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Clinical significance of human kallikrein 12 gene expression in gastric cancer - PubMed

  • ️Sun Jan 01 2012

Clinical significance of human kallikrein 12 gene expression in gastric cancer

En-Hao Zhao et al. World J Gastroenterol. 2012.

Abstract

Aim: To investigate whether the expression of kallikrein 12 (KLK12) is related to the development of gastric cancer (GC) and to determine the role of KLK12 in gastric cancer cells growth, invasion and migration.

Methods: Between September 2007 and March 2008, 133 patients with histologically confirmed GC were recruited for the study. Expression of KLK12 was detected in samples from GC patients by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry. The relationship between KLK12 protein expression and clinicopathological features of GC was analyzed. The difference in 5-year survival rates between the high KLK12 protein expression group and the low KLK12 expression group was compared. Additionally, the expression of KLK12 was examined in various human GC cell lines, including MKN-28, SGC-7901 and MKN-45. Small interfering RNA (siRNA) was used to inhibit KLK12 expression in MKN-45 cells. Cell clones stably transfected with KLK12 siRNA were tested for KLK12 expression by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. Furthermore, a series of functional assays were performed in this study to assess the biological features of transfected cells. Cell proliferation was assessed using the methylthiazolyltetrazoliumassay. Finally, cell migration and invasion were assessed using transwell chamber assays.

Results: Of the 133 GC patients included in the study, 126 (94.7%) showed a higher expression level of KLK12 mRNA when compared to noncancerous tissue specimens. Expression of KLK12 mRNA was significantly higher in GC tissues than in normal tissue (P < 0.001). KLK12 protein expression was detected in 96 of 133 (72.2%) GC samples with moderate or strong staining primarily in the cytoplasm. In contrast, negative immunostaining for KLK12 protein was observed in the corresponding normal gastric mucosal tissue. Overexpression of KLK12 protein was significantly associated with lymph node metastasis (P = 0.001), histological type (P < 0.001) and tumor-node-metastasis stage (P = 0.005), while no significant correlation was observed between expression of KLK12 protein and sex, age, depth of invasion, tumor size or lymphatic invasion. Furthermore, patients with high KLK12 expression had a significantly poorer 5-year survival rate than those with low KLK12 expression (P = 0.002). Expression of KLK12 mRNA was significantly higher in MKN-45 GC cells compared to normal mucosal cells or two other GC cell lines (P < 0.01). Expression of KLK12 in MKN-45 cells was downregulated after transfection with siRNA. Knockdown of KLK12 markedly decreased the proliferation of MKN-45 cells when compared with parent or mock-transfected cells (P = 0.001), especially from the 3rd to the 5th day of the assay. In migration assays, fewer KLK12 siRNA cells migrated through the chambers (22.00 ± 1.81) when compared to the parent (46.47 ± 2.42) or mock-transfected cells (45.40 ± 1.99); these differences were statistically significant (P < 0.001). However, in the invasion assay, the number of KLK12 siRNA cells that invaded the chambers was 18.40 ± 1.12, closely similar to both the parent (18.67 ± 0.98) and mock-transfected cells (18.53 ± 0.92). There was no significantly difference between the three groups in the invasion assay (P = 0.054).

Conclusion: The KLK12 gene is markedly overexpressed in GC tissue, and its expression status may be a powerful prognostic indicator for patients with GC. KLK12 might serve as a novel diagnosis and prognosis biomarker in GC.

Keywords: Gastric cancer; Human kallikrein 12; Immunohistochemistry; Invasion; Migration; Prognosis; Small interfering RNA.

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Figures

Figure 1
Figure 1

Upregulation of kallikrein 12 mRNA expression in gastric cancer. Quantitative real-time reverse transcription polymerase chain reaction showed that the mean expression value of kallikrein 12 (KLK12) mRNA in cancer tissues was significantly higher than the value in relevant normal tissues. Data are shown as mean ± SD, using the Student’s t test (bP < 0.01 between tumor and normal group, horizontal bars represent medians).

Figure 2
Figure 2

Expression of human kallikrein 12 protein in gastric cancer and noncancerous mucosal tissues detected by immunohistochemistry. A: Strong positive human kallikrein 12 protein (hK12) immunostaining in gastric cancer (GC) tissues, hK12 staining was observed in the cytoplasm of cancer cells; B: Weak positive hK12 immunostaining in GC tissues; C: Negative hK12 immunostaining in relevant normal tissues; D: Negative hK12 immunostaining in GC tissues (original magnification A, B, D ×200, C ×100).

Figure 3
Figure 3

Overall survival of patients with gastric cancer according to human kallikrein 12 protein expression in the cancer tissues. Patients in the high human kallikrein 12 protein (hK12) expression group had a significantly poorer prognosis than those in the low hK12 expression group. Survival curves are drawn according to the Kaplan-Meier method, using the log-rank test to compare the survival rates (P = 0.002).

Figure 4
Figure 4

Expression of kallikrein 12 mRNA in gastric cancer cell lines and normal gastric mucosal cell line. Gastric cancer cell lines show higher levels of kallikrein 12 (KLK12) mRNA expression than normal gastric mucosal cell line, while MKN-45 cells expressed the highest level of KLK12 across the four cell lines. Data are shown as mean ± SD, using the one-way analysis of variance test (bP < 0.01 vs other cell lines).

Figure 5
Figure 5

Efficiency of small interfering RNA in silencing the kallikrein 12 mRNA, and protein expression in MKN-45 cells. A: MKN-45 cells transfected with small interfering RNA targeting human kallikrein 12 (KLK12) gene showed a remarkable decrease in the level of KLK12 mRNA compared to mock-transfected or parent MKN-45 cells. Data are shown as mean ± SD, using the one-way analysis of variance test (bP < 0.01 vs other cell lines); B: Western blotting analysis showed a reduced protein expression in KLK12 small interfering RNA (siRNA) transfected cells. The protein levels are measured by Image J software (National Institutes of Health, United States) with β-actin protein normalization.

Figure 6
Figure 6

Reduction of cell proliferation by methylthiazolyltetrazolium assay after silencing the kallikrein 12. The proliferative ability significantly decreased in MKN-45 cells after transfection with kallikrein 12 small interfering RNA (siRNA), especially from the 3rd to 5th days of the assay. Data are shown as mean ± SD, using the one-way analysis of variance test (P = 0.001). MTT: Methylthiazolyltetrazolium.

Figure 7
Figure 7

Effect of cell migration by kallikrein 12 knockdown. A: 24-well transwell chambers with upper and lower culture compartments separated by polycarbonate membranes with 8-μm pores were used for migration or invasion assay. The chambers were stained by 0.09% crystal violet and cells were counted using light microscopy under high magnification (magnification ×10); B: The migration or invasion cells were counted in 5 individual fields per insert. Values were the number of cells. Data are shown as mean ± SD, using the one-way analysis of variance test (aP < 0.05 vs other cell lines).

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