Relationship Between Expression of Gastrokine 1 and Clinicopathological Characteristics in Gastric Cancer Patients

Gastric cancer, one kind of the most common malignant tumors worldwide, is the second leading cause of cancer death. It was estimated there were about 989 thousand new gastric cancer cases and 738 thousand gastric cancer deaths globally one year (Ferlay et al., 2010). Human gastric mucosa suffers from various wound continually, such as gastric acid, alcohol, too cold or too hot food and so on. Normally because of self repair and protection of gastric mucosa protective factors, injured gastric mucosa can heal soon. However, lessening or lack of protective factors may lead gastric mucosa more exposion to harmful substance, which may induce expression changes of gastric cancer genes, and then promote happening or development of gastric cancer at last. In the past few years one gene named Gastrokine 1, which had the functions such as protecting gastrointestinal mucosa, promoting mucosa repairment, suppressing cancer and so on, was found (Toback et al., 2003). Reducing or lack of its expression


Introduction
Gastric cancer, one kind of the most common malignant tumors worldwide, is the second leading cause of cancer death.It was estimated there were about 989 thousand new gastric cancer cases and 738 thousand gastric cancer deaths globally one year (Ferlay et al., 2010).Human gastric mucosa suffers from various wound continually, such as gastric acid, alcohol, too cold or too hot food and so on.Normally because of self repair and protection of gastric mucosa protective factors, injured gastric mucosa can heal soon.However, lessening or lack of protective factors may lead gastric mucosa more exposion to harmful substance, which may induce expression changes of gastric cancer genes, and then promote happening or development of gastric cancer at last.In the past few years one gene named Gastrokine 1, which had the functions such as protecting gastrointestinal mucosa, promoting mucosa repairment, suppressing cancer and so on, was found (Toback et al., 2003).Reducing or lack of its expression

Relationship Between Expression of Gastrokine 1 and Clinicopathological Characteristics in Gastric Cancer Patients
Jiang-Wei Xiao 1& , Jia-Hui Chen 2& , Ming-Yang Ren 2 , Xiao-Bing Tian 1 , Chong-Shu Wang 1 ﹡ plays a significant role in happening and development of gastric cancer (Shiozaki et al., 2001;Rippa et al., 2010).The expression of Gastrokine 1 in gastric cancer and corresponding non-cancerous gastric tissues was detected with RT-PCR and INH.Finally, the relationship between its expression and clinicopathological characteristics of gastric cancer was analysed.

Patient characteristics
After inform consent forms were signed, the specimens including gastric cancer and corresponding non-cancerous gastric tissues in general surgery of affiliated hospital of North Sichuan Medical College during April to July in 2011 were collected.All cases were diagnosed as gastric adenocarcinoma with pathological biopsy both preoperation and postoperation.They who had taken anti-HP drug or chemotherapy or radiotherapy lately were removed.The clinicopathological characteristics of patients are summarized in Table 1.

Samples
All samples consisted of gastric cancer and corresponding non-cancerous gastric tissues were obtained intraoperation just after they were cut off.Then part were put into -80℃ refrigerator quickly used for total RNA extraction.The left were dipped in formalin and then embedded with paraffins for INH.

Detection of HP
Patients were arranged to take 14 C-urea breath test to detect HP value preoperation.They whose HP values lower than 100 dpm were regarded as negative, and the others positive.Due to cardia complete obstruction or poor compliance, 11 patients did not accept 14 C-urea breath test.

RT-PCR
104 gastric cancer and corresponding non-cancerous gastric tissues were ground into fine powder with mortars and pestles in liquid nitrogen.Then total RNA was extracted with TRizol Reagent (Tiangen, China).Later the quality of total RNA was assessed with ultraviolet spectrophotometer (SHIMADZU, Japan) and agarose gel electrophoresis (agarose from Sigma, America; electrophoresis apparatus from BIO-RAD, America).cDNA was synthesized using reverse transcription kit (BioBRK, China) according to instruction.RT-PCR thermocycler (ABI, America) and kit (Tiangen, China) including Pre Mix, Dye and DNase/RNase free ddH2O were used for cDNA amplification.1μl cDNA, 10μl Pre Mix, 2μl Dye, forward and reverse primer both 0.6μl (10 pmol/μl) and 5.8μl DNase/RNase free ddH 2 O were consisted in amplification system.Gastrokine 1 and β-actin primers were synthesized by invitrogen company.The primer sequence, reaction condition and product size are all in Table 2.All samples were tested duplicately.After amplification finished, dissociation curve was analysed to identify the uniqueness of product. 2 -rCT was used as relative expression value.

INH
Paraffins embeded 104 gastric cancer and corresponding non-cancerous gastric tissues were sectioned to 3μm in thickness.After the slices were dipped in dimethylbenzene twice each for 10 min, they were put into 100% alcohol, 85% alcohol and 75% alcohol successively, and then washed with running water for deparaffinization.3% H 2 O 2 covered the whole tissues on slices for 20 min at room temperature and away from light in order to block endogenous peroxidase activity.Heat antigen retrieval was performed in 0.01M citrate buffer (ph 6.0) at 95℃ for 20 min.Then slices were incubated with mouse anti-Gastrokine 1 monoclonal antibody (ABCAM, England) at a dilution of 1:150 (final concentration: 6.67 μg/ml) at 4℃ overnight.At the next moring the slices were incubated with common secondary antibody (Zhongshan Jinqiao, China) at room temperature for 15 min.After colorated with diaminobenzidine and counterstained with hematoxylin, the reaction products were visible.All slices were assessed by two professors of pathology who had no knowledge of any clinicopathological characteristics of the patients.More than 30% epithelial cell cytoplasm stained positively were considered positive (Yoon et al., 2011).

