Iscove's modified Dulbecco's medium (IMDM), Opti-MEM^® I reduced serum medium and fibronectin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Matrigel and Transwell chambers were purchased from BD Biosciences (Franklin Lakes, NJ, USA). The antibodies used were against LPXN (dilution, 1:500; ab67571; Abcam, Cambridge, UK), β-actin (dilution, 1:3,000; #4967S; Cell Signaling Technology, Inc. Danvers, MA, USA) and p-mitogen-activated protein kinase (p-MAPK; dilution, 1:1,000; #9910; Cell Signaling Technology, Inc.). The small interfering RNA (siRNA) sequences of fluorescein amidite (FAM)-siRNA, LPXN-siRNA and luciferase-siRNA were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China).

The leukemia cell line SHI-1 was gifted by the Jiangsu Institute of Haematology, First Affiliated Hospital of Soochow University (Suzhou, China) and was originally derived from the mononuclear cells of the bone marrow from a patient with acute monocytic leukemia in relapse. The cells were maintained as a stable cell line in vitro (11). SHI-1 cells were incubated in IMDM with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Cells in the exponential growth phase following passaging every 3–4 days were used for the subsequent experiments.

Specific siRNAs of the LPXN gene were designed according to a previously described protocol (12). Transfection of SHI-1 cells was accomplished using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol with the following different LPXN gene-specific siRNA duplexes: L1-siRNA, sense, 5′-UAUUCCAACCCAGCUCCUC-3′ and antisense, 5′-GAGGAGCUGGGUUGGAAUA-3′; L2-siRNA, sense, 5′-GGCGCAGCUCGUGUAUACUACCAAU-3′ and antisense, 5′-AUUGGUAGUAUACACGAGCUGCGCC-3′ (Shanghai GenePharma Co., Ltd.). The negative control group was transfected with siRNA duplex oligonucleotides against the firefly luciferase gene (N-siRNA) (Shanghai GenePharma Co., Ltd.) and the positive control group was transfected with FAM-siRNA in SHI-1 cells.

To improve the transfection efficiency, several modifications to the transfection protocol were performed. Briefly, prior to plating the cells in 400 µl Opti-MEM^® I in 24-well dishes, 4×10⁵ SHI-1 cells were washed with serum-free IMDM. Prior to combining the reagents, the siRNA oligomer and Lipofectamine 2000 were diluted in 50 µl Opti-MEM^® I. The transfection efficiency was assessed with FAM-siRNA using flow cytometry (FCM), as previously described by Wang et al (11).

According to the manufacturer's protocol of Membrane and Cytosol Protein Extraction kit (Beyotime Institute of Biotechnology, Nanjing, China), total cellular extracts were collected and the protein concentration was determined after transfection 48 h. Equal amounts of extracted proteins were subjected to western blotting and the specific experiments were conducted as previously described (13). Following SDS-PAGE (5% concentration gel and 10% separation gel) electrophoresis and membrane transfer to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA), the proteins were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 2 h and then incubated with the primary antibodies against LPXN (Abcam), phosphorylated (p)-c-Jun, N-terminal kinase (p-JNK), p-p38 MAPK, p-extracellular-signal-regulated kinase (p-ERK) (Phospho-MAPK Family Antibody Sampler kit; dilution, 1:1,000; cat. no. 9910) and β-actin (dilution, 1:3,000; cat. no. 4967S) (both from Cell Signaling Technology, Inc.) at 4°C overnight separately. The membranes were washed three times in TBS with 0.1% Tween-20 and subsequently probed with HRP-conjugated goat anti-mouse IgG (dilution, 1:10,000; cat. no. 7072; Cell Signaling Technology, Inc.) at room temperature for 2 h. Following incubation, the blots were washed three times in TBS with 0.1% Tween-20, enhanced chemiluminescent reagents (Beyotime Institute of Biotechnology) were added and scanned with an ImageQuant LAS 4000 Mini (GE Healthcare, Chicago, IL, USA).

