The Effect Of Hypoxia On IL-6, TGF-β1 And The Epithelial Mesenchymal Transition In The Esophageal Squamous Carcinoma Cells

Background and PurposeThe morbidity and mortality of esophageal cancer in China rank the first in the world. In the early stage of esophageal carcinogenesis, tumor infiltration to adjacent tissues and metastasis are the major cause of ineffective treatment and death in cancer patients. Therefore,that much more research on the molecular mechanism of invasion and metastasis of malignant tumor can develop specific drugs that inhibit or terminate this process.This has become a hotpoint in current medical study.Tumor metastasis is an important cause of recurrence and difficult to cure,which progress through histopathologically distinct stages. Epithelial mesenchymal transition(EMT) plays a potential important role for this process. EMT plays crucial roles in embryonic development, morphogenesis of various tissues and organs, injury repair and organ fibrosis. It also is an important event in tumor invasion and metastasis. Many celluar signal transduction pathway involved in the EMT process causing the changes of many gene expression. The classic cellular signal transduction pathways include STAT3 signaling pathway, Snail signaling pathway, Wnt signaling pathway and so on.In recent years, dozens of studies show that hypoxia inducible factor(HIF)signaling pathway also plays a very significant role in the process of EMT.Hypoxia inducible factor 1(HIF-1) is common in many malignant solid tumors such as liver cancer, breast cancer, gastric cancer, and glioma. However, in normal oxygen environment it is degraded by the oxygen dependent ubiquitin-proteasome pathway in cells.When tumors grow to a certain size and cancer cells divide uncontrollably,they form larger tumors. As a consequence, there is limited availability of nutrients and oxygen in the microenvironment and cancer cells are exposed to intermittent hypoxic conditions and HIF-1 is stably expressed.HIF-1α regulated by hypoxia signal is the active subunit of HIF-1 and can form a heterodimer within the cell nucleus under hypoxia. When cancer cells are exposed to intermittent hypoxic conditions HIF-1α is overexpression at both m RNA and protein levels with the down-regulation of E-cadherin and the up-regulation of Vimentin.This phenomenon has been confirmed in the process of occurrence and development of a variety of solid tumors.In recent years, many scholars have made great progress in the research of gene transcription regulation mediated by HIF-1α.Interleukin-6(IL-6) is a multifunctional cytokine produced by monocytes /macrophages, fiber mother cell, T lymphocyte, glial cells, epithelial cells.It can stimulate the proliferation and differentiation of cells in the immune response to enhance their function. Previous studies found that IL-6 was involved in the body’s immune response, inflammation, hematopoiesis and other physiological and pathological processes and was also high expression in the breast cancer, prostate cancer, lung cancer and other solid tumors.The high expression of IL-6 can significantly activate signal transducer and activator of transcription-3(STAT-3)regulating the expression of the downstream gene of STAT-3 through the IL-6/GPl30 receptor system and induce epithelial mesenchymal transition.Transforming growth factor-β(TGF-β) regulating the growth and differentiation of normal cells and cancer cells significantly is widely existed in various tissues from drosophila to human.TGF-β includes three subtypes:TGF-β1,TGF-β2 and TGF-β3 in the human tissues.The expression of TGF-β1 was the highest in somatic cells and plays an important role in many physiological and pathological processes, such as embryonic development, tissue repair, inflammatory response, and the occurrence andprogression of tumors.TGF-β1 can induce epithelial mesenchymal transition in a variety of tumor cells and is