| Colorectal cancer (CRC) is the third most common tumor only after lung cancer and breast cancer in the world. From a global perspective, the incidence of CRC in malignant tumors ranks third in men, and second in women. In recent years, this incidence is rising. Therefore, in-depth researches on the pathogenesis of colorectal cancer are of important realistic significance in improving the diagnosis and the treatment level of CRC and estimating the prognosis.In the last century, investigations of CRC were focus on the tumor cells themselves, and the gene and epigenetics changes causing occurrence and development of tumors have been widely accepted. However, an increasing number of evidences have demonstrated that the tumor microenvironment played an important role in tumorigenesis, tumor infiltration and metastasis. Cancer-associated fibroblasts (CAFs) in colorectal cancer tissues are the major cells in the tumor microenvironment and are related closely to occurrence and development of CRC. Colorectal cancer cells produce transforming growth factor (TGF-β), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and other vital cytokines through paracrine mechanisms to induce activation of CAFs. Activated CAFs can up-regulate matrix metalloproteinase (MMPs) and plasminogen activator inhibitor-1(PAI-1), ultimately play a role of breaking through basilar membrane, invasion and metastasis of tumor cells. According to experiments, the cancer cells co-cultured with CAFs and colorectal cancer cells obviously became spindle-shape to increase the metastasis capacity. Meanwhile, CAFs secrete matrix metalloproteinase (MMP) to specifically degrade ECM, preparing for cancer cell migration. All these indicated that CAFs can directly increase the invasion ability of tumor cells and promote their metastasis, and they were directly involved in the development process of CRC.The interaction between CAFs and tumor cells involved many signaling pathways, such as TGF-β, PDGF, SDF-1/CXCR4 and HGF, of which TGF-β had a critical role. TGF-β can first induce fibroblast activation, and these activated fibroblasts could promote malignant transformation of epithelial cells. Activated fibroblasts have been proved to promote tumor cell growth, while the normal fibroblasts inhibited tumor cell growth. This was achieved mainly by secreting various factors or changing extracellular matrix components and accelerating the epithelial cells cancerization, but the specific mechanism is not clear.Studies have shown that TGF-β can induce CAFs to express PGP9.5( Protein gene product). PGP9.5 is an ubiquitin hydrolase, also known as ubiquitin C terminal hydrolase-L1 (UCH-L1). It regulates cellular progresses in cell hydrolysis pathway including cell division cycle and death. Current relevant researches indicated a high expression of PGP9.5 in many tumors including colorectal cancer, medullary thyroid carcinoma, pancreatic cancer, esophageal cancer, bladder cancer. From recent studies, in tumor tissues, PGP9.5 induced an increase of cyclin ubiquitination to result in uncontrolled growth of undifferentiated cells, which was one of the key factors leading to oncogene activation. Through an analysis of many patients with ulcerative colitis, PGP9.5 was demonstrated to have a key function in the malignant transformation process from ulcerative colitis to carcinoma. In colorectal cancer, UCHL1 hypomethylation is closely associated with lymphatic metastasis, and in the colorectal cancer patients with lymphatic metastasis, methylation status of UCHL1 significantly reduced.According to current researches, PGP9.5 expressed by CAFs under the induction effect of TGF-β acted on CRC, which may be an important mechanism of the interaction between CAFs and tumor cells. We need to investigate the specific action process. Accordingly, the present study investigated specially the possible mechanisms above, thus contributing to a better understanding of tumor growth environment and helping find new targets for cancer therapy.Research object The role of CAFs in tumor growth has attracted sufficient attention, and the role of TGF-beta in the activation process of CAFs also has a number of studies, and the important role of PGP9.5 in CRC is also relatively clear. Studies have indicated that TGF- can induce PGP9.5 expression in CAFs, but the specific molecular mechanism has not been reported. Therefore, this study aimed at the specific mechanism of action between TGF-beta/CAFs/PGP9.5, and the pathway through which TGF- beta promotes the secretion of PGP9.5 in CAFs, and thus play its role in promoting cancer.Methods and results Part oneObjective To verify that TGF-β1 induce CAFs activation and promote PGP9.5 expression.Methods Normal fibroblasts were cultured with colorectal cancer cells to obtain CAFs and immunofluorescence assay was used to determine the expression of α-SMA. After TGF-β1 was added in CAFs, cells and medium were collected at different time points, and western blot was used to detect the expression of PGP9.5. TGF beta 1 specific siRNA was designed and synthesized, and TGF beta 1 expression vector was constructed to be transfect into CAFs. Western blot was used to detect the expression of PGP9.5 of CAFs.Results1. The results showed that a-SMA expression of CAFs increased significantly when TGF-β1 was added.2. With the prolonged duration of action of TGF-β1, the expression of PGP9.5 of CAFs gradually increased and there was a