| 其他摘要 | Gastric cancer and colorectal cancer are common gastrointestinal tumors
threatening human health, which are related to many factors during its development.
The protease activated receptors (PARs) and trefoil factor protein (TFFs) family
expressed in the gastrointestinal tract are involved in the development. In
physiological conditions, PARs is related to the secretion of digestive juice and the
contraction and relaxation of muscle. Meanwhile, PARs also plays a role in cancer
development, invasion and metastasis besides platelet aggregation. Aberrant
expression of PAR4 had been detected in ulcerative colitis, colorectal cancer and
certain cancer cell lines, which might be important in initiating colorectal
cancer.formation. TFFs is in involved in protecting gastrointestinal tract against
mucosal damage and play an important role in the subsequent repair. During the
development of cancer, TFFs was reported as a tumor suppressor factor in some cases,
but as a potential tumor promoting factor in others. TFF2, consisting of two trefoil
factor domains, is mainly expressed in the neck cells of gastric glands. The level of
TFF2 was in line with the degree of cell differentiation and and decreased in gastric
ulcer, chronic atrophic gastritis and gastric cancer. As reported, the expression of
TFF2 is related to gastric mucosal cell proliferation and malignant transformation. In
intestine, TFF2 inhibited the development of colitis induced by the NO. In mice
colitis model, TFF2 reduced the degree of inflammation and ulcers which suggested
TFF2 is involved in the immune response.
Based on our previous studied on the interaction between PARs and Bo-TFF2,
which could induced human platelet aggregation, we were prompted to study the
relationship between hTFF2 and PARs. Immunohistochemistry staining showed that
both hTFF2 and PAR4 are distributed in the normal gastric mucosa from base to the
middle position. Partial sequences of the second Loop of hTFF2, are conservative and
shwows high similarity with tethered-ligand of PAR4. In the study, we invesgated
whether there exists an interaction between TFF2 and PAR4, and the biological activity of PAR4 is regulated by TFF2 or not. The variant expression of TFF2 and
PAR4 in gastrointestinal cancer and the chemotaxis of TFF2 to cells overexpression
PAR4 were discussed.
At first, We tested the expression of TFF2 and PAR4 in gastric and colorectal
cancer by semi-quantitative PCR. The results suggested that both gene’s expression
were two gene expression was significantly lower in gastric cancer tissue than in the
normal tissue but higher in colorectal cancer tissue. Similar results were shown by
Western blotting. To further define the variant quantity of TFF2 and PAR4 in gastric
cancer and colorectal cancer, two genes’ expression in 28 gastric cancers and 38
colorectal cancers were were quantifiedf. The results showed that mRNA level of both
genes in cancer tissue were significantly lower than in normal gastric tissue (P<0.001),
but significantly higher in colorectal cancer (P<0.001). Clinical pathology results
suggested that the level of PAR4 in gastric cancer patients with lymph node metastasis
was lower than without lymph node metastasis.The level of PAR4 gene expression
was lower in gastric antrum than in no-gastric antrum cancer (P <0.05). However, the
level of TFF2 and PAR4 in colorectal cancer patients with lymph node metastasis was
higher than no lymph node metastasis, and was also significantly higher in poorly
differentiated colorectal carcinoma than in well-differentiated cancer (P<0.001). By
immunohistochemical analysis, the protein level of TFF2 and PAR4 in gastric
adenocarcinoma was significantly lower than in the surrounding normal mucosa
(P<0.001), but higher in colorectal carcinoma accordingly (P<0.001). And the results
showed that both TFF2 and PAR4 were expressed consistently in the gastrointestinal
tract cancer.
The migration of lovo-PAR4 was evidently increased comparing with lovo-mock
cell after induced by same concentration of hTFF2, the activity performed in a
dose-dependent manner accompanied by increasing phosphorylation of ERK1/2. The
proliferation activity of PAR4 comparing with the mock cell, but the activity
decreased after induced by TFF2, which demonstrated the anti-proliferative capacity domain truncation. Since having three disulfide bonds, we
expressed recombinant fusion proteins by pET32a expression system, removed
thioredoxin by Xa factor, and purified free recombinant proteins by affinity
chromatography and reverse high pressure liquid chromatography column. The
recombinant Bm-TFF2 had platelet aggregation activity, and the first domain of
Bm-TFF2 only induced platelet deformities. However, three recombinant proteins
have the function of inducing AGS migration, and the migration activity was in a
dose-dependent, and there is no evident activity difference among three recombinant
proteins. The facts provided a convenient and reliable means for studying the
relationships of structure and function of TFFs protein family. |
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