one of the most widely studied factors that could induce EMT.In the previous studys, the exogenous TGF-β1 and IL-6 were added to induce EMT in the cancer cells. After induction,the cancer cells can secrete TGF-β1 and IL-6to maintain EMT status.The results of previous studies in our laboratory showed that hypoxia can induce EMT in esophageal squamous carcinoma cell Eca-109 and the exogenous TGF-β1 and IL-6 were added to induce EMT in esophageal squamous carcinoma cell EC-1.We hypothesized that the process of EMT transformation in esophageal cancer cells under hypoxic conditions may be associated with the production and secretion of more TGF-β1 and IL-6.This research will adopt scientific research methods to establish a chemical hypoxia model, block the expression of HIF-1α gene,detect the m RNA expression and protein secretion of TGF-β1、IL-6,the m RNA and protein expression level of E-cadherin and Vimentin in hypoxia condition.Finally, detect the effect of HIF-1α si RNA transfection on the invasion and migration ability of Eca-109 cells.Part I. Establishment of cellular hypoxia model and the HIF-1αgene disruptionMethods:1. Establish a chemical hypoxia model by incubating esophageal squamous carcinoma cell Eca-109 in medium with a final concentration of 150μmol/L Co Cl2 to simulate hypoxic environment. Observe the changes of morphology of Eca-109 cells and expression of HIF-1α under the condition of chemical hypoxia.1.1 An inverted microscope was used to observe the changes in morphology of cultured Eca-109 cells to select the optimal time for Co Cl2 action.1.2 Real-time PCR was used to examine the effect of hypoxia on expression of HIF-1α m RNA in Eca-109 cells under hypoxia at 0h(normoxic), 3h, 6h, 12 h, 24 h.1.3 Western blotting was used to examine the effect of hypoxia on expression of HIF-1α protein in Eca-109 cells under hypoxia at 0h(normoxic), 3h, 6h, 12 h, 24 h.2. HIF-1α si RNA transfected in esophageal squamous carcinoma cell Eca-109 and observed the transfection efficiency,the changes of morphology of Eca-109 cells and the expression status of HIF-1α m RNA to select the optimal transfection concentration and sequence.2.1 An inverted microscope was used to observe the transfection efficiency,the changes of morphology of Eca-109 cells in different transfection concentration(20n M、50n M、80n M、100n M) to determine the optimal transfection concentration(100n M).2.2 Real-time PCR was used to examine the expression of HIF-1α m RNA in different transfection sequence and determined the optimal transfection sequence(HIF-1α-homo-1217).Result:1.Establishment of cellular hypoxia model and expression of HIF-1α at different hypoxia time.1.1 Observed under an inverted microscope, Eca-109 cells were multipolar and presented a cobblestone-like appearance after fusion to monolayer at the bottom of the 6-wells plate under normoxic conditions.With the prolongation of the time of hypoxia,Eca-109 cells gradually began to shuttle, brown particles inside the cells increased, the refractive index decreased, the cell debris and suspended death of cells gradually increased. The cell death was not obvious under hypoxia at 0h(normoxic),3h, 6h.There were a small amount of spindle cells and suspended dead cells under hypoxia at 12 h and the growth-condition of cells was good,but the spindle cells and suspension cells were significantly increased after 24 hours hypoxia comparing with12 h hypoxia group.More than 50% cells have died under hypoxia at 48 h.In order to avoid the effect of Co Cl2 induced apoptosis and cell injury, 24 h incubation was chosen as the optimal time for subsequent hypoxia experiments.1.2 Real-time PCR results: The relative expression level of HIF-1α m RNA was1±0.048 under hypoxia at 0h(normoxic), decreased in hypoxia groups to 0.732± 0.102 at 3h and 0.613±0.114 at 6h, but increased in hypoxia groups to 2.216±0.215 at12 h and 3.512±0.712 at 24 h.The difference in expression level of HIF-1α m RNA between normoxia group and each hypoxia group was statistically significant(P<0.05).1.3 Western bloting results: The relative expression levels of HIF-1α protein was0.631 ± 0.086 under the hypoxia condition at 0h(normoxic) in Eca-109 cells,increased to 1.692±0.166 at 3h, 1.802±0.131 at 6h, 1.804±0.094 at 12 h, 2.099±0.095 at 24 h, they were significantly higher than the normal oxygen group(P<0.05).2.HIF-1α si RNA transfected in esophageal squamous carcinoma cell Eca-109 to block the expression of HIF-1α gene.2.1 Observed under an inverted fluorescence Microscope,there were a little of green fluorescent labeled substances and the number of green cells were rare, the growth of tumor cells were well, the suspension cells were not obvious using 20 n M Lipofectamine?2000 and 20 n M fluorescence labeled HIF-1α si RNA to transfect Eca-109 cells.The green fluorescent labeled substances and the number of green cells which were transfected successfully gradually increased with the increase of transfection concentration. The number of green cells which were transfected successfully was the most and the transfection efficiency was the highest,when we used 100 n M Lipofectamine?2000 and 100 n M fluorescence labeled HIF-1α si RNA to transfecte Eca-109 cells.The cells grew well and the dead cells were rare.So we will used 100 n M Lipofectamine?2000 and 100 n M HIF-1α si RNA to transfect Eca-109 cells.2.2 Real-time PCR results: The relative expression levels of HIF-1α m RNA were1±0.091 in the blank control group, 0.912±0.214 in negative control group, 0.802±0.122 in HIF-1α-homo-934 group, 0.571±0.074 in HIF-1α-homo-1067 group and0.182±0.020 in HIF-1α-homo-1217 group.Compared with the blank control group,the relative expression levels of HIF-1α m RNA was no significant difference(P>0.05)and significantly reduced in the rest of the experimental group(P<0.05).In HIF-1α-homo-1217 group,the expression of HIF-1α m RNA was the lowest,so the HIF-1αsi RNA sequence in HIF-1α-homo-1217 group was the optimal transfection sequence.Part II The effect of hypoxia on the production of IL-6,TGF-β1 in Eca-109 cellsMethods:1. The effect of hypoxia on the production of IL-6,TGF-β1 in Eca-109 cells1.1 Real-time PCR was used to examine the effect of hypoxia on expression of IL-6 m RNA and TGF-β1 m RNA in Eca-109 cell under hypoxia at 0h(normoxic),3h,6h, 12 h, 24 h.1.2 Enzyme-linked immunosorbent assay(Elisa) was used to examine the level of IL-6,TGF-β1 concentration in the culture supernatants of Eca-109 cells under hypoxia at 0h(normoxic), 3h, 6h, 12 h, 24 h.2.The effect of transfection of HIF-1α si RNA on the production of IL-6,TGF-β1in Eca-109 cells2.1Real-time PCR was used to examine the expression of IL-6 m RNA and TGF-β1 m RNA in Eca-109 cells in the normoxic group(only added to the culture medium), the hypoxia group(150μmol/L Co Cl2), the HIF-1α si RNA group(HIF-1αsi RNA sequence) and the dual role of group(150μmol/L Co Cl2+HIF-1α si RNA sequence) at 24 h.2.2 Enzyme-linked immunosorbent assay(Elisa) was used to examine the level of IL-6,TGF-β1 concentration in the culture supernatants of Eca-109 cells in the normoxic group(only added to the culture medium), the hypoxia group(150μmol/L Co Cl2), the HIF-1α si RNA group(HIF-1α si RNA sequence) and the dual role of group(150μmol/L Co Cl2+HIF-1α si RNA sequence) at 24 h.Result1. The changes of the production of IL-6,TGF-β1 in Eca-109 cells at different hypoxia time.1.1 Real-time PCR results: The relative expression levels of IL-6 m RNA was1±0.216 at 0h(normoxic), increased to 1.154±0.169 at 3h, 1.249±0.215 at 6h,1.970±0.359 at 12 h and 2.874±0.448 at 24 h.They were significantly higher than the normoxic group(1±0.216)(P<0.05). The relative expression levels of TGF-β1m RNA was 1±0.024 at 0h(normoxic), increased to 1.286±0.210 at 3h, 1.637±0.147 at 6h,1.913±0.578 at 12 h and 2.557±0.194 at 24 h.They were significantly higher