The Significance Of Human Telomerase Reverse Transcriptase And Gli1、Gli2 In Gastric Cancer And Its Molecular Mechanism

Background and objectiveGastric cancer is one of the most malignant tumors in China. As is well known, early diagnosis of gastric cancer is difficult, and most of the patients had been in the middle-late stage at diagnosis. Although there are many methods to treat gastric cancer, the mortality rate is still high and the 5 years of survival rate is still low. The main reason of gastric cancer patients’ poor prognosis is invasion and metastasis. In the clinical work, we found that even some early gastric cancer also could occur metastasis in the adjacent or distant tissue.Therefore, there has an important clinical significance to study the molecular mechanisms of invasion and metastasis in gastric cancer.Human telomerase reverse enzyme(h TERT), as a speed limit of telomerase components, its activity may represent the activity of telomerase. In the previous study, researchers often focused on h TERT how to promote cell proliferation, but also was involved in regulate the transfer and invasion of the tumor.Our previous study showed that the inhibition of telomerase activity in gastric cancer can reduce the invasion ability of cancer in vitro. The overexpression of hTERT in the osteosarcoma cell U2 OS,which telomerase expression is negative may promote the invasion and metastasis. By Meta analysis, many researchers found that the high expression of h TERT was related with the size of gastric cancer, infiltration depth, lymph node metastasis and distant metastasis. These conclusions suggested that h TERT played a very important role on the metastasis and invasion of gastric cancer. But the mechanism of invasion and metastasis is still not very clear.Wnt signaling pathway plays an important role in invasion and metastasis of gastric cancer. Studies have shown that the classic Wnt signaling pathways was activated by h TERT interaction with β-catenin, then promoted β-catenin into nuclear and enhanced its transcriptional activity. In this way, Wnt signaling pathway enhanced the invasion and metastasis of gastric cancer cells. However, our group found that overexpression of h TERT in HCT116 which Wnt signal pathway has not been actived, the invasion and metastasis ability was still enhanced. This result showed prompting hTERT may also promote the invasion and metastasis of gastric tumor cells by other ways.The hedgehog signaling pathway plays an important role in the development of the embryonic developmentand organ formation. The classic Hh signaling pathway was mainly composed of ligand Hedgehog, transmembrane protein receptor patched(Ptch) and smoothened(Smo) and Gli family of transcription factors. Gli family is the core of the Hh signaling pathway. Gli activation will lead to the downstream transcription of a series of molecules, and then complete the Hh signaling pathway for the readjustment of the various processes of life.Studies have shown that activation of Hh signaling pathway was involved in a wide variety of tumors, including gastric cancer, especially in the process of invasion and metastasis. There was few literatures reported whether Hh/Gli signaling pathway was involved in the hTERT regulation of invasion and metastasis in gastric cancer.Therefore, this topic of research studied the expression of h TERT, Gli1 and Gli2 in gastric cancer tissue and the relationship between the patients with gastric cancer clinical pathology. In addition,we explored the correlation of hTERT and Gli1、Gli2.At last,we tested the expression of Gli1 and Gli2 after overexpression of hTERT in gastric cancer cell,to expound the molecular mechanism of invasion and metastasis in gastric cancer.Methods:The first part: the expression of h TERT and Gli1、Gli2 in gastric cancer and their correlation.1. Using immunohistochemical to test the expression of hTERT, Gli1, Gli2 in 72 gastric carcinoma and their adjacent tissues;2. According to the expression of hTERT and Gli1、Gli2,analysis the relationship between the expression of h TERT and Gli1, as well as the expression of h TERT and Gli2 in 72 cases with gastric carcinoma;3. The relationship between clinical and pathological with the expression of h TERT and Gli1、Gli2;4. The relationship between the prognosis of the patients with the expression of h TERT、Gli1、Gli2.The second part: hTERT can regulate the expression of Gli1 in gastric cancer cells and its molecular mechanism.1. Western-blot to test the expression of hTERT and Gli1、Gli2 in gastric cancer cell;2. Q-PCR to test the change of Gli1 after transfected h TERT;3. Western-blot to detect the expression of Gli1 after over-expression of hTERT.Results:The first part: the expression of h TERT and Gli1、Gli2 in gastric cancer and their correlation.1. The results of immunohistochemistry showed that, the expression of hTERT、Gli1、 Gli2 in gastric carcinoma were significantly higher than their adjacent cancer tissue(p<0.01);2. The relative expression of hTERT in gastric cancer have positive-relationship with Gli1、Gli2, The correlation coefficients are r=0.502(p<0.01)、r=0.6246(p<0.01);3. The high expression of hTERT、Gli1、Gli2 all have business with lymph node metastasis(p<0.05),forever the high expression of Gli1 relating to distant metastasis(p<0.05);4. The expreesion of h TERT and Gli1、Gli2 is higher(p<0.05、p<0.05), the shorter survival time of gastric cancer patients(p<0.01).The second part: h TERT can regulate the expression of Gli1 in gastric cancer cells and its molecular mechanism.1. Western-blot indicated the relatively low-expression of BGC-823, SGC-7901 from all gastric cancer cell;2. The mRNA level of Gli1 was decreased in the gastric cancer cell BGC-823, SGC-7901 after transfected with hTERT plasmid after 24 hours;3. Use Western-blot detected BGC-823 and SGC-7901 increase the level of Gli1 protein which transfected with h TERT plasmid after 24 hours.Conclusions:1. The clinical study,the expression of h TERT and Gli1、Gli2 in gastric cancer were higher than Tissue adjacent to cancer,the expression of h TERT was positive-relation with Gli1 or Gli2, they all were associated with lymph node metastasis. hTERT、Gli1 higher expression group and h TERT、 Gli2 higher expression group have poor prognosis, the highest expression group have the worst prognosis;2. Over expression of h TERT, the expression of Gli1 is increase in mRNA and protein level,promote the invasion and metastasis of gastric cancer.In a word, h TERT can positively regulate the expression of the transcription factor Gli1 in gastric cancer.