The Molecular Pathogenesis Mechanism Of Gastric Cancer And Its Clinical Significance

Aim:To study the rule and feature of the expression of DNA mismatch repair (MMR) genes and hypermethylation status of the 5'CpG island locating in the promoter region in gastric cancer and adjacent mucosa, detect their role in gastric cancer pathogenesis and development, and evaluate the possible effects and mechanism of LMP2A (EBV latent membrane protein 2A) on human gastric cancer cell line SGC-7901, and establish a theoretical foundation for further understanding pathogenesis mechanism of gastric cancer.Methods:Immunohistochemical staining, western-blotting, RT-PCR, and methylation specific PCR (MS-PCR) was used to detect the expression of MMR genes and hypermethylation status of the 5'CpG island locating in the promoter region in gastric cancer and adjacent mucosa. The recombinant adenovirus particles vAd-2A carrying LMP2A was used to infect human gastric cancer cell line SGC-7901. Then cell viability was assayed by MTT, the apoptosis of SGC cells was evaluated by cell cycle analysis using flow cytometer, the level of cyclin E and GFP was detected by laser confocal fluorescence microscopy.Results:(1) The hMLH1 and hPMS2 proteins were predominantly expressed in the cytoplasm, whereas hMSH6 protein was mainly located in the nucleus. The negative expression rates of hMLH1, hPMS2 and hMSH6 in adjacent mucosa were 40%,56.67% and 53.33% respectively, which were correspondingly higher than those in gastric cancer (26.67%,53.23% and 43.33%) although no significant difference was observed (P>0.05), either of them in both tissues respectively higher than those in the gastric mucosa of non-cancer patients with chronic atrophic gastritis (10%,15% and 10%) (P<0.01).(2) There was respectively no obvious correlation of negative expression of hMLHl, hPMS2 and hMSH6 in gastric cancer and adjacent mucosa. If the negative expression of the same MMR gene in either gastric cancer or the adjacent mucosa was defined as negative expression of the gene of the patient, the negative expression rates of hMLH1, hPMS2和hMSH6 were 61.67%, 78.33% and 75% respectively, which were significantly higher than those in gastric cancer (P<0.01). The negative expression rates of hPMS2 and hMSH6 were also higher than those in the adjacent mucosa tissues (P<0.05). In both gastric cancer and adjacent mucosa, the negative expression rate of hPMS2 gene was the highest among the three MMR genes examined, while hMLHl was the lowest. There was significant difference in gastric cancers (P＝0.01), but not in the adjacent mucosa tissues or in both of them considered together (P>0.05).(3) If the negative expression of anyone of hMLH1, hPMS2 and hMSH6 gene in gastric cancer and adjacent mucosa was considered as negative expression of the corresponding gene in gastric mucosa of the patient, the frequencies of the negative expression of MMR genes were 66.67% and 76.67% respectively, which were obviously higher than those of the corresponding gene in gastric cancer and adjacent mucosa respectively (26.67%-56.67%). In the gastric cancer patients with negative expression of MMR genes, the negative expression of single MMR gene was 37.5%, whereas the rate of negative expression of multiple genes (two or three MMR genes) was 62.5%. In the adjacent mucosa the rates of negative expression of single MMR gene and multiple genes were 32.61% and 67.39% respectively. If the negative expression of anyone of MMR genes in either gastric cancer or the adjacent mucosa in one patient was considered as negative expression of the corresponding MMR gene, the negative expression of MMR genes occurred in 90% of the cases, and the negative expression of multiple genes was 88.89% in the gastric cancer patients with negative expression of MMR genes. Among di-gene negative expression of three genes, the negative expression of hPMS2 and hMSH6 was higher than that of hMLHl and hMSH6 respectively.(4) The relative density of hMSH2 protein (0.28±0.09) in gastric cancer group was significantly higher than that (0.23±0.07) in the corresponding adjacent group, and much higher than that in gastritis group (0.11±0.10) (P<0.01). The relative density of hMSH2 protein in both of the gastric cancer and the adjacent mucosa in patients with gastric cancer much higher than that in the gastric mucosa of non-cancer patients with chronic atrophic gastritis (P<0.001).(5) Though no statistical difference was observed between the relative density of hMLH1 protein in gastritis and adjacent mucosa (0.25±0.08 and 0.26±0.06 respectively), those were slightly higher than that (0.22±0.06) in gastric cancer (P>0.05), and there was significant difference between the gastric cancer and adjacent mucosa (P<0.01).(6) The level of hMLH1 mRNA in gastric cancer, adjacent mucosa and the chronic gastritis was 7.23±11.91,6.59±11.58and 2.07±1.28 respectively, there was no significant difference among them (P>0.05). The level of the hMLH1 mRNA in gastric cancer and adjacent mucosa was similar, but they were markedly higher than that in the chronic gastritis mucosa (P<0.05).(7) The level of hPMS2 mRNA in gastric cancer, adjacent mucosa and chronic gastritis was 0.55±0.30,0.63±0.40 and 0.33±0.16 respectively (F =3.349, P=0.039). The relative density of hPMS2 mRNA in adjacent mucosa was slightly higher than that in gastric cancer (P>0.05), but both of them in gastric cancer and adjacent mucosa were markedly higher than that in the chronic gastritis mucosa (P<0.05).(8) The methylation of the 5'CpG island locating in promoter region of hMSH2 was detected in 73,3% gastric cancer,46.7% adjacent mucosa,30.0% chronic atrophic gastritis (P<0.01). The methylation status in gastric cancer was markedly higher than that in other tissues, adjacent mucosa and chronic gastritis mucosa (P<0.05). No significant difference was detected between the non-cancer tissues, adjacent mucosa and chronic gastritis mucosa (P>0.05).