Clinicopathological Significance Of The Expression Of Smad4, Kindlin‐2 And Anxa2 In Gastric Cancer

Objective: To study the expression of Smad4, Kindlin-2, Anxa2 in gastric cancer(GC). To assess the correlation of Smad4, Kindlin-2, Anxa2 expression with clinical pathological characteristics and MMP-9 expression in GC. To explore the role of Smad4, Kindlin-2 and Anxa2 expression in the occurrence and development of GC.Method:1. To investigate the level of Smad4, Kindlin-2 and Anxa2 m RNA in 50 pairs of GCs and corresponding adjacent non-neoplastic tissues by using Real time quantitative polymerase chain reaction(RT-q PCR). 2. Using Western Blot to quantitatively detect the protein expression of Smad4, Kindlin-2, Anxa2 in 20 pairs of GCs and corresponding adjacent non-neoplastic tissues. 3. We made gastric cancer tissue microarray and used immunohistochemistry(IHC) technique to positioning analyze the expression of Smad4, Kindlin-2, Anxa2 in 139 pairs of GCs and corresponding adjacent non-neoplastic tissues as well as to detect the expression of MMP-9 in GC tissues. Then we evaluated the correlation of the expression of Smad4, Kindlin-2, Anxa2 with GC clinicopathological parameters. The relationships between Smad4, Kindlin-2, Anxa2 protein expression and MMP-9 protein expression in GCs were also analyzed.Result:1. Clinicopathological significance of Smad4 expression in GC.RT-q PCR showed that the relative quantity(RQ) of Smad4 m RNA was 0.911±0.795 in GCs, and 1.222±0.806 in corresponding adjacent non-neoplastic tissues. There was no statistically significant difference between the two groups(P= 0.060). Western Blot showed that the level of Smad4 protein in GCs was 0.714±0.341, which was lower thancorresponding adjacent non-neoplastic tissues(1.149±0.596), with statistically difference(P=0.039). Immunohistochemistry staining showed Smad4 protein located in nucleus and(or) cytoplasm of tumor cells or adjacent non-neoplastic epithelial cells. Low or loss expression of Smad4 was observed in 61.9%(86/139) GCs, But consistent strong expression of Smad4 was found in all corresponding adjacent non-neoplastic tissues. Low or loss expression of Smad4 was correlated with adenocarcinoma of esophagogastric junction(P=0.016) and intestinal type adenocarcinoma(P=0.021). The spearman rank correlation test showed no apparent correlation between the Smad4 expression and MMP-9 expression in GCs(r=-0.025,P=0.772).2. Clinicopathological significance of Kindlin-2 expression in GC.RT-q PCR showed that the relative quantity of Kindlin-2 m RNA was 1.400±1.474 in GCs, and 1.167±1.522 in corresponding adjacent non-neoplastic tissues. There was no statistically significant difference between the two groups(P= 0.359). Western Blot test showed that the level of Kindlin-2 protein in GCs was 1.086±0.334, which was significantly higher than corresponding adjacent non-neoplastic tissues(0.665±0.178)(P=0.001). Immunohistochemistry staining showed Kindlin-2 protein located in stromal fibroblasts. The positive expression of Kindlin-2 was observed in 57.6%(80/139) GCs, but negative expression of Kindlin-2 was found in almost all corresponding adjacent non-neoplastic tissues. Kindlin-2 overexpression showed a significant positive correlation with tumor stromal invasion, lymph node metastasis, and TNM stage. The spearman rank correlation test showed positively correlation between the Kindlin-2 expression and MMP-9 expression in GCs(r=0.233, P=0.006).3. Clinicopathological significance of Anxa2 expression in GC.6 RT-qPCR showed that the relative quantity of Anxa2 mRNA was 2.072±1.477 in GCs, and 1.271±0.907 in corresponding adjacent non-neoplastic tissues. There was statistically significant difference between the two groups(P= 0.010). Western Blot showed that the level of Anxa2 protein in GCs was 2.144±1.226, which was higher than corresponding adjacent non-neoplastic tissues(1.269±0.736)(P<0.001). Immunohistochemistry staining showed Anxa2 protein located in membrane and(or) cytoplasm of tumor cells or adjacent non-neoplastic epithelial cells. Overexpressionof Anxa2 was observed in 49.6%(69/139) GCs, And the rate was significantly higher than corresponding adjacent gastric tissues(18.7%, 26/139)(P<0.001). Anxa2 overexpression showed a significant positive correlation with intestinal type adenocarcinoma, lymph node metastasis, and TNM stage. The spearman rank correlation test showed positively correlation between the Anxa2 expression and MMP-9 expression in GCs(r=0.188, P=0.024).Conclusion:1. All of Smad4, Kindlin-2, Anxa2 are closely related to the occurrence and progression of GC. 2. Smad4 may play different roles in GC with different anatomical sites and histological types. 3. Both Kindlin-2 and Anxa2 may participate the degrading of extracelluar matrix through activating MMP-9 or regulating MMP-9 expression and then promote the invasion and metastasis of GC. 4. Both Kindlin-2 and Anxa2 may be potential new therapeutic targets and prognosis indexes in GC.