Expression of Gastrokine 1 gene by RT-PCR
The mean rCT value of Gastrokine 1 in gastric cancer and corresponding non-cancerous gastric tissues were in Figure 1, and the expression variation between them was 8.22±5.01(t=11.83,P<0.01).The difference was of statistical siginificance.The amplification curve and dissociation curve are in Figure 2.

Relationship between Gastrokine 1 gene expression and HP, clinicopathological characteristics of gastric cancer patients
The mean rCT value of Gastrokine 1 in gastric cancer tissues of HP positive and negative patients were 11.14±5.19and 7.67±4.18respectively.The variation was siginificant (t=2.33,P<0.05; Table 3).The CEA data of 3 patients and CA19-9 of 2 patients were lost, so they were removed when made those two statistics analysis.No siginificant relationship was found between Gastrokine 1 and clinicopathological characteristics, including gender, age, tumor's location, depth of invasion, differentiation, lymph node metastasis, stage and CEA, CA19-9 level in peripheral blood preoperation (all P>0.05; Table 3).

Discussion
Gastrokine 1, once named CA11, AMP-18 or foveolin, is located in chromosome 2p13.3.Its full length is about 6 kb, including 6 exon and 5 intron, whose corresponding protein is made up of 185 amino acid.Gastrokine 1 is one part of gastric mucous layer, which is possessed of the functions of promoting gastric mucosa epithelial cells division and migration, maintaining integrity of gastric mucosa, promoting injured mucosa renovation, suppressing neoplasm and so forth (Lacy et al., 1993;Shiozaki et al., 2001;Martin et al., 2003;Toback et al., 2003;Rippa et al., 2010;Yoon et al., 2011).Researches found that from chronic gastritis, atrophic gastritis, gastric mucosa intestinal metaplasia to gastric cancer, the expression of Gastrokine 1 in gastric mucosa reduced gradually (Nardone et al., 2008).So it is thus clear that the expression of Gastrokine 1 changes throughout the gastric canceration, and it plays an important role in the process of formation and development of gastric cancer.If some relationship between Gastrokine 1 expression in gastric cancer tissues and some clinicopathological characteristics can be found, biopsy through gastroscope preoperation may contribute to preliminary assessment of gastric cancer and guide clinical treatment.However, related researches are too few.Whether expression of Gastrokine 1 is related to gastric cancer clinicopathological characteristics and HP or not, past researches can not give a final conclusion.Reports about relationship between expression of Gastrokine 1 and gender, age, CEA and CA19-9 level in peripheral blood preoperation can not be found up to now.Therefore, it is worth lucubration further.In this study, expression of Gastrokine 1 gene and protein in gastric cancer and corresponding non-cancerous tissues were detected using RT-PCR and INH followed with statistics analysis to try to clarify the questions above.
Our experiment results, both Gastrokine 1 gene and protein expressing in gastric cancer tissues were significantly lower than in corresponding non-cancerous tissues, confirmed it is an anti-oncogene (Shiozaki et al., 2001;Rippa et al., 2010;Yoon et al., 2011).Nevertheless, no relationship was found between Gastrokine 1 expression in gastric cancer tissues and pathological characteristics including tumor's location, depth of invasion, differentiation, lymph node metastasis and stage, which conformed to the past two research results (Shiozaki et al., 2001;Yoon et al., 2011).HP infection is related to gastric canceration (Gisbert, 2011).It is an important initiating factor of chronic atrophic gastritis, a precancerous disease of gastric cancer, and can induce the sequential event chronic atrophic gastritis developing to gastric mucosa intestinal metaplasia and to gastric cancer (Ohata et al., 2004;Weck, 2009).Nardon et al. (2008) speculated throught research result that HP infection could down regulate expression of Gastrokine 1, and participate in the early stage of gastric canceration.Our results, Gastrokine 1 expressing notably lower in gastric cancer tissues of HP positive patients than HP negative ones, accorded with their conclusion.In addition, there was no relation between Gastrokine 1 expression and gender, age, CEA and CA19-9 level in peripheral blood preoperation.CEA and CA19-9 level in peripheral blood can be used to assess prognosis of gastric cancer patients (Ucar et al., 2008).So it can be speculated that Gastrokine 1 is unable to act as an indicator of gastric cancer prognosis, but Moss' s research result considered Gastrokine 1 to be related to poorer prognosis of intestinal gastric cancer (Moss et al., 2008).So follow-up to the patients in this study would be very important to make it clear.

Figure 1 .
Figure 1.The Mean rCT Value of Gastrokine 1 Gene in Gastric Cancer and Corresponding Non-cancerous Gastric Tissues (Note: The negative numbers of rCT value were too many, so the data in figure 1 were initial data plus 10)

Figure
Figure 2. The Amplification Curve (A) and the Dissociation Curve (B)