| BackgroundGlucose uