangen Biotech [Beijing], Beijing, China). Escherichia coli DH5a from our laboratory was prepared for screening of recombinant plasmid. Primer synthesis and PrimeScript RT-PCR kit (TaKaRa Biotechnology [Dalian], Dalian, China) were prepared for Reverse transcriptaseepolymerase chain reaction analysis. 2.2. Short hairpin RNA and transfection assays The shRNAs targeting PLCE1 (referring to GenBank NM_016341) were prepared by method of chemical synthesis according to principle of design. The length of the hairpin loop construction in the sequence was 9 bp, and the end of the sequence was determined by BamHI and HindIII incision enzyme site besides a 6-T polyT site, the transcription termination of RNA polymerase III. Meanwhile, we designed a pair of nonspecificity sequences for a negative blank control. Finally, the expression plasmid targeting PLCE1 was constructed and identified. After the objective gene fragment was synthesized with annealing buffer and became a synthetic double-stranded oligonucleotide, pGenesil expression plasmid containing promoter U6 was digested by BamHI and HindIII. The linear pGenesil plasmid was connected with double strands of shRNA transcription template with T4 DNA ligase. 5 mL expression vector of ring-shaped pGenesil plasmid product was added into 200mL DH5a competent cells, then incubated in ice bank for 30 min. The clonal coenobium was screened with lysogeny broth (LB) plate cultivation containing kanamycin (30 mg/L). After conventional culture of the T24 human bladder cancer cell line, the cells were divided into four groups: positive plasmid pGenesil-PLC31 (P1), positive plasmid pGenesil-PLC32 (P2), blank control, and negative plasmid (HK-A). Compounds of plasmids and liposome were transfected with lipofectamine 2000 transfection reagent at the ratio of 1:2. The cells after transfection were cultivated in medium containing G418 selective antibiotic (400 mg/mL), and the concentration of G418 was decreased to 200 mg/mL.

2.3. Reverse transcriptaseepolymerase chain reaction analysis for PLCE1 mRNA First, RNA isolation was completed, total RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. After being washed with 75% ethanol, the final RNA extracts were eluted in a 20-mL volume of distilled water treated with diethyl pyrocarbonate. The concentration and purity of RNA were measured with a spectrophotometer. All the RNA preparations had an optical density OD 260:OD 280 ratio of 1.9e2.0. The RT-PCR was performed as the reference described [24]. In brief, RPMI 1640 containing 10% serum cultures were harvested, and total cellular RNA was extracted by a Trizol method. Reverse transcription was performed by using 0.5 mg of total RNA in a first-strand cDNA synthesis reaction with PrimeScript RT-PCR kit as recommended by the supplier (TaKaRa). Oligonucleotide primers were synthesized on the basis of the entire coding region of human PLCE1 (GenBank NM_016341) as follows: forward primer, 50 -CATGGAAGGATAAGCGTTGGT-30 ; reverse primer, 50 -CCCAAGTCCCGTGTTAAGA-30 . b-Actin was amplified as internal control: forward primer, 50 -GTGGA CATCCGCAAA-30 ; reverse primer, 50 -AAAGGGTG TAACGC AATCAA-30 . The PCR (35 cycles) was conducted in a Mastercycler thermal cycler (Eppendorf, Hamburg, Germany). Each cycle included denaturation (94 C, 30 seconds), annealing (56 C, 30 seconds), and extension (72 C, 30 seconds). The initial denaturation period was 2 minutes, and the final extension was 5 minutes. The size of the amplified PLCE1 fragment was 395 bp, and the length of amplified b-actin was 543 bp. Amplified products were analyzed by DNA gel electrophoresis in 1.5% agarose and were visualized by ethidium bromide staining under ultraviolet illumination. The result of gel electrophoresis was analyzed by Quantity one 4.5.0 software for the optical density.

