| Backgroud:Esophageal cancer is a common malignant tumor of digestive tract.More than 90%of esophageal cancer in China is squamous cell carcinoma.Despite the obvious improvement of modern treatment technology,the mortality rate of esophageal cancer patients still ranks the 4th among malignant tumors,and the 5-year overall survival rate is only about 15%-25%.Clinically,local invasion or distant metastasis has occurred in 40%-50%of patients at the time of diagnosis.Chemoradiotherapy is the most important method for the treatment of advanced esophageal cancer,but the median survival period of patients is only 5-10 months.There are progressive changes of polygenic genetic material in esophageal squamous cell carcinoma,and gene overexpression caused by multiple factors is an important mechanism leading to the occurrence and development of esophageal carcinoma.Through the study of the molecular biological mechanism of the occurrence,development,recurrence and metastasis of esophageal cancer,molecular targeted therapy of esophageal cancer has emerged.Currently,some molecular targeted drugs for esophageal cancer have entered phase Ⅱ clinical trials,but the clinical results are not satisfactory.The tumor heterogeneity of esophageal cancer and the generation of drug resistance in the process of treatment may lead to the poor efficacy of molecular targeted drugs.Therefore,it is of great significance for early diagnosis and improvement of prognosis of esophageal cancer to look for molecular targets that are more likely to produce good curative effect,explore new biomarkers to predict curative effect and prognosis,and screen the population with advantageous benefits.Leucine-rich-alpha-2-glycoprotein1(LRG1),one of leucine-rich repeat(LRR)family,is made up of Haupt and Baudner first in 1977 of trace proteins found in human serum,LRG1 outside the pathological tissue,secrete,abnormally high expression of the place such as blood,cerebrospinal fluid associated with the development of a variety of malignant tumors,and this may indicate poor prognosis in patients with abnormal expression.However,it is not clear whether it is involved in the progression of esophageal squamous cell carcinoma(ESCC).In this study,we discussed the expression level of LRG1 in esophageal squamous cell carcinoma and its biological significance.Methods:LRG1 and esophageal squamous cell carcinoma were studied in three parts.1.Part Ⅰ:LRG1 is highly expressed in esophageal squamous cell carcinoma,suggesting a poor clinical prognosis.(1)Real-time fluorescence quantitative PCR(qRT-PCR)and Western blot were used to detect the expression of LRG1 in esophageal squamous cell carcinoma and normal esophageal tissue.Immunohistochemical staining(IHC)was used to detect the difference in the expression of LRG1 in different esophageal squamous cell carcinoma tissues.(2)Pearson’s chi-square test was used to analyze the correlation between the expression level of LRG1 and the pathological parameters of patients with esophageal squamous cell carcinoma.Kaplan-meier method was used to draw the survival curve and analyze the prognostic value of LRG1 in patients with esophageal squamous cell carcinoma.(3)Univariate Cox analysis was used to perform univariate analysis on the clinical characteristics of the included cases and the expression level of LRG1.Multivariate Cox analysis was performed on variables with prognostic value in univariate analysis to evaluate whether LRG1 was an independent predictor of survival in patients with esophageal squamous cell carcinoma.2.Part Ⅱ:The effect of LRG1 on the proliferation,apoptosis and invasion of esophageal squamous cell carcinoma cells in vitro.