A CCK-8 assay was used to determine proliferation according to the manufacturer's protocol. SHI-1 cells were transfected with siRNA against LPXN and the luciferase gene, serving as the negative control, as aforementioned. Following incubation with the transfection medium at room temperature. for 24 h, cells were harvested and seeded in a 96-well plate at 4×10⁵ cells/well with three wells of each group. Cells were incubated for an additional 48 h and the spectrophotometric absorbance of each sample was determined at a wavelength of 450 nm, following the addition of 10% CCK-8 for 3 h.

A Costar Transwell chamber system was used to determine cell invasion. Membrane filters with a pore size of 8 µm and a diameter of 6.5 mm were coated with 50 µl Matrigel and subsequently placed into the wells forming the upper chamber. Following transfection for 24 h, the siRNA-treated SHI-1 cells were suspended in 200 µl serum-free IMDM and were seeded at a final density of 2.0×10⁵ cells/well in the upper chamber. The lower chamber of invasion was filled with 800 µl IMDM, supplemented with 10% human serum, containing 100 ng/ml stromal-cell-derived factor 1-α as a source of chemoattractants. The chambers were incubated for 24 h at 37°C with 5% CO₂. The cells which had migrated into the lower compartment were counted by inverted optical microscope (magnification, ×400; Leica Microsystems GmbH, Wetzlar, Germany) and the invasion rate was calculated by comparing the cell number in the lower compartment medium with the total number of leukemia cells, 2.0×10⁵ cells, loaded into the upper compartment.

Cell culture supernatants were analyzed for the presence of the secreted MMP-2 and MMP-9 gelatinases using zymography. SHI-1 cells were transfected with siRNA against LPXN and the luciferase gene, serving as the negative control, as aforementioned. Following incubation with the transfection medium for 24 h, SHI-1 cells were harvested and seeded in a 96-well plate at 4×10⁵ cells/well with serum-free IMDM. The supernatant of each sample was harvested after the 24 h incubation. The samples were separated by SDS-PAGE (10% gels) copolymerized with 0.1% gelatin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C and were incubated at 37°C with developing buffer containing 0.02% Brij-35 (Sigma-Aldrich; Merck KGaA), according to our previously published protocol (13). Gelatinase-digested regions were visualized as light bands against a blue background at 37°C under natural light.

All experiments were repeated at least three times and the data were expressed as the mean ± standard deviation. The image processing software Photoshop CS3 (Adobe Systems Inc., San Jose, CA, USA) and statistical software SPSS (version 15.0; SPSS, Inc., Chicago, IL, USA) was used for analysis. Student's t-test was used to compare the means of two groups and one-way analysis of variance was used to compare means of multiple samples followed by the Least-Significant-Difference and Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Among the repeated experiments, the highest transfection efficiency reached 74.5%, when the SHI-1 cells were seeded at a density of 4×10⁵ cells/ml and the mixing ratio of FAM-siRNA/Lipofectamine 2000 was 200 pmol/1 µl (Fig. 1). Therefore, these conditions were used for transfection of LPXN siRNA in SHI-1 cells.

After 48 h of transient transfection of L1-siRNA and L2-siRNA, the protein expression of LPXN was detected using western blot analysis. As indicated in lane 1 (200 pmol/1 µl) and lane 2 (100 pmol/1 µl) in Fig. 2, transfection of L1-siRNA had no effect on the expression of the LPXN protein. Similarly, lane 3 (200 pmol/1 µl) and lane 4 (100 pmol/1 µl) of the N-siRNA, negative control group, did not change LPXN expression levels. In contrast, transfection of L2-siRNA, indicated in lanes 5 (200 pmol/1 µl) and 6 (100 pmol/1 µl), downregulated LPXN expression in SHI-1 cells compared with the negative control group. In particular, when the transfection ratio of L2-siRNA/Lipofectamine 2000 was 200 pmol/1 µl, LPXN expression was effectively downregulated, as indicated in lane 5. Therefore, in subsequent experiments, L2-siRNA was transfected at the optimal ratio of 200 pmol L2-siRNA/1 µl Lipofectamine 2000 to investigate the effect of LPXN on the proliferation and invasion of SHI-1 cells.

