Abstract Objective To investigate Cks1 role in gastric cancer and its regulation of p27Kip1 in Skp2 degradation process. Methods measured by flow cytometry in normal gastric mucosa and gastric cancer tissue Cks1 expression of p27Kip1, Skp2 protein expression. Results gastric carcinoma Cks1 Expression of Skp2 expression was significantly higher than that in normal gastric mucosa (χ2 = 22.69, P <0.05; χ 2 = 13.42, P <0.05), and p27Kip1 expression is lower than the normal gastric mucosa (χ 2 = 14.83, P <0.05) . Gastric Cks1, Skp2 expression and p27Kip1 expression was negatively correlated (r = -0.649, P <0.05; r = -0.732, P <0.05); while the the Cks1 protein expression Skp2 protein lack of correlation (P> 0.05). Cks1 expression and tumor differentiation in gastric cancer (χ2 = 5.05, P <0.05), with the depth of tumor invasion, lymph node metastasis and clinical stage are not related (P> 0.05). Conclusion Cks1 may be involved in gastric carcinogenesis; gastric Cks1 may be involved in the process of the Skp2 regulating p27Kip1 ubiquitination and degradation.
Key words gastric tumor; the Cks1 protein; Skp2 protein; p27Kip1 protein
Many malignant cells periodically protein p27Kip1 expression is reduced, F box protein Skp2 (S phase kinase protein 2, Skp2) to participate in the p27Kip1 ubiquitination and degradation process. Cks1 (Cyclin kinase subunit 1, Cks1) is one of the highly conserved cell cycle regulatory proteins Suc1/Cks family members, and its role in the tumor tissue and their correlation with p27Kip1 and Skp2 is not very clear, we use streaming cytometric detection Cks1, p27Kip1, Skp2 expression in normal gastric mucosa and gastric cancer tissue below to find answers to this problem.
1 Materials and methods
1.1 Materials and reagents
Access to surgical resection, clinical pathology confirmed 64 cases of gastric cancer specimens and 20 cases of normal gastric mucosa. All specimens no preoperative radiotherapy, chemotherapy, or immunotherapy. Gastric cancer patients, 34 males and 30 females; aged 22 to 78 years, with an average age of 54.7 years. TNM staging: Ⅰ 12 cases, Ⅱ 23 cases, Ⅲ of 19 cases, Ⅳ of 10 cases. Degree of differentiation: well differentiated in 17 cases, in differentiation 19 cases, poorly differentiated 18 cases, 10 cases of undifferentiated. Cancer metastasis: lymph node metastasis in 31 cases without lymph node metastasis 33 cases; 10 cases of distant metastasis, distant metastasis 54 cases. Of invasion depth: infiltration does not exceed serosa (T1 + T2), 32 cases, infiltration over serosa (T3 + T4) 32 Li. The Cks1 rabbit anti-human monoclonal antibody and of Skp2 mouse anti-human monoclonal antibodies were purchased from American ZYMED expression of p27Kip1 mouse anti-human monoclonal antibodies were purchased from Denmark DAKO company.
Surgical removal of fresh tissue to take no bleeding to some organizations block, PBS wash bloodstains, mechanical crushing method into a single cell suspension. Cell suspension added two small test tube 0.5ml PBS were washed once, centrifuged, the supernatant was removed, added Cks1 (or p27Kip1, Skp2), rabbit anti-human in vitro fluorescence polyclonal antibody 10μl, control tube PBS, and mix 4 ℃ for 30min, PBS washed once, centrifuged, and remove excess unbound antibody-on testing. Every 1 specimens 10 000 cells. Cks1 (or expression of p27Kip1, of Skp2) positive rate = [test tube Cks1 (or p27Kip1, Skp2) positive cells – negative control, the number of positive cells] / 10 000 x 100%. Let ≥ 10% positive rate for positive expression, the positive rate of ≥ 30% for high-expression .
1.3 Statistical analysis using SPSS 10.0 statistical analysis software χ2 test and Spearman rank correlation analysis, P <0.05 for significant differences.
2.1 Cks1, p27Kip1, Skp2 expression
The gastric carcinoma Cks1, Skp2 positive rate in LSCC significantly higher than normal gastric mucosa (χ2 = 22.69, P <0.05; χ 2 = 13.42, P <0.05), while p27Kip1 positive rate lower than the normal gastric mucosa (χ 2 = 14.83, P <0.05), see Table 1. Table 1 Cks1 expression of p27Kip1 Expression of Skp2 expression in different gastric tissue specimens NOTE: comparison between groups, P <0.05 gastric carcinoma Cks1 expression associated with the degree of tumor differentiation, low, undifferentiated group Cks1 expression were significantly higher than high, differentiation group (χ2 = 5.05, P <0.05), and gastric cancer patients age, sex, tumor depth of invasion, lymph node metastasis, and clinical stage are not related (P> 0.05), as shown in Table 2.