(1)sh-LRG1 plasmids that down-regulated the expression of LRG1,namely sh-LRG1-1 and sh-LRG1-2,were constructed,and sh-NC was used as negative transfection control to down-regulate the expression level of LRG1 in esophageal squamous cell carcinoma cell lines by plasmid transfection.The expressions of LRG1 mRNA and protein in the corresponding sh-LRG1-1 group,sh-LRG1-2 group and sh-NC group of Eca109 and KYSE150 esophageal squamous cell carcinoma lines were detected by RT-PCR and Western blot,respectively,to determine the low expression of LRG1 in the transfected esophageal cancer cells.(2)Changes in proliferation,apoptosis and invasion ability of esophageal squamous cell carcinoma cells after down-regulation of LRG1 expression were detected by CCK8 assay,clonal colony formation assay,Hoechst staining assay and Transwell invasion assay.3.Part Ⅲ:Effect of LRG1 on angiogenesis of esophageal squamous cell carcinoma and its related mechanism.(1)The supernatant of esophageal squamous cell carcinoma Eca109 was obtained and mixed with fresh medium in equal volume.The obtained mixture was used to culture human umbilical vein endothelial cells(HUVEC)and detect the change of HUVEC cells’ tube-forming ability.(2)To detect the expression level of vascular endothelial growth factor(VEGF)in Eca109 cell line of esophageal squamous cell carcinoma after down-regulating the expression of LRG1,and to preliminarily explore the molecular mechanism of LRG1 affecting the angiogenesis of esophageal squamous cell carcinoma.Result:1.Part Ⅰ:LRG1 is highly expressed in esophageal squamous cell carcinoma,suggesting an adverse clinical prognosis.(1)Compared with normal esophageal squamous cell,the expressions of LRG1 mRNA and protein in esophageal squamous carcinoma cell,were significantly increased(mRNA:0.95 ± 0.37 vs.0.28 ± 0.12,P<0.001;Protein:LRG1/GAPDH 0.61±0.14 vs.0.30±0.13,P<0.001).LRG1 is mainly expressed in the cytoplasm of esophageal squamous cell carcinoma cells,and its expression is different in different patients with esophageal squamous cell carcinoma.LRG1 expression in esophageal squamous cell carcinoma was significantly higher than that in paracancerous tissue(2=41.558,P<0.001).(2)Among the 120 patients with esophageal squamous cell carcinoma collected in this study,77 patients(64.2%)showed high expression of LRG1,and the expression level of LRG1 was significantly correlated with the invasion depth and vascular invasion of esophageal squamous cell carcinoma(P<0.001),but had no significant correlation with age,gender,tumor differentiation degree and lymph node metastasis.Compared with patients with low LRG1 expression,the overall survival time(OS)and progressive-free survival time(PFS)of patients with high LRG1 expression were significantly shortened(P = 0.003 and 0.002).(3)Univariate and multivariate analyses confirmed that LRG1 expression level was an independent prognostic factor of OS(HR = 1.586,95%CI:0.976-2.578,P =0.026)and PFS(HR = 1.467,95%CI:0.909-2.368,P<0.001)in patients with esophageal squamous cell carcinoma.2.Part Ⅱ:Down-regulating the expression of LRG1 can inhibit the proliferation and invasion ability of esophageal squamous cell carcinoma cells and promote apoptosis.(1)RT-PCR results showed that the expression of LRG1 mRNA in the sh-LRG1-1 group was 58.3%lower than that in the sh-NC group,and the expression rate of LRG1 mRNA in the sh-LRGl-2 group was 55.5%lower than that in the sh-NC group.After the transfection,the expression of LRG1 mRNA of KYSE150 cells in the sh-LRG1-1 group and sh-LRG1-2 group was reduced by 54.6%and 53.3%,respectively.Western blot results showed that after the transfection of sh-LRG1-1 and sh-LRG1-2 in Eca109 and KYSE150 cells,compared with the sh-NC group,the stained bands of LRG1 protein were weakened,and the optical density values of LRG1 and GAPDH internal reference protein bands were calculated.The expressions of LRG1 in the sh-LRG1-1 group and sh-LRGl-2 group were significantly down-regulated.