2.4. Western blot analysis of PLCE1 protein Total protein was prepared after cell disruption, and the concentration of protein was detected. The proteins of the samples in each group were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The method for transferring the protein was electroblotting, which uses an electric current to pull proteins from the gel into the polyvinylidene difluoride membrane. Blocking of nonspecific binding is achieved by placing the membrane in a dilute solution of protein (nonfat dry milk), with a minute percentage of Tween 20. After blocking, a dilute solution of primary antibody (1:200) named antiPLCe (goat polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with the membrane under gentle agitation. In the next step, the membrane was exposed to secondary antibody (1:5000),Go IgG (HþL)/ horseradish peroxidase (Zhongshan Goldenbridge Biotechnology,

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

Beijing, China), and then a 3,30 -diaminobenzidine horseradish peroxidase color development kit was used to accomplish coloration. The result was displayed by using semiquantitative image analysis. 2.5. Transwell (Boyden chamber) cell migration assay A transwell unit (BD, Franklin Lakes, NJ; Becton Dickinson Medical Devices, [Shanghai], Nanjing, China) was prepared first, containing 12 wells per plate, with 8.0-mm pore size polycarbonate microporous membrane, precoated with matrigel diluted at the ratio of 1:5; transwell units (Falcon; BD) were coated with 40 mL matrigel per chamber. Cells were grown as monolayers in RPMI 1640 culture medium containing 10% fetal calf serum. After dilution of cells to 5105 cells/mL, 0.2 mL of each cell line was added to the upper chamber of the transwell unit. The membrane was cut from the upper chamber of each unit and then fixed in 10% unbuffered formalin saline. Each was stained with hematoxylin and eosin. Experiments were performed in triplicate. Invasion power of the cells was determined by counting the number of cells that have migrated to the lower side of the membrane. Five visual fields (400) were determined in each chamber. Finally, the mean value of fields was calculated. 2.6. Gelatin enzymography assay of MMP-2 and MMP-9 The expression of matrix metalloproteinases (MMPs) is often associated with invasiveness or grade of tumors. Concentration of MMP-9 and MMP-2 could be detected by gelatin enzymography. When each group of T24 cells had covered 80% of the culture flask bottom, the culture fluid with serum was removed and new culture fluid without serum was added, to wash the cells three times. Then the cells were cultured for 4 hours and viable cells were counted. The culture fluid was collected to be centrifuged at 4 g and filtered. The supernatant of the samples in each transfection group (per 106 cells) was separated using 8% SDS-PAGE (gelatin, 1 mg/mL) for 3 hours in an ice bath with constant voltage (160 V). After electrophoresis, the gel was rocked in 2.5% Triton-X100 for 1 hour and then was incubated in buffer solution of Gelatinase for 16 hours (50 mmol/L Tris, pH 7.5; 200 mmol/L NaCl; 5 mmol/L CaCl2; 0.02% Brij-35) in 37 C. The gel was stained with 0.5% Coomassie brilliant blue and decolorized; transparent bands that showed a blue background indicated MMP-2 (72 kD) and MMP-9 (92 kD). 2.7. Immunocytochemical analysis of MMP-2 and MMP-9 Sterile coverslips were placed in each pore of 24-pore plates. Cells were collected in the exponential phase of growth and were inoculated at approximately 5104 cells per pore. After the cells ideally adhered the wall, make

113

the cells grow on coverslips. The cells on coverslips were fixed with formaldehyde and washed with phosphatebuffered saline, then incubated in 3% H2O2 deionized water, goat serum blocking fluid, and a dilute solution (1:50) of primary antibody anti-MMP-2 and anti-MMP-9 and biotinylation secondary antibody. A type of 3,30 -diaminobenzidine horseradish peroxidase color development kit was used to accomplish coloration. 2.8. RT-PCR analysis of BCL2 mRNA levels among different T24 cell line groups Total RNA was isolated with Trizol reagent according to the manufacturer’s instructions (Invitrogen). All primers and reagents for the PCR were produced by Shanghai Sangon Biological Engineering Technology & Services company (Shanghai, China). For BCL2, the primers were designed as follows: forward primer, 50 -GTC TGG GAA TCG ATC TGG AA-30 ; reverse primer, 50 -CCT AGC AAC GGA ATA CGT AA-30 . Meanwhile, we chose one constant structural protein gene, GAPDH (glyceraldehyde phosphate dehydrogenase), which is expressed in various kinds of cells. This worked as internal control gene. For GAPDH, the primers were designed as follows: forward primer, 50 -ACC ACA GTC CAT GCC ATC AC-30 ; reverse primer, 50 -ATG TCG TTG TCC CAC CT-30 . The specific size of the amplified BCL2 fragment was 131 bp, and the length of amplified GAPDH was 450 bp. The PCR (30 cycles) was conducted in a Mastercycler thermal cycler (Eppendorf). Each cycle included denaturation (94 C, 45 seconds), annealing (61 C, 45 seconds), and extension (72 C, 45 seconds). The initial denaturation period was 3 minutes, and the final extension was 5 minutes. Amplified products were analyzed by DNA gel electrophoresis in 1.5% agarose and were visualized by ethidium bromide staining under ultraviolet illumination. The result of gel electrophoresis was analysed by Quantity one 4.5.0 software for the optical density. 2.9. Statistical analysis All data were analyzed by analysis of variance with the SPSS 10.0 statistical software package (SPSS, Chicago, IL). The interclass means were compared by a Studente NewmaneKeuls q-test method, as previously described in Ref. [25]. In all cases, the level for statistical significance was set at P ! 0.05.