2.2 gastric Cks1 expression and association of Skp2, p27Kip1
The 64 cases of gastric cancer were divided into three groups according to the Cks1, Skp2 protein expression: Cks1, Skp2 while high expression of the 21 cases; Cks1, Skp2 and low expression of the 27 cases; Cks1 expression, Skp2 low expression or Cks1 low expression, Skp2 high expression 16 cases. 21 cases Cks1 of Skp2 simultaneously low high expression in 18 cases of p27Kip1 expression; 27 cases Cks1, Skp2 simultaneously low expression in 23 cases of p27Kip1 expression. Cks1 Expression of Skp2 expression in gastric carcinoma and p27Kip1 protein expression negative correlation (r = -0.649, r = -0.732, P <0.05), but there are six cases of Skp2 and p27Kip1 protein and high expression, four cases of Skp2 and p27Kip1 protein low expression; while the Cks1 protein expression of Skp2 lack of correlation (P> 0.05).
This study shows that Skp2 expression in the gastric cancer tissues was significantly higher than that relations are in Table 2 gastric cancer of Cks1 expression with the clinicopathological factors (%)
Category number of cases Cks1P age (years old) 603,574.3 (26/35) ≤ 602,972.4 (21/29) P> 0.05 △ Gender Male 3473.5 (25/34) female 3073.3 (22/30) P> 0.05 △ degree of differentiation + + undifferentiated differentiation 3661.1 (22/36) 2889.3 (25/28), P <0.05 ※ the invasion depth T1 + T23271.2, (23/32) T3 + T43275.0 (24/32) P> 0.05 △ lymph node metastasis 3177.4 (24 / 31) 3369.7 (23/33) P> 0.05 △ clinical stage I + II 3568.6 (24/35) Ⅲ + Ⅳ 2979.3 (24/35) P> 0.05 △
0.05,※ χ2=5.05,P＜0.05常胃粘膜，而p27Kip1表达则低于正常胃粘膜；胃癌组织中Skp2蛋白与p27Kip1蛋白负相关。">Groups △ P> 0.05, * χ2 = 5.05, P <0.05 regular gastric mucosa, and p27Kip1 expression below normal gastric mucosa; Skp2 protein p27Kip1 protein in gastric carcinoma negative correlation. This result supports p27Kip1, Skp2 involved in gastric carcinogenesis and Skp2 involved in p27Kip1 ubiquitination and degradation process of inference. However, this experiment also found 10 cases of Skp2 expression of p27Kip1 high expression of Skp2 expression of p27Kip1 low expression. Dowen et al  found that 50% of cervical tissue expression of p27Kip1 of Skp2 and high expression or low expression. Hara T  found that even lack Skp2 cytosolic p27Kip1 ubiquitination activity detected p27Kip1 ubiquitination process may exist within the cytoplasm.
Sitry et al , either in vivo or in vitro, Skp2 only combined with Cks1 can identify p27Kip1 ubiquitinated protein degradation. The study found that Cks1 Expression of Skp2 simultaneously high expression of p27Kip1 low expression; Cks1 Expression of Skp2 the same time, high low expression of p27Kip1 expression, Cks1, Skp2 expression are negatively correlated with p27Kip1 protein expression, indicating that Cks1 Expression of Skp2 may participate p27Kip1 ubiquitination degradation process. Kitajima et al  found that the Cks1 of Skp2 individually transfected cells Cks1 does not make The p27Kip1 the levels, while Cks1 Expression of Skp2 simultaneously transfected cells induced p27Kip1 degradation Cks1 expression can p27Kip1 expression by siRNA inhibition and can inhibit the growth of tumor cells. Masuda et al  found that Cks1 Expression of Skp2 simultaneously transfected cells not the p27Kip1mRNA level to reduce, but to reduce the level of p27Kip1 protein, which proved once again that Cks1 Expression of Skp2 the p27Kip1 protein ubiquitination and degradation process may be involved.
This study found that Cks1 expression in gastric cancer tissues was significantly higher than that in normal gastric mucosa associated of Cks1 expression of the degree of tumor differentiation, This shows that Cks1 may be involved in gastric carcinogenesis Cks1 protein expression increased, so that the gastric cancer cells from the normal growth cycle more regulation, was the uncontrolled growth of the state, Cks1 expression, cell differentiation, the lower the higher the degree of malignancy, or license as a poor prognostic indicator, but we found that is not related with the clinical stage and lymph node metastasis, probably due to Cks1 expression in gastric cancer and biological effects related to a variety of factors, and by a variety of other genes in gastric cancer progression is a multi-gene, the result of many factors, it is only Cks1 indicators, and does not reflect the of tumor invasion and metastasis.