(2)CCK8 results showed that,compared with the sh-NC group,the proliferation activity of Eca109 and KYSE150 cells transfected with sh-LRG1-1 and sh-LRG1-2 were inhibited.After 72h transfection with Eca109 cells,the cells growth activity of sh-LRG1-1 group and sh-LRG1-2 group was only 38.0%and 34.1%,while that of sh-NC group was 57.2%.After 72h transfection with KYSE150 cells,the growth activity of sh-LRG1-1 group,sh-LRG1-2 group and sh-NC group were 33.8%,29.9%and 57.8%,respectively.Moreover,the growth activity of esophageal squamous cell carcinoma lines in the two groups showed a good time relationship from 12 to 72 hours after transfection,which was statistically different from that in the sh-NC group(P<0.05).(3)After 14 days of transfection of esophageal squamous cell carcinoma cells with plasmids,the clonal colony formation rates of sh-LRG1-1,sh-LRG1-2 and sh-NC groups were 0.26 ±0.07,0.31 ±0.03 and 0.62 ±0.14,respectively.KYSE150 cells:0.28 ±0.02,0.29±0.06 and 0.71 ±0.17.Compared with the sh-NC group,the colony formation rates of Eca109 and KYSE150 cells transfected with sh-LRG1-1 and sh-lRG1-2 were significantly reduced,P<0.01.(4)After LRG1 was down-regulated,the apoptosis rates of the two types of esophageal squamous cell carcinoma cells in the sh-LRG1-1 group and sh-LRG1-2 group were 22.8%and 21.7%,respectively.KYSE150 cells:19.7%and 23.4%.In the negative transfection control group,the apoptosis rates of Eca109 cells and KYSE150 cells were only 3.7%and 2.5%,respectively.LRG1 down-regulation can promote apoptosis of esophageal squamous cell carcinoma cells,P<0.01.(5)After LRG1 was down-regulated,the number of cells passing through the Tranwell chamber in the two groups of esophageal squamous cell carcinoma cells(sh-NC group,sh-LRG1-1 group and sh-LRG1-2 group)was 465.4 ±28.1,207.2 ±19.1 and 189.3±21.0,respectively.KYSE150 cells:357.4±38.2,143.7±23.1,156.6± 18.2.Compared with the negative control group,the down-regulation of LRG1 can reduce the invasion ability of esophageal squamous cell carcinoma cells,P<0.01.3.Part Ⅲ:Down-regulation of LRG1 expression inhibited the angiogenesis of esophageal squamous cell carcinoma.(1)After HUVECs cells were cultured in Eca109 cell culture medium,the mean number of single high-power field tubes in the sh-LRG1-1 group,sh-LRG1-2 group and sh-NC group were 29±5.16,23±2.08 and 42±3.74,respectively.The total tube lengths were 1907 ± 139 dpi,1379± 102 dpi and 2994±215 dpi,respectively.The tube-forming ability of HUVECs cells induced by supernatant of Eca109 cells in sh-LRG1-1 group and sh-LRG1-2 group was significantly lower than that of the corresponding sh-NC group(P<0.01).The results suggested that the ability of LRG1 to induce vascular endothelial tubule formation in esophageal squamous cell carcinoma was decreased after LRG1 expression was down-regulated,and LRG1 may have the ability to promote abnormal angiogenesis in esophageal squamous cell carcinoma.(2)Quantitative analysis of density value and correction of internal reference protein GAPDH showed that,compared with sh-NC group,protein expression levels of VEGF in sh-LRG1-1 group and sh-LRG1-2 group of Eca109 cells were significantly decreased(P<0.01).Conclusion:(1)High expression of LRG1 in esophageal squamous cell carcinoma is significantly correlated with clinical and pathological parameters such as invasion depth and vascular invasion,and its high expression significantly shortens the overall survival time and progressive-free survival time of patients,which is expected to be an effective predictor of clinical prognosis of patients with esophageal squamous cell carcinoma.(2)Down-regulating the expression of LRG1 in esophageal squamous cell carcinoma cells can effectively reduce the proliferation and invasion ability of tumor cells and improve the apoptosis rate,indicating that LRG1 plays an important role in the biological progress of esophageal squamous cell carcinoma.(3)Down-regulation of LRG1 expression in esophageal squamous cell carcinoma cells may reduce HUVEC’s tubulogenic ability and inhibit the formation of new blood vessels in esophageal squamous cell carcinoma by affecting the expression of VEGF in tumor cells. |
