| Colorectal cancer (CRC), including colon, rectum and appendix cancer, is theworld’s third most common malignancy of the digestive system. Every year655000people die of CRC in the world. The incidence of CRC is quite different in differentregions. North America and Oceania have the highest incidence, but lower in Asiaand Africa. Although the incidence of colorectal cancer in China has been low, withthe improvement of people’s living standards, changes in diet structure and geneticfactors influence, the incidence shows an increasing trend year by year. Accordingto the Cancer Registry2012Annual Report’s report, the incidence of colorectalcancer in China has been ranked No.3in all kinds of malignant cancer, and themortality has been ranked No.5. The incidence is even higher in some metropolissuch as Beijing, Shanghai and Guangzhou.The incidence of such cities may beranked No.2or3. Therefore, looking for the new targets to heal CRC andimproving the patients’ quality of life is the key to study CRC.The current research suggests that the process of invasion and metastasis ofmalignant tumors includes: tumor cells separate from the primary tumor, through theextracellular matrix of basement membrane, migrate into the surrounding bloodvessels and lymphatic vessels, reach the distant organs and then adhesion endothelialcell, invade the artery and stay there, then finally format metastases. In this process,angiogenesis is generally considered to be the key factors among tumor growth,invasion and metastasis. And vascular endothelial growth factor (VEGF) is crucialto promote angiogenesis. VEGF is currently considered to be the strongest role topromote angiogenic found by human and it is expressed in both normal and tumor cells. At present, a large number of studies have confirmed an increased expressionof VEGF during ovarian cancer, lung cancer, liver cancer, breast cancer, colorectalcancer, gastric cancer and other solid tumors. Moreover, the expression of VEGF isclosely related with tumor blood vessel density, invasion, metastasis and prognosis.More and more evidence shows that chemotactic inflammation response intumor microenvironment is related with tumor invasion and metastasis. Interleukin-17(IL-17) is mainly secreted by TH17cells, which is a newly discovered T helpcell subsets. IL-17has a strong pro-inflammatory effect and as the core of theinflammatory cytokines, participates not only in a variety of inflammatory andautoimmune disease, but also plays an important role in tumor development.However, whether IL-17can induce tumors produce VEGF to promote tumorinvasion and metastasis, has not been reported. Therefore, in this experiment we willresearch the influence between IL-17and colorectal cancer angiogenesis and explorethe possible molecular mechanisms, hoping to provide new evidence for finding anew anti-angiogenic target.[Objective]Explore the influence between IL-17and colorectal cancer angiogenesis and studythe possible molecular mechanisms, hoping to provide new evidence for finding anew anti-angiogenic target.[Methods]1. Collected50cases of surgery specimens including cancer tissues, adjacent tissuesand normal tissues from colorectal cancer patients. QRT-PCR and Western Blotmethods were used to detect the expression of IL-17mRNA and IL-17protein incolorectal cancer tissues situation.2. Collected100cases of colorectal cancer patients withbiopsy.Immunohistochemistry was used to detect the expression of IL-17incolorectal tissues and then analyze the relationship between IL-17distribution andmicrovessel density (MVD) and the clinic pathological factors. 3. Cultured the colon cancer cell lines in vitro (SW480, LOVO), add50ng/LrhIL-17to them, then observe the influence of rhIL-17to colon cancer cellsline proliferation by CCK-8assay and invasion and migration effects by Transwell.4. Cultured the colon cancer cell lines in vitro (SW480, LOVO), add50ng/LrhIL-17for its intervention, QRT-PCR and Western Blot were used to detect theexpression of VEGF in both two colon cancer cell lines.5. Cultured the colon cancer cell lines in vitro (SW480, LOVO), add50ng/LrhIL-17to them, then QRT-PCR was used to detect the expression of JAK mRNAand STAT mRNA in vitro colon cancer cell lines (SW480, LOVO), were added to50ng/L rhIL-17for its intervention, Western Blot was used to detect theexpression of IL-17R, JAK, p-JAK, STAT, p-STAT protein. ELISA was used todetected the expression of IL-6and IL-8.[Result]1, In colorectal cancer tissue, we found that the expression level of IL-17mRNA andIL-17protein is cancer tissues, adjacent tissues and normal tissues successively thedifference was statistically significant (P<0.05).2, In colorectal cancer tissue，positive staining of IL-17showed brown particles,mainly around the cancer nests. Among100cases of colorectal cancer tissue biopsy,there are75cases of positive expression,25cases of negative expression. Among50cases of normal mucosa biopsy, there are16cases of positive expression and34cases of negative expression. The expression of the difference was statisticallysignificant (2=11.191，P=0.001). We use CD34as the vascular endothelial markers,and reference the method of Weidner et al to statistical tumor MVD and thenanalyze the correlation between IL-17expression and MVD. We found that theMVD value was (15.60±3.65) in the team of high expression of IL-17and （11.15±1.44） in the team of low expression. Independent sample t test statistic showedthat tumor MVD and IL-17expression was positively correlated (t=-5.920, r=0.567, p=0.000). Furthermore we analyzed the relationship between IL-17expression and MVD and clinic pathological factors,knowing that high expressionof IL-17was related with the degree of cancer swelling, TNM stages, and whether there are lymph nodes metastasis or not (P <0.05)), and the MVD value was relatedwith the degree of tumor differentiation, histological type, TNM stages(P <0.05).3, CCK-8experiments showed that in the absence of exogenous IL-17stimulation,SW480and LOVO cell proliferation can be maintained independently, after adding50ng/L of IL-17cultured24h,48h,72h, SW480and LOVO cells performance asignificant proliferative response.SW480cell proliferation rate (%) was (1.18±0.07)%,(1.42±0.09)%,(1.62±0.08) and LOVO cell proliferation rate (%) was(1.13±0.02)%,(1.32±0.05)%,(1.73±0.02)%, showing a tendency to promoteproliferation. There was a significant difference (P <0.05) for the control group.Transwell invasion experiments showed that in the absence of exogenous IL-17stimulation, almost no cells permeates the membrane; After adding IL-17into thetwo cell lines, cell number increased significantly. Compared with the controlgroup, the number of SW480cells was (34.00±0.45) and LOVO cells was (41.60±0.51) after adding IL-17. The cells of both teams were significantly enhanced.Transwell migration assay showed that in the absence of IL-17, there was only a fewpenetrating cells; After adding IL-17into the two cell lines, cell number increasedsignificantly. Compared with the control group, the number of SW480cells was(36.40±0.0.51) and LOVO cells was (46.40±0.68) after adding IL-17. The cells ofboth teams were significantly enhanced.4. QRT-PCR method was used to detect the VEGF mRNA expression and WesternBlot was used to detect the VEGF protein expression. After adding IL-17into thetwo cells line, the VEGF mRNA and protein were increased in the experimentalgroup compared with the control group.5. QRT-PCR method was used to detected the JAK gene and STAT gene expressionand Western Blot was used to detected the JAK protein, p-JAK protein, STATprotein, p-STAT protein expression. ELISA method was used to detect the IL-6andIL-8protein expression. After adding IL-17into the two cells line, the gene andprotein were increased in the experimental group compared with the control group. [Conclusion]The expression of interleukin-17in colorectal cancertissue is associated with tumormalignancy. The possible mechanisms of IL-17promoting CRC development is thatIL-17could activate the JAK-STAT signaling pathway in colon cancer cells andthen up regulate the expression of VEGF to promote tumor angiogenesis, thuscontributing to the proliferation,invasion and metastasis of tumor. |
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