3. Results 3.1. Successful construction of recombinant plasmid containing shRNA The pGenesil plasmid was identified using a digestion method with SalI, and multiple clone sites of pGenesil were identified as follows: HindIII, ShRNA, BamHI, U6

114

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

promoter, EcoRI, SalI, XbaI, DraIII. A site for SalI was designed in the objective gene fragment, and this site was inserted between BamHI and HindIII of the pGenesil plasmid. After the identification, a DNA segment of 400 bp was cut off from the recombinant plasmid by SalI, and the two positive plasmids and one negative plasmid were ideally designed. The transformation bacterium fluid was sent to Invitrogen Corporation for sequencing identification. The result was a match; the two positive plasmids were named pGenesil-PLC31 (P1) and pGenesil-PLC32 (P2); the one negative plasmid was named HK-A. The stable clonal cells were then screened by culture fluid contained RPMI 1640, 10% fetal calf serum, and G418 (400 mL/mL) and the stable clone was observed under an inverted research microscope in 30 days. The cells that had clear structure and form took on full green fluorescent protein in cytoplasm and nucleus. After transfection of recombinant plasmid through lipofectamine 2000, the T24 cells took on clostridial form and became irregular: the cell bodies and nuclei all displayed enlargement, the granules in cytoplasm increased in number, and the cells swelled slightly. Changes in transfection efficiency after 6e12 hours were determined through the observation of green fluorescence; the strongest green fluorescence occurred at 48 hours. The most suitable ratio of plasmid to liposome was 1:2.

3.2. Expression of PLCE1 mRNA in T24 cells The RT-PCR analysis of T24 RNA detected expression of PLCE1 mRNAs (Fig. 1). In terms of brightness of the bands, the blank control and HK-A groups exhibited stronger expression than did the P1 and P2 groups. Under analysis with the Bio-Rad image system (Hercules, CA), expression of PLCE1 mRNAs in the two positive plasmids was lower than in the blank control and HK-A groups, and the difference was significant (P ! 0.01). Relative to the blank control group, which was transfected with liposome, the inhibition ratio was 4.2% for the HK-A group, 76.0% for P1 and 73.9% for P2. Expression of the internal standard b-actin did not differ significantly between the groups (P O 0.05). These data show that both recombinant plasmids, P1 and P2, specifically inhibited the expression of PLCE1 in T24.

Fig. 1. Reverse transcriptaseepolymerase chain reaction (RT-PCR) analysis of the intracellular mRNA expression of the PLCE1 gene in T24 human bladder cancer cell line transfection groups. After 35 cycles, 5 mL of each sample was separated electrophoretically through agarose gel, and an expected product at 395 bp for PLCE1 was stained with ethidium bromide. Lane M, marker (100 bp); lane 1, blank control; lane 2, negative plasmid HK-A; lane 3, positive plasmid pGenesil-PLC31 (P1); lane 4, positive plasmid pGenesil-PLC32 (P2).