(9)The incidence of the hMLHl methylation of the 5'CpG island locating in the promoter region was 36.7% in gastric cancer,20.0% in adjacent mucosa,5.0% in chronic atrophic gastritis (P<0.01). The methylation status in gastric cancer was markedly higher than that in chronic gastritis mucosa only (P=0.01).(10) The methylation status of the 5'CpG island locating in promoter region of hMSH2 was markedly higher than that of hMLHl in the same kind of tissue respectively (P＜0.05), especially in gastric cancer (P<0.01). Though the methylation status of the 5'CpG island locating in promoter region of hMSH2 or hMLHl was found in gastric cancer or adjacent mucosa, the no methylation status of the 5'CpG island locating in promoter region of the same kind of gene, hMSH2 or hMLH1, was still detected in the same tissue, gastric cancer or adjacent mucosa. That is the no methylation status and methylation status of the same kind of gene existing in one kind of tissue.(11) The level of hMLHl mRNA in gastric cancer with or without methylation of the 5'CpG island locating in the promoter region of hMLH1 was higher than that in adjacent mucosa in the same condition (3.43 and 3.94 vs.0.71 and 1.35), and also the level of hMLH1 mRNA in gastric cancer and adjacent mucosa without methylation was higher than that in the same kind of tissue with methylation respectively, but there was no significant difference between them (P>0.05).(12) The level of hMLH1 protein in adjacent mucosa with or without methylation of the 5'CpG island locating in the promoter region of hMLH1 was higher than that in gastric cancer in the same condition (P>0.05), there was significant difference between two kind of tissues without methylation only (P<0.05). The level of hMSH2 protein in gastric cancer with or without methylation of the 5'CpG island locating in the promoter region of hMSH2 was higher than that in adjacent mucosa in the same condition(P>0.05), there was significant difference between two kind of tissues with methylation only (P<0.05). There was no significant difference at the level of protein of hMSH2 or hMLHl between the gastric cancer and adjacent mucosa with or without methylation respectively (P >0.05).(13) The protein expression of hMLH1 in gastric cancer has positive relationship with methylation of the 5'CpG island locating in the promoter region of hMLH1 (r=0.066, P=0.617). The methylation of the 5'CpG island locating in the promoter region of hMLH1 or hMSH2 in gastric cancer and adjacent mucosa show positive relationship also, but there was significant difference in hMLH1 only (r=0.484, P=0.007). clinic pathological characteristics of gastric carcinoma (r=0.484, P =0.007).(14) The methylation of the 5'CpG island locating in the promoter region of hMLH1 and the negative expression of hMLH1, hPMS2 and hMSH6 did not correlate with clinical pathological characteristics of gastric cancer (P>0.05).(15) The growth of SGC cells expressing LMP2A was apparently improved. The anti-apoptosis ratio was 16.7%-25.0% at.24h-120h (P＜0.001). The apoptosis was inhibited. Both cyclin E expression and S phase ratio in SGC cells expressing LMP2A were up-regulated respectively. The S phase ratio in SGC cells expressing LMP2A was 35.2% and 25.6% at 24h and 48h respectively (P<0.001).Conclusions:(1) As in the gastric cancer tissues, the variety of negative expression of MMR genes occurs in the adjacent mucosa, in which negative expression of polygene dominates, and the negative expression of MMR genes in the adjacent mucosa and gastric cancer was higher than that in the gastric mucosa of non-cancer patients with chronic atrophic gastritis respectively. It is demonstrated that the MMR gene negative expression, which correlates with the pathogenesis of the gastric cancer, may be the molecular basis of postoperative relapse of gastric cancer, and different gastric cancer patients require different gene diagnosis, precaution and treatment.(2) Abnormal MMR genes in gastric cancer were more common than that in non-cancer patients'gastritis tissues and did not correlate with clinical pathological characteristics of gastric cancer. It is demonstrated that abnormal MMR genes in gastric tissue may be one of potential molecular makers of gastric cancer or histological maker predicting gastric cancer occurrence, but did not play an important role in the development of the gastric cancer.(3) The methylation of the 5'CpG island locating in the promoter region of hMLHl or hMSH2 and active transcription of hPMS2 and hMLHl in gastric cancer and adjacent mucosa were found, but they can not be used to predict the expression of MMR genes.(4)The methylation status of the 5'CpG island locating in promoterregion of hMSH2 was more common than that of hMLHl in gastric cancer and adjacent mucosa, the positive relationship was found in either hMSH2 or hMLHl between two kinds of tissues. No methylation status and methylation status of the same kind of gene existing in one kind of tissue. The methylation status of the 5'CpG island locating in promoter region of MMR genes was one of reasons of the protein expression of them and may be a main mechanism for the dysfunction of MMR genes, but not the only way. Except that the mechanism for the dysfunction of MMR can be on the level of translation and transcription. It may be a main mechanism for gastric cancer pathogenesis and early molecular event.(5) The recombinant adenovirus vector vAd-2A expressing LMP2A can regulate the growth of SGC cells and inhibit their apoptosis. The mechanism was that LMP2A upregulate the expression of cyclin E and S phase to increase GC cell proliferation and inhibit apoptosis of SGC cells. These studies have provided a more complete understanding of the role of EBV in tumorigenesis of gastric cancer and established a foundation for further study on gastric cancer and its gene therapy.