| BackgroundColorectal cancer (CRC), threatening health of the population, is one of the most common solid tumors in the world. In China, the morbidity and mortality of CRC is increasing during 2000 and 2011. According to the latest data published by National Central Cancer Registry of China, it is estimated that 3763 thousand people were diagnosed as CRC and 1910 thousand people died of CRC in 2015. Radical surgery still is the main measure to cure CRC. However, for those who have no possibility for radical surgery, medical therapy is the first choice to suppress tumor and prolong survival, in which anti-angiogenesis therapy is truly important. Therefore, studying the mechanism of angiogenesis and searching for the target of therapy are meaningful in theory and practice.LRG1, found in 1977, is expressed in tissue and serum. Recently, people found that LRG1 was highly expressed in multiple kinds of tumors, and was associated with bad behavior. Some other researches indicated that LRG1 can promote angiogenesis in tumor and other diseases. Nevertheless, it is less studied that whether in CRC, LRG1 can also promote angiogenesis. As a result,we try to explore the role of LRG1 in CRC and angiogenesis, at the meanwhile, explain the mechanism.Method1. Clinical cases study: To build the database and tissue chips of stage III CRC who received radical surgery from 2005 to 2009 in the Chinese PLA General Hospital.We tested LRG1 and CD34-MVD in cancer and matched normal tissues, analyzed the relationship between LRG1 and clinicopathological feathers and survival and searched for the association between LRG1 and CD34-MVD.2. In vitro study: To build the LRG1 overexpression and knockdown cell lines.Using invasion assay and wound healing assay tested the role of LRG1 to cancer cell invasion and migration. We cultured cancer induced HUVECs and tested the ability of proliferation, migration and tube formation by MTT assay, Transwell migration assay and tube formation assay.3. In vivo study: Utilizing the xenograft mice model to explore the role of LRG1 in vivo angiogenesis. Overexpression cells and control cells were injected subcutaneously in nude mice. We measured the volume of xenograft tumor each 3 days and on the 30th day sacrificed the mice to get the tumor body. Using HE stain, CD34 and VEGFA fluorescence staining to establish the impact of LRG1 to in vivo angiogenesis.4. Initial study on mechanism of LRG1 promoting angiogenesis: Testing the expression and secretion level of VEGFA in overexpression cells; measuring the the level of main proteins in PI3K/AKT and MEK/ERK signaling pathway; detecting the distribution of HIF-la in cytoplasm and nucleus; looking for the downregulation of VEGFA and ability to tube formation under stimulation of pathway inhibiors.Result1.312 stage III CRC cases were included. IHC results showed 60.9% cancer tissues and 11.9% matched normal tissues expressed high level of LRG1. The expression level of LRG1 in cancer tissues was significantly higher than normal ones (X2=35.412, P <0.001). Univariate analysis indicated that LRG1 was associated with poor differentiation,T4, blood vessels invasion (P=0.035, 0.028, 0.007). Multivariate analysis suggested LRG1 was an independent worse prognostic factor for OS and DFS of stage III CRC(HR=1.517, P=0.02; HR=1.754, P=0.013). LRG1 is in positive correlation with MVD.2. Choose DLD1 cell lines to establish overexpression (OE) cells and use HT29 cells to build knockdown (KD) cells. It is demonstrated that mRNA level of RNA was upregulated to 6.7 folds in OE group and was downregulated by 35% to 96% in KD groups. In vitro experiments showed the possibility that LRG1 can promote cancer cell invasion and migration, and also HUVECs proliferation, migration and tube formation (P<0.01).3. Growth curve of xenograft mice model indicated that LRG1 promoted tumor growth (P < 0.01). The mean weight of OE tumors was heavier than control tumors(P <0.05). HE staining showed LRG1 induced tumor to deeper invasion. CD34 and VEGFA fluorescence staining demonstrated LRG1 can increase angiogenesis and VEGFA expression.4. The expression level and also the secretion level of VEGFA were increased in LRG1 OE cells and decreased in KD cells. Results of Western Blot showed that LRG1 induced PI3K/AKT/HIF-la and also Ras/MEK/ERK pathway. LRG1 can also translocate HIF-1? from cytosol to nucleus. Under the stimulation of pathway inhibitors, VEGFA expression was inhibited and ability of tube formation was weaken in LRG1 OE group.ConclusionLRG1 was highly expressed in CRC tissues than in normal ones and was associated with T stage,differentiation,vessel invasion. It is also an independent prognostic factor for OS and DFS. LRG1 was positive correlated with MVD. It can not only promote cancer cell invasion and migration, HUVECs proliferation, migration and tube formation in vitro, but also enhance tumor growth and angiogenesis in vivo. CRC cells can induce angiogenesis by VEGFA upregulated through PI3K/AKT/HIF-1? and Ras/MEK/ERK pathway. |
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