significant difference.3. PGP9.5 in the cells and media showed a consistent expression trend.4. PGP9.5 expression reduced obviously after adding siRNA specifically targeting TGF-β1 or its inhibitor; while TGF-β1 overexpression vector was transfected, the effect was similar to that after directly adding TGF-β1 factor, both of which showed a significant increase of PGP9.5 expression.5. When TGF-β1 overexpression vector was transfected and TGF-β1 inhibitor was added simultaneously, PGP9.5 expression reduced remarkably compared with simple TGF-β1 overexpression vector transfected group.Conclusion1. TGF-beta 1 can promote CAFs activation.2. TGF-beta 1 can induce CAFs expression and secretion of PGP9.5 in a time dependent pattern.3. TGF-beta 1 siRNA and its inhibitor LY2109761 inhibited the expression of PGP9.5 in CAFs.4. TGF-beta 1 over expression also induced PGP9.5 expression in CAFs.Part twoObjective To investigate the pathway by which TGF-β1 promoted PGP9.5 expression in CAFs of colorectal cancer cells..Methods Specific siRNAs targeted to Smad2 and Smad3 were designed and synthesized, and then were transfected into CAFs. After TGF-β1 and the specific inhibitors of ERK1/2, PI3K, JNK and p38 pathways were added simultaneously, the expression of PGP9.5 was detected.Results1. Smad2/3 siRNAs have significant inhibitory effect on expression of PGP9.5 inCAFs 。2. After the specific siRNA was used to down-regulate Smad2 and Smad3 expression, the promotion effect of TGF-β1 on PGP9.5 expression was decreased obviously, but still up-regulated when compared with no TGF-β1 stimulation.3. The results indicated that after blocking ERK1/2 and PI3K pathways, promotion effect of TGF-β1 on PGP9.5 expression was suppressed significantly.4. After blocking JNK and p38 pathways, whereas, PGP9.5 expression displayed a less significant decrease than that of TGF-β1 addition group.Conclusion1. TGF-β1 regulated PGP9.5 expression through Smad pathway, but it is not the only one.2. In CAFs of colorectal cancer cells, TGF-β1 promoted PGP9.5 expression also through ERK1/2 and PI3K pathways.Part threeObjective To validate whether one of the mechanisms for promoting the growth of cancer cells by CAFs is through TGF-β1/PGP9.5.Methods CAFs and colorectal cancer cells were cultured on the upper and lower chambers of transwell device, respectively, thereby establishing a co-culture model. When TGF-β1 was added into CAFs, Western blot was used to determine PGP9.5 expression of of colorectal cancer cells in the lower chamber, MTT assay was used to detect cancer cells proliferation, Flow cytometry was used to detect cycle and apoptosis and Transwell assay was used to detect cancer cell invasive capability.Results1. According to the results, with the extension of TGF-β1 action duration, PGP9.5 expression was enhanced significantly in the lower layer media containing colorectal cancer cells.2. MTT assay showed a gradual and significant increase in the proliferation of colorectal cancer cells as the concentration of TGF-β1 was rising. When TGF-β1 was added into CAFs and PGP9.5 inhibitor was added into colorectal cancer cells at the same time, the proliferation was increased compared with the control group, but the proliferation rate was reduced obviously compared with simple TGF-β1 addition group.3. When TGF-β1 was added into CAFs, the proportion of the apoptosis peak (Sub G1 peak) of colorectal cancer cells significantly reduced. After TGF-β1 inhibitor was added into the colorectal cancer cells, the proportion of Sub G1 peak also decreased, but was higher than that of TGF-β1 addition group. Similarly, after PGP9.5 inhibitor was added into the colorectal cancer cells, the proportion of Sub G1 peak also declined compared with the control group, but was still higher than that of TGF-β1 addition group.4. After TGF-β1 was added into CAFs, the invasion ability of colorectal cancer cells increased significantly. When TGF-β1 and PGP9.5 inhibitor was added into CAFs and colorectal cancer cells, respectively, however, the invasion ability of the cells increased compared with the control group, but was suppressed obviously compared with simple TGF-β1 addition group.Conclusion1. PGP9.5 expressed by CAFs under the induction effect of TGF-β1 can promote the proliferation of colorectal cancer cells; while PGP9.5 was inhibited, the promotion effect of TGF-β1-activated CAFs on colorectal cancer cells was suppressed obviously.2. PGP9.5 did play a role in the inhibition process of TGF-β1-activated CAFs against colorectal cancer cells apoptosis, however, the effect of TGF-β1-activated CAFs against tumor cells was not completely blocked by inhibiting the function of PGP9.5, and TGF-β1-activated CAFs may also secrete other factors to play a role.3. After CAFs was activated by TGF-β1, the invasion of tumor cells was promoted significantly, in which PGP9.5 have a key function as inhibition of PGP9.5 effect blocked the promotion of TGF-β1-activiated CAFs on the invasion of colorectal cancer cells to some extent.Research conclusion:1. TGF- beta 1 can induce CAFs secretion and expression of PGP9.5 and showed a time dependent pattern.2. In CAFs, TGF- beta 1 regulates the expression of PGP9.5 through Smad, ERK1/2 and PI3K pathways.3. TGF- beta 1 induced expression of PGP9.5 in CAFs can promote the proliferation and invasive capability of colorectal cancer cells, and inhibit its apoptosis. |
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