The effective L2-siRNA and N-siRNA negative control were transfected into SHI-1 cells at a ratio of 200 pmol siRNA/1 µl Lipofectamine 2000. The proliferation of SHI-1 cells was determined using the CCK-8 assay followed by transient transfection for 48 h. The inhibition proliferation rate of the L2-siRNA transfection group was 27.043±2.051%, compared with that of the negative control group of 8.247±1.003% (Fig. 3). In addition, there was a significant difference between these two groups (P<0.05), which indicated that transfection with L2-siRNA decreased the proliferation rate in SHI-1 cells by downregulating LPXN expression.

Furthermore, following transfection with L2-siRNA and N-siRNA for 24 h, the SHI-1 cells were collected and plated in Transwell chambers to determine the transmembrane invasion rate of each group. The transmembrane invasion rate of the L2-siRNA transfection group was 25.270±2.145 and the invasion rate of the N-siRNA control group was 36.112±2.103 (Fig. 3). The transfection efficiency of siRNA in suspension cells was not high; however, the results of the present study indicated that downregulating the expression of LPXN by L2-siRNA decreased the invasion of SHI-1 cells.

To investigate further the effect of LPXN on invasion, the culture supernatant of SHI-1 cells transfected with L2-siRNA and N-siRNA was collected, and the gelatinase level was analyzed by gelatin zymography. There were two white bands in the gelatin, which were identified as MMP-2 (72 kDa) and MMP-9 (92 kDa) (Fig. 4). The gelatinase levels of MMP-2 and MMP-9 between the negative control group in lane 2, transfected with N-siRNA, and the blank control group in lane 1, without any siRNA, remained relatively unchanged (Fig. 4). However, the gelatinase levels in the supernatant of the L2-siRNA transfection group in lane 3 were decreased compared with the aforementioned control groups (Fig. 4). Previous studies have indicated that MMP-2 and MMP-9 serve important functions in the invasion process of various tumors (11,13). Therefore, these results indicated that downregulating LPXN expression by L2-siRNA potentially decreased the secretion of MMP-2 and MMP-9, weakening the invasion of SHI-1 cells.

MAPKs are serine/threonine protein kinases that have been reported to serve an important function in extrinsic and intrinsic signal transduction pathways, and regulate proliferation, apoptosis and invasion (13). In the present study, the protein expression of p-ERK, p-JNK and p-p38 MAPK were detected by western blotting after 48 h of transient transfection with L2-siRNA and N-siRNA. In the blots presented in Fig. 5A, lane 1 was the blank control group without any siRNA, lane 2 was the negative control group with N-siRNA transfection and lane 3 was the L2-siRNA transfection group. The expression level of p-ERK in these three groups remained unchanged; however, the expression level of p-p38 MAPK and p-JNK was increased in the L2-siRNA transfection group (lane 3) compared with the aforementioned control groups (lanes 1 and 2).

In addition, Photoshop CS3 and SPSS software were used to analyze the results for the expression level of p-MAPK. No difference in the expression levels of ERK, JNK and p38 MAPK between the negative group and the blank control group was identified. However, there were significant differences in the expression level of p-JNK and p-p38 MAPK between the negative group and the L2-siRNA transfection group (P<0.05; Fig. 5B). The increase in the p-JNK and p-p38 MAPK expression level may not be apparent, due to the fact that the transfection efficiency of siRNA in SHI-1 cell suspensions was not high; however, the changes remained statistically significant. Therefore, it was hypothesized that downregulating the expression of LPXN by L2-siRNA activated p-JNK and p-p38 MAPK to inhibit SHI-1 proliferation and invasion.