Expression of Signal-induced Proliferation-associated Gene 1 (SIPA1), a RapGTPase-activating Protein, Is Increased in Colorectal Cancer and Has Diverse Effects on Functions of Colorectal Cancer Cells

Materials and Methods

Colorectal tissues and cell lines. Colorectal cancer tissues (n=94) and normal background tissues (n=80) were collected immediately after surgery from patients with colorectal cancer (with the local Research Ethic Committee approval) and stored at –80°C until use. Two human colorectal adenocarcinoma cell lines, HT115 and Caco-2 were obtained from the European Collection for Animal Cell Culture (ECACC, Porton Down, Salisbury, UK).

RNA extraction. RNA extraction kits, reverse transcription kits and Reverse Transcription Polymerase Chain Reation Mix were purchased from Promega (WI, USA) and Bio-Rad (CA, USA). Conventional PCR primers were designed using Beacon Designer (PREMIER Biosoft) and synthesized by Invitrogen (Paisley, Scotland, UK). Following the manufacturer’s protocol, total RNA was isolated using a standard guanidine isothiocyanate method. The concentration of RNA was determined using UV spectrophotometry at 260 and 280 nm. cDNA samples were synthesized in a total volume of 20 μl of reaction mixture. Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) primers were used as house keeping genes.

Real-time quantitative polymerase chain reaction (QPCR). Real-time quantitative PCR was used for verifying the level of mRNA expression of SIPA1 from the prepared cDNA samples. All colorectal cDNA samples were synchronously examined for SIPA1 along with an applicable set of plasmid standards. QPCR primers for SIPA1 were designed using the Beacon Design software (PREMIER Biosoft). Sequences of primers used in the current study are provided in Table I. Real-time PCR was carried out using IcyclerIQ™ (Bio-Rad, Hemel Hempstead, UK), following the cycling conditions: 94°C for 5 min, 80-90 cycles of: 94°C for 10 s, 55°C for 35 s and 72°C for 20 s.

Ribozyme transgene targeting human SIPA1. Anti-human SIPA1 hammerhead ribozymes were designed using the Zuker NA mFold program, on the basis of the secondary structure of SIPA1 mRNA (14). The ribozymes were synthesized using touchdown PCR and cloned into the pEF6/V5-His TOPO TA Expression plasmid vector (Invitrogen), according to the protocol provided. Ribozyme transgenes and empty plasmids were transfected into the two colorectal cell lines HT115 and Caco-2, respectively, utilizing an Easyjet Plus electroporator (EquiBio, Kent, UK). Following selection of cells using blasticidin, verified transfectants which had lost the expression of SIPA1 were used in the subsegment experiments.

In vitro cell growth assay. Cells were seeded into a 96-well plate at 2,500 cells/well, which were cultured using normal media (10% fetal cattle serum, 0.1% antibiotics). The cells were cultured in triplicate for 1, 3 and 5 days. After incubation the cells were fixed in 4% formalin and stained by 0.5% crystal violet (w/v). The stained crystal violet was then extracted using 10% (v/v) acetic acid, and the absorbance was determined using a spectrophotometer (Bio-Tek, ELx800), at a wavelength of 540 nm.

In vitro cell adhesion assay. A 96-well plate was pre-coated with 5 μg of Matrigel (CollaborativeResearch Products, Bedford, MA, USA) and allowed to air dry. Following rehydration using serum-free media, 40,000 cells were seeded into each well. After 40 min of incubation, non-adherent cells were washed-off using balanced salt solution. The adherent cells were then fixed with 4% formalin and stained using 0.5% crystal violet. The number of adherent cells was counted under a microscope.

In vitro invasion assay. Transwell inserts (with 8-μm pores) were pre-coated with 50 μg of Matrigel and air dried. Following rehydration, 40,000 cells were seeded into each insert. After incubation for 3 days, cells which had invaded through the matrix and adhered to the other side of the insert were fixed in 4% formalin, and stained with 0.5% (w/v) crystal violet. The number of invaded cells was then counted under a microscope.

In vitro wounding assay. Cells were seeded into a 24-well plate at a density of 200,000 per well and allowed to form a monolayer of cells. The monolayer of cells was then scraped to create a wound. Migration of the cells at wounding edges was monitored over a period of up to 18 hours. Optimas 6.0 Motion Analysis (Meyer Instruments, Houston, TX, USA) was used to track the leading edge of the cells, in order to measure the distance of the migration.

Immunohistochemical staining of SIPA1 protein. The frozen sections of colorectal tumors and adjacent background tissues were sectioned at a thickness of 6 μm using a cryostat (15). The samples were mounted onto Super Frost Plus microscope slides. After air-drying, the samples were fixed in a mixture of 50% acetone and 50% methanol and then air-dried once again. After rehydration and blocking with 0.6% solution of horse serum, the sections were probed with a rabbit polyclonal anti-SIPA1 antibody (Santa Cruz Biotechnology, Inc., CA, USA) and subsequently a peroxidase-conjugated anti-rabbit antibody (Sigma, Dorset, UK). Following the instructions, the avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) was applied before staining with diaminobenzidine chromogenI. Nuclei were counterstained in Gill’s haematoxylin.

Data analysis. The relationship between SIPA1 expression and tumor grade, TNM staging and nodal status was respectively analysed using the Mann-Whitney U and Kruskal-Wallis tests. Survival analysis curves were drawn using Kaplan Meier survival analysis. Quantitative data were analysed using the Student’s t-test, and chi-square test, where appropriate. Differences were considered to be statistically significant at p<0.05.