3.4. Transwell (Boyden chamber) cell migration assay The tumor cell migration assay indicated that transfection of PLCE1 shRNA could reduce the invasion power of the T24 cell line. The number of invasive cells in P1 and P2 groups was lower than in the blank control and HK-A groups (Fig. 3), and the difference was significant (P ! 0.01). There was no significant difference between the blank control and the HK-A group (P O 0.05) (Table 1).

3.3. Western blot analysis of PLCE1 protein The PLCE1 protein was expressed in all four transfection groups of the T24 cell line (Fig. 2). The optical density value for the bands for each of the four groups was compared with that of b-actin. The ratios of P1 and P2 groups were apparently lower than for the other two groups, and the difference was significance (P ! 0.01). The inhibition ratio was 65.4% for the P1 group and 64.8% for the P2 groups; this difference was not significant (P O 0.05).

Fig. 2. Western blot analysis of PLCE1 protein expression (PLC3) in T24 human bladder cancer cell line transfection groups. (A) Integrated optical density (IOD) analysis of PLC3 expression in Western blot. (B). PLC3 expression in T24 cells was measured by immunoblotting, normalized to b-actin expression in T24 cells. Lane 1, control; lane 2, shRNA pGenesilPLC3 plasmid 1; lane 3, HK-A; lane 4, shRNA pGenesil-PLC3 plasmid 2.

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

115

3.5. Gelatin enzymography assay of MMP-2 and MMP-9

Table 1 Effect of RNA interference on migrant cells, by T24 human bladder cancer cell line transfection group

The gelatin enzymography assay indicated that cells of all four transfection groups could secrete MMP-2 and MMP-9, but the level of secretion was different in each group (Fig. 4). The brightness of the band in the blank control and HK-A groups was stronger than in the P1 and P2 groups, and the difference was significant (P ! 0.01). There was no significant difference between the blank control and the HK-A group (P O 0.05).

Group

Visual fields, no. No. of cells/field, mean 6 SD P-valuea

P1 P2 Blank control HK-A

5 5 5 5

3.6. Immunocytochemistry analysis of MMP-2 and MMP-9 After immunocytochemistry analysis, photos were taken of each group of T24 cells. These images showed that the samples were full of T24 cells with uniformity, and positive granules were mainly distributed over cytoplasm of tumor cells, maybe covering the nucleus; the positive granules were stained buffy (Fig. 5). The blank l and HK-A groups both had strong expression MMP-2 and MMP-9 in cells, with no significant difference between them (P O 0.05). By contrast, the level of expression in P1 and P2 groups, compared with negative plasmid HK-A group, was clearly weakened (P ! 0.01) (Table 2).

25.8 26.8 34.8 33.8

6 6 6 6

6.2 5.8 6.9 5.7

!0.01 !0.01 d O0.05

Abbreviations: HK-A, negative plasmid; P1, positive plasmid pGenesilPLC31; P2, positive plasmid pGenesil-PLC32; SD, standard deviation. a The P-value is for comparison with the blank control. For P1 vs. P2, P O 0.05.

3.7. RT-PCR analysis of BCL2 mRNA levels among different T24 cell line groups Expression levels of the BCL2 mRNA were analyzed by RT-PCR in the blank control, HK-A, and the RNA interference-plasmid transfected cell lines (P1 and P2). Expression of BCL2 mRNA was lower in the two positive plasmid-transfected cell groups, compared with the blank control and HK-A groups, at almost the same levels; by contrast, expression of BCL2 mRNA was high in both the blank control and in HK-A (Fig. 6). The optical density values of all straps were analyzed by Quantity One 4.4.0 software; optical density for BCL2 had a tendency toward

Fig. 3. Cells of T24 human bladder cancer cell line transfection groups, hematoxylineeosin stain in Boyden chamber assay. These cells are the ones that have migrated to the lower side of the membrane. (A) Blank control (400). (B) shRNA pGenesil-PLC3 plasmid 1 (400). (C) Blank control (100). (D) shRNA pGenesil-PLC3 plasmid 1 (100).