than the normoxic group(1±0.216)(P<0.05).1.2 Enzyme-linked immunosorbent assay(Elisa) results:The concentration of IL-6 was 30.201±2.197 pg/ml at 0h(normoxic), decreased to 19.115±2.005 pg/ml at3 h, 23.313±1.820pg/ml at 6h, increased to 31.434±10.558 pg/ml at 12 h, 43.403±7.824 pg/ml at 24 h.The difference in protein expression between anormoxia and hypoxia groups was statistically significant(P<0.05). The concentration of TGF-β1was 80.424±9.228 pg/ml at 0h(normoxic), 75.412±0.305 pg/ml at 3h,84.251±7.014pg/ml at 6h, 74.539±23.117 pg/ml at 12 h,no significant difference(P>0.05). But,it increased to 207.551±9.154 pg/ml at 24h(P<0.05).2.Transfection of HIF-1α si RNA to inhibit the expression of HIF-1α able to partially reduce the production of IL-6,TGF-β1 in Eca-109 cells2.1 Real-time PCR results: The relative expression levels of IL-6 m RNA in the HIF-1α si RNA group(0.314±0.147),the dual role of group(0.847±0.162) was significantly lower than the normoxic group(1±0.126), the hypoxia group(1.601±0.296), and of HIF-1α si RNA group IL-6 m RNA expression was significantly lower than the result in the dual role of group(P<0.05).The relative expression levels of TGF-β1 m RNA in the HIF-1α si RNA group(0.198±0.052), the dual role of group(0.522± 0.294) were significantly lower than the normoxic group(1±0.208), the hypoxia group(3.122±0.317), and of HIF-1α si RNA group TGF-β1 m RNA expression was significantly lower than the result in the dual role of group(P<0.05).2.2 Enzyme-linked immunosorbent assay(Elisa) results:The concentration of IL-6 protein in the HIF-1α si RNA group(15.543±2.143 pg/ml) was significantly lower than the normoxic group(27.796±1.896pg/ml), the hypoxia group(43.002±3.518 pg/ml), the dual role of group(29.042±1.257pg/ml),and of HIF-1αsi RNA group IL-6 concentration was significantly lower than in the dual role of group(P<0.05).The concentration of TGF-β1 protein in the HIF-1α si RNA group(86.737±15.457pg/ml) was significantly lower than the normoxic group (171.757±24.773 pg/ml), the hypoxia group(230.293±2.677pg/ml), the dual role of group(153.667±35.704 pg/ml), and of HIF-1α si RNA group IL-6 concentration was significantly lower than in the dual role of group(P<0.05).Part III The effect of hypoxia on the epithelial mesenchymal transition in Eca-109 cellsMethods:1. The changes in morphology of cultured Eca-109 cells after HIF-1α si RNA transfection An inverted microscope was used to observe the changes in morphology of cultured Eca-109 cells in the normoxic group(only added to the culture medium), the hypoxia group(150μmol/L Co Cl2), the HIF-1α si RNA group(HIF-1α si RNA sequence) and the dual role of group(150μmol/L Co Cl2+HIF-1α si RNA sequence) at24 h.2.The effect of HIF-1α si RNA transfection on the expression of HIF-1α 、E-cadherin、Vimentin2.1 Real-time PCR was used to examine the expression of HIF-1α m RNA,E-cadherin m RNA and Vimentinin m RNA in Eca-109 cells in the normoxic group,the hypoxia group, the HIF-1α si RNA group and the dual role of group at 24 h.2.2 Western blotting was used to examine the expression of HIF-1α、E-cadherin and Vimentin protein in Eca-109 cells in the normoxic group,the hypoxia group,the HIF-1α si RNA group and the dual role of group at 24 h.3.The effect of HIF-1α si RNA transfection on the invasion and migration ability of Eca-109 cells3.1 Transwell in vitro migration assay was used to examine the changes of Eca-109 cells migration ability in each experimental group.3.2 Transwell in vitro invasion assay was used to examine the changes of Eca-109 cells invasive ability in each experimental group.Result1. The changes in morphology of cultured Eca-109 cells after HIF-1α si RNA transfection Observed under an inverted microscope, Eca-109 cells were multipolar and presented a cobblestone-like appearance after fusion to monolayer at the bottom of the 6-wells plate. The normoxic group,the HIF-1α si RNA group and the dual role of group in esophageal squamous cell carcinoma cell Eca-109 reduced cell gap, the cells unite with each other,compared to the number of spindle-shaped cells and floating dead cells relative reduction than the hypoxia group,and of the HIF-1α si RNA group the number of spindle-shaped cells was least.2.2.The effect of HIF-1α si RNA transfection on the expression of HIF-1α 、E-cadherin、Vimentin2.1 Real-time PCR results: The relative expression levels of HIF-1α m RNA in the HIF-1α si RNA group(0.148±0.087),the dual role of group(0.252±0.091) were significantly lower than the normoxic group(1±0.011), the hypoxia group(2.417±0.104), and of HIF-1α si RNA group HIF-1α m RNA expression was significantly lower than the result in the dual role of group(P<0.05). The relative expression levels of E-cadherin m RNA in the HIF-1α si RNA group(1.711±0.357),the dual role of group(1.221±0.129) were significantly higher than the normoxic group(1±0.126),the hypoxia group(0.201±0.226), and of HIF-1α si RNA group HIF-1α m RNA expression was significantly higher than the result in the dual role of group(P<0.05).The relative expression levels of Vimentin m RNA in the HIF-1α si RNA group(0.617±0.076) was significantly lower than the normoxic group(1±0.241), the hypoxia group(2.283±0.348), the dual role of group(1.247±0.196)(P<0.05).2.2 Western bloting results:The relative expression levels of HIF-1α protein in the HIF-1α si RNA group(0.343±0.019), the dual role of group(0.420±0.021) were significantly lower than the normoxic group(1.121±0.094), the hypoxia group(1.553±0.135), and of HIF-1α si RNA group HIF-1α protein expression was significantly lower than in the dual role of group(P<0.05). The relative expression levels of E-cadherin protein in the HIF-1α si RNA group(3.971±0.097),the dual role of group(3.673±0.017) were significantly higher than the normoxic group(3.270±0.020), the hypoxia group(1.978±0.035), and of HIF-1α si RNA group E-cadherin protein expression was significantly higher than in the dual role of group(P<0.05).The relative expression levels of Vimentin protein in the HIF-1α si RNA group(1.849±0.155),the dual role of group(2.163±0.183) were significantly lower than the normoxic group(2.506±0.132), the hypoxia group(4.376±0.097), and of HIF-1α si RNA group Vimentin protein expression was significantly lower than in the dual role of group(P<0.05).3.HIF-1α si RNA transfection can inhibit the invasion and migration ability of Eca-109 cells3.1 Transwell in vitro migration assay results:the hypoxia group(475.33±21.54)cells through the largest number of membrane(P<0.05), the normoxic group(312.18±12.32), the HIF-1α si RNA group(157.28±15.68) and the dual role of group(202.37±15.26) with the hypoxia group compared to a significant reduction(P<0.05), and of HIF-1α si RNA group, the number of cell-penetrating below the dual role of group(P<0.05).3.2 Transwell in vitro invasion assay results:the hypoxia group(143.28±11.52)cells through the largest number of membrane(P<0.05), the normoxic group(85.43±8.03), the HIF-1α si RNA group(36.16±10.34) and the dual role of group(69.34±10.50) with the hypoxia group compared to a significant reduction(P<0.05), and of HIF-1α si RNA group, the number of cell-penetrating below the dual role of group(P<0.05).Conclusions:1. Hypoxia can increase the m RNA expression and protein secretion of TGF-β 1and IL-6 in esophageal squamous carcinoma cell Eca-109.2. Hypoxia can induce EMT and enhance cell in vitro invasion and metastasis in esophageal squamous carcinoma cell Eca-109.3. The process of EMT in esophageal squamous carcinoma cells under hypoxic conditions may be associated with the production and secretion of more TGF- β 1 and IL-6.Transfection of HIF-1α si RNA to inhibit the expression of HIF-1α can partially block the expression of IL-6 and TGF-β1, inhibite EMT and reduce the migration and invasion ability of the esophageal squamous carcinoma cells.