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.
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.
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).