116

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

Fig. 4. Gelatin enzymography assay of MMPs expression level in T24 human bladder cancer cell line transfection groups. (A) Integrated optical density (IOD) analysis of matrix metalloproteinases MMP-2 and MMP-9 from gelatin enzymography. (B) Gelatin enzymogram: lane 1, MMP-2, MMP-9 expression level of group pGenesil-PLC31; lane 2, MMP-2, MMP-9 expression level of group pGenesil-PLC32; lane 3, MMP-2, MMP-9 expression level of group HK-A; lane 4, MMP-2, MMP-9 expression level of group control.

reduction. The difference between positive plasmids and negative plasmids was significant (P ! 0.05).

4. Discussion The most frequently detected alterations in oncogenes in animal and tumor models of human cancer are mutations in the RAS family of oncogenes. All RAS gene products have GTPase activity and regulate cell growth and differentiation. Mutations in members of the RAS gene family are found in a wide variety of human cancers. There is an association between the mutated RAS gene family (HRAS, KRAS, and NRAS ) and 30% of all human cancers, including bladder cancer. Some studies substantiate the concept that activation of the HRAS oncogene by point mutation and the activation of all RAS genes (mainly KRAS and NRAS ) by overexpression are frequent events in bladder carcinoma cases. Increased RAS mRNA levels may indicate bladder carcinoma development, but they do not appear to be responsible for its progression. Currently, little is known about the molecular changes that lead to development of bladder tumors. The PLCE1 protein was identified as a direct downstream effector of small GTPases Ras, Rap1 and Rap2. According to Boulalas et al. [8], a significant percent of HRAS gene codon 12 mutations are present in bladder carcinoma cases; they detected 9 of 30 bladder transitional-cell carcinomas (30%) bearing HRAS mutations (G¡A and G¡T) in codon 12, which are the most commonly reported point mutations.

RNA interference regulates gene expression by the cleavage of messenger RNA, by mRNA degradation, and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNAinduced silencing complex) [26]. A short hairpin RNA (shRNA) or short interfering RNA (siRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. Although, the requirement for an effective siRNA are not completely understood, experience indicates that choice of siRNAs based on published guidelines and on our own experience will result in approximately two sequences being effective at downregulation, at least to some extent [27]. Our objective in this study was to examine the effects on metastasis and invasion of specific inhibition of the PLCE1 expression in tumor cells, to determine if PLCE1 is a valid therapeutic target. Przybojewska and colleagues found the HRAS mutation in 84% of patients with bladder cancer using a PCR-restriction fragment length polymorphism assay, the relationship of oncogene H-ras with carcinogenesis and progress of bladder cancer has been confirmed by them [28]. The PLCE1 protein should, therefore, as a novel bifunctional phospholipase C, have important effect on regulation of tumor cell after activation by mutein products of HRAS. In this study, we used plasmid pGenesil working as vector of shRNA expression. After transfection into tumor cells, the plasmids could express shRNA directly. The shRNA included two short inverted repeat sequences and had a structure of loop to separate in the middle; the hairpin was constructed and it was further elaborated for siRNA to produce a marked effect. Through this mechanism of action, specific shRNA expression vector aiming at PLCE1 successfully expressed the siRNA and brought about the desired interfering effect. The oligofectamine reagent used in the study had two key points: one was that the complex of DNAelipofectamine could be added into the nutrient medium directly; the other was that the reagent had no influence on the experiment result. Transfection caused profound cell deformities. Cells took on fusiform or irregular shape, the cell body and nucleus enlarged, granules in cytoplasm grew in number, and cells slightly swelled. When the ratio of plasmid to liposome was 1:2, the green fluorescence intensity reached the highest level at 48 hours. The number of cells having the green fluorescence should be increased through the screening of positive clone. We applied fluorescently labeled expression vector, so coefficient of transfection at different times in the different groups was easily detected through fluorescent labeling. The expression of fluorescein was independent of expression of shRNA, and did not influence the effectiveness of gene silencing. We demonstrated initially that shRNA specific for PLCE1 could be successfully transfected into T24 tumor cells, resulting in significantly reduced gene transcription or expression level of protein.

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

117

Fig. 5. Immunocytochemical analysis of MMP-2 expression in T24 human bladder cancer cell line transfection groups. (A) Blank control (400). (B) pGenesil-PLC3 plasmid 1 (400). (C) Blank control (100). pGenesil-PLC3 plasmid 1 (100).

The successful construction of expression vector for gene silencing about PLCE1 which had stable and lasting induction effect may establish basis for study of genetic mechanism for bladder carcinoma. We have demonstrated that levels of PLCE1 mRNA expression in bladder tumor cells follow the transfection of recombinant plasmids, with significantly higher levels of expression in blank control T24 cells than in transfected cells. In fact, it was very important for us to validate the high expression level of PLCE1 in T24 cells before we started this study and the result was just in line with expectations. Among 31 bladder cancer cell lines, the T24 cell line is known to have high invasion power. Levels of PLCE1 transcripts derived from expression array data for four groups of T24 cells showed higher levels in groups

of blank control and negative plasmid, but in the positive plasmid groups P1 and P2 the levels were both significantly lower. The two positive plasmids had almost equal inhibition ratio for PLCE1 mRNA in RT-PCR analysis. The negative plasmid group had very low inhibition ratio, compared with the positive P1 and P2 groups. We achieved almost stable interference of PLCE1 using plasmid-delivered shRNA. This method has the advantage that the plasmids transfected in a very high percentage of cells, yielding mass populations of transfected cells and so avoids potential problems with interclone differences that arise when using stable clones of transfected cells. This approach reduced PLCE1 transcript levels by up to 76.01%. To confirm the result of RT-PCR, we completed Western blot analysis and got almost the same inhibition

Table 2 Optical density of MMP-2 and MMP-9 in T24 human bladder cancer cell line transfection group OD of unit area, mean 6 SD Group

MMP-2

P1 P2 Blank control HK-A

0.021 0.034 0.134 0.100

6 6 6 6

0.001 0.005 0.006 0.006

P-valuea

MMP-9

!0.01 !0.01 d O0.05

0.023 0.024 0.095 0.094

6 6 6 6

P-valuea 0.008 0.001 0.007 0.004

!0.01 !0.01 d O0.05

Abbreviations: HK-A, negative plasmid; OD, optical density; P1, positive plasmid pGenesil-PLC31; P2, positive plasmid pGenesil-PLC32; SD, standard deviation. a The P-value is for comparison with the blank control. For P1 vs. P2, P O 0.05.

118

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119

Fig. 6. RT-PCR analysis of BCL2 mRNA expression in T24 human bladder cancer cells. After 30 cycles, 5 mL of each sample was separated electrophoretically through agarose gel, and an expected product at 395 bp for PLC3 was stained with ethidium bromide. Lane 1, marker (100 bp); lane 2, T24 pGenesil-PLC31 (P1); lane 3, T24 pGenesil-PLC32 (P2); lane 4, T24 blank control; lane 5, T24 HK-A. The specific size of the amplified BCL2 fragment was 131 bp, and the length of amplified GAPDH was 450 bp.

ratio, up to 76.60% for positive plasmids. Importantly, we have shown that interference of PLCE1 in T24 cells inhibits not only proliferation but also invasion. This shRNA was effective at downregulating PLCE1 in bladder cancer T24 cells. From MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] of cell viability after transfection, we observed that inhibition ratio of cell proliferation was 1.48% for negative plasmid, and 24.48%e25.29% for positive plasmids [28]. Decrease in metastasis was found following PLCE1 gene downregulation. According to many previous studies, MMPs have important function in tumor metastasis and vasoformation. We tried to apply immunocytochemistry and gelatin enzymography for detection of MMPs expressed. Then, we conducted a transwell chamber experiment to determine the invasive power of tumor cells. We confirmed the result for MMPs, with parallel results for both array assay methods. The expression of BCL2 mRNA was analyzed, as a reflection of malignant power of each T24 cell line group. Some previous studies had indicated enhancement of malignant potential of bladder cancer cells in vivo by overexpression of BCL2 protein, revealing a crucial role of antiapoptotic activity during tumor progression. In the present study, the findings for BCL2 were similar. Through RNA interference of the PLCE1 gene, the expression of BCL2 was significantly reduced in the two transfected cell clones. This result suggests that silencing of PLCE1 might be a useful adjuvant to chemotherapy for bladder cancer. In brief, this was considered to be a feasibility study, and we know of no such previous research. We demonstrated that a potent shRNA directed to PLCE1 with inhibition effect could be selected from a library of PLCE1 target sequences. The shRNA was dependent on the double-strand structure of

the RNA and has target site specificity. Most importantly, our preexperiment algorithm predicted potent shRNA expressed using efficient plasmid vectors or direct transfection of chemically synthesized siRNA. In this experiment, high levels of siRNAs were present within the cells. We designed independently and constructed shRNA directed to PLCE1 and imported recombinant plasmid into bladder cancer T24 cells. The result showed that this transfection could decrease PLCE1 gene and protein expression; MMP-2 and MMP-9 activity decreased also, and the invasive power of T24 tumor cells were effectively inhibited. The result of our research suggests that RNA interference of PLCE1 may inhibit metastasis of T24 cells through inactivation of protein kinase C (the downstream signaling molecule of the Ras signal pathway mediated by the PLCE1 protein). Thereby, the possibility of inhibition of metastasis through the RasePLC3 signal pathway was confirmed, and the RasePLC3 signal pathway might serve as a new target point for gene therapy of carcinoma of the urinary bladder. The PLCE1 gene is closely associated with tumor development and metastasis, but its invasive power should be decreased by shRNA directed to PLCE1. This discovery may provide a fresh theoretical basis and research direction for therapy of bladder carcinoma. Questions remain in understanding of the PLCE1 gene, such as the concrete effect channel, the interrelationship, molecule mechanism of regulations, and so on. Nevertheless, further studies could confirm that effective shRNAs can be obtained for long-term therapeutic purposes, such as for use in gene therapy. Through transfection of eukaryotic cells, the shRNA was imported into human bladder cancer cell line T24, and the regulatory effect on gene expression of this procedure was observed. An influence on the invasive power of T24 was observed, suggesting a crucial role of PLCE1 in bladder cancer development downstream of Ras signaling. shRNAbased gene therapy for bladder cancer remains an intriguing potential future therapy for bladder cancer. Additional advances in gene delivery and the establishment of novel vector safety will be required to move this therapeutic option out of the laboratory and into the clinical setting.

Acknowledgments The research team recognizes the support provided by grants from the Project Foundation of Chongqing Municipal Education Committee (KJ080306).

References [1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics. CA Cancer J Clin 2002;2005(55):74e108. [2] Tran E, Souhami L, Tanguay S, Rajan R. Bladder conservation treatment in the elderly population: results and prognostic factors of muscle-invasive bladder cancer. Am J Clin Oncol 2009;32:333e7.

L. Ou et al. / Cancer Genetics and Cytogenetics 200 (2010) 110e119 [3] Brendler CB, Steinberg GD, Marshall FF, Mostwin JL, Walsh PC. Local recurrence and survival following nerve-sparing radical cystoprostatectomy. J Urol 1990;144:1137e40. [4] Raghavan D, Huben R. Management of bladder cancer. Curr Probl Cancer 1995;19:1e64. [5] Frazier HA, Robertson JE, Dodge RK, Paulson DF. The value of pathologic factors in predicting cancer-specific survival among patients treated with radical cystectomy for transitional cell carcinoma of the bladder and prostate. Cancer 1993;71:3993e4001. [6] Levesque P, Ramchurren N, Saini K, Joyce A, Libertino J, Summerhayes IC. Screening of human bladder tumors and urine sediments for the presence of HRAS mutations. Int J Cancer 1993;55:785e90. [7] Bonner RB, Hemstreet GP 3rd, Fradet Y, Rao JY, Min KW, Hurst RE. Bladder cancer risk assessment with quantitative fluorescence image analysis of tumor markers in exfoliated bladder cells. Cancer 1993; 72:2461e9. [8] Boulalas I, Zaravinos A, Karyotis I, Delakas D, Spandidos DA. Activation of RAS family genes in urothelial carcinoma. J Urol 2009;181: 2312e9. [9] Song C, Hu CD, Masago M, Kariyai K, Yamawaki-Kataoka Y, Shibatohge M, et al. Regulation of a novel human phospholipase C, PLCe, through membrane targeting by Ras. J Biol Chem 2001; 276:2752e7. [10] Li M, Edamatsu H, Kitazawa R, Kitazawa S, Kataoka T. Phospholipase Ce promotes intestinal tumorigenesis of ApcMin/þ mice through augmentation of inflammation and angiogenesis. Carcinogenesis 2009;30:1424e32. [11] Wing MR, Bourdon DM, Harden TK. PLC-e: a shared effector protein in Ras-, Rho-, and Gabg-mediated signaling. Mol Interv 2003;3:273e80. [12] Matsubara K, Kishida S, Matsuura Y, Kitayama H, Noda M, Kikuchi A. Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation. Oncogene 1999;18:1303e12. [13] Chen H, Chen Y, Zhang Z, Ding Y, Lin X, Zhang G, et al. Study of the mechanism of regulating effect of protein kinase C on the metastasis and invasion of large intestine carcinoma cells [In Chinese]. Practical J Cancer 2004;19:361e4. [14] Hojilla CV, Mohammed FF, Khokha R. Matrix metalloproteinases and their tissue inhibitors direct cell fate during cancer development. Br J Cancer 2003;89:1817e21. [15] Bjo¨rklund M, Koivunen E. Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta 2005;1755:37e69. [16] Gerhards S, Jung K, Koenig F, Daniltchenko D, Hauptmann S, Schnorr D, et al. Excretion of matrix metalloproteinases 2 and 9 in

[17]

[18]

[19]

[20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28]

119

urine is associated with a high stage and grade of bladder carcinoma. Urology 2001;57:675e9. Gohji K, Fujimoto N, Komiyama T, Fujii A, Ohkawa J, Kamidono S, et al. Elevation of serum levels of matrix metalloproteinase-2 and -3 as new predictors of recurrence in patients with urothelial carcinoma. Cancer 1996;78:2379e87. St-Pierre Y, Van Themsche C, Este`ve PO. Emerging features in the regulation of MMP-9 gene expression for the development of novel molecular targets and therapeutic strategies. Curr Drug Targets Inflamm Allergy 2003;2:206e15. Kader AK, Shao L, Dinney CP, Schabath MB, Wang Y, Liu J, et al. Matrix metalloproteinase polymorphisms and bladder cancer risk. Cancer Res 2006;66:11644e8. Miyake H, Hara I, Yamanaka K, Gohji K, Arakawa S, Kamidono S. Overexpression of Bcl-2 enhances metastatic potential of human bladder cancer cells. Br J Cancer 1999;79:1651e6. Ruifa H, Liwei L, Binxin L, Ximing L. Additional gene therapy with rAAV-wt-p53 enhanced the efficacy of cisplatin in human bladder cancer cells. Urol Int 2006;77:355e61. Bochner BH. Gene therapy in bladder cancer. Curr Opin Urol 2008; 18:519e23. Zhang T, Li X, Yu W, Yan Z, Zou H, He X. Overexpression of thymosin beta-10 inhibits VEGF mRNA expression, autocrine VEGF protein production, and tube formation in hypoxia-induced monkey choroid-retinal endothelial cells. Ophthalmic Res 2009;41:36e43. Wu D, Tadano M, Edamatsu H, Masago-Toda M, YamawakiKataoka Y, Terashima T, et al. Neuronal lineage-specific induction of phospholipase C expression in the developing mouse brain. Eur J Neurosci 2003;17:1571e80. Cross KJ, Bomsztyk ED, Weinstein AL, Teo EH, Spector JA, Lyden DC. A novel method for targeted gene therapy in ischemic tissues through viral transfection of an expression cassette containing multiple repetitions of hypoxia response element. Plast Reconstr Surg 2009;123(2 Suppl):76Se82S. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000;404:293e6. Shimizu S, Kamata M, Kittipongdaja P, Chen KN, Kim S, Pang S, et al. Characterization of a potent non-cytotoxic shRNA directed to the HIV-1 co-receptor CCR5. Genet Vaccines Ther 2009;7:8. Przybojewska B, Jagiello A, Jalmuzna P. H-RAS, K-RAS, and N-RAS gene activation in human bladder cancers. Cancer Genet Cytogenet 2000;121:73e7.