| Objective:Human lens epithelial cells(HLECs) are crucial for lens to maintain normal physiological functions. The homeostasis of lens will be broken when HLECs are damaged by oxidative stress, which may accelerate the development of cataract. There are lots of studies about oxidative damage on HLECs, but many questions are still uncertain, such as the source of oxidative damage, link between oxidative damage and pathogenesis of cataract, and detailed molecular mechanism. Recent studies indicate that in physiological condition, HLECs are living in an environment of low oxygen: the partial pressure of oxygen is only about 3~7mm Hg(1k Pa=7.5mm Hg), which equals 1% volume fraction, and HLECs are sensitive to changes of oxygen concentration in physiological hypoxia. However, previous studies about HLECs are completed in common oxygen concentration of 21% volume fraction, which is totally different from physiological condition. In this study, we compared the discrepant abilities of HLECs cultured in physiological hypoxia and common oxygen conditon to resist oxidative damage, and found the key genes associated with oxidative stress in these two different environments. Methods:1. Human lens epithelial cell line HLEB3 was revived and cultured stably. Then the cells were respectivly incubated for 24 h in common oxygen concentration of 21% volume fraction and low oxygen concentration of 1% volume fraction. The cells were digested and centrifugated, and supernatant was collected for assays of superoxide dismutase(SOD) activity, catalase(CAT) activity and glutathione(GSH) content. Besides, the cells cultured in different oxygen conditions as mentioned before was planted in 96-well plates and respectively incubated with hydrogen peroxide in different concentrations of 0μM, 25μM, 50μM, 75μM and 100μM for 24 h, then the cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.2. The cells cultured in different oxygen conditions as mentioned were incubated with 75μM hydrogen peroxide for 24 h and total RNA were collected and determined for micro Array analysis and m RNA chip analysis. Both micro RNAs and m RNAs of significantly altered expression among different samples were screened and part of the results were confirmed by quantitive real-time PCR. Gene Ontology(GO) analysis was used for m RNA funtion classification. Results:1. The SOD activity, CAT activity and GSH content of the cells in physiological hypoxia group were 124.36±6.48U/mg protein, 13.28±0.74U/mg protein and 136.63±5.21μg/ml, whereas the SOD activity, CAT activity and GSH content of the cells in common oxygen group were 168.78±7.51U/mg protein,15.02 ±0.86U/mg protein and 163.96±4.76μg/ml. The SOD activity is 35.72% higher in common oxygen group(P<0.01, n=3), CAT activity is 13.10% higher in common oxygen group(P<0.05, n=3) and GSH content is 20.02% higher in common oxygen group(P<0.001, n=3). The proliferation activity of cells treated with hydrogen peroxide in concentrations of 0μM, 25μM, 50μM, 75μM and 100μM in common oxygen group were 100±4.98%, 95.60±3.57%, 86.40±7.02%, 74.09±8.50% and 44.59±5.25%, while the proliferation activity of cells treated with hydrogen peroxide in the same concentration gradient in physiological hypoxia group were 100±6.66%, 86.40±4.12%, 68.64±4.66%, 53.63±6.03% and 9.26±2.04%. At the concentration of 25μM, cell viability is 10.64% higher in common oxygen group(P<0.001, n=5); at 50μM, cell viability is 25.88% higher in common oxygen group(P<0.001, n=5); at 75μM, cell viability is 38.15% higher in common oxygen group(P<0.01, n=5); at 100μM, cell viability is 381.44% higher in common oxygen group(P<0.001, n=5).2. The microarray results of micro RNA showed that mi R-34b-5p, mi R-630 and mi R-222-5p were up-regulated, mi R-15b-3p was down-regulated under oxidative damage in normoxia; mi R-630 was up-regulated and mi R-335-3p was down-regulated under oxidative damage in physiological hypoxia; mi R-210 was up-regulated in physiological hypoxia without oxidative stress; mi R-145-5p, mi R-193b-3p, mi R-630 and mi R-210 were up-regulated when oxidative damage happened in physiological hypoxia compared with normoxia. The q RT-PCR results confirmed that the microarray results were positive.3. The results of m RNA chip and Gene Ontology analysis revealed that:1) 367 up-regulated genes clustered in 310 gene categories and 154 down-regulated genes clustered in 110 gene categories when oxidative damage happened in normoxia;2) 867 up-regulated genes clustered in 430 gene categories and 466 down-regulated genes clustered in 185 gene categories when oxidative damage happened in physiological hypoxia;3) 319 up-regulated genes clustered in 202 gene categories and 83 down-regulated genes clustered in 35 gene categories when the hypoxia group compared with the normoxia group without oxidative damage;4) 501 up-regulated genes clustered in 352 gene categories and 119 down-regulated genes clustered in 125 gene categories when the hypoxia group compared with the normoxia group under oxidative damage;5) The q RT-PCR tests verified that the discrepant expression level of DDIT4, EDN1, EGLN1, VLDLR and VEGFA presented in the m RNA array was real.Conclusions:HLECs have weaker anti-oxidative ability in physiological hypoxia. A variety of differences appeared on the expression level of m RNA when oxidative damage happened on HLECs cultured in two different environments, indicating the underlying discrepancy of anti-oxidative mechanism. Mi R-630 was up-regulated in both environments when subjected to oxidative stress, showing that this micro RNA involved in the process of oxidation. Mi R-210 was up-regulated when HLECs cultured in hypoxia. Moreover, mi R-630 was up-regulated when oxidative damage happened in hypoxia compared with the normoxia group, indicating that the high level of mi R-210 might have changed the process of oxidation which mi R-630 was involved. The target genes of the two micro RNAs and the interplay between the two micro RNAs will be the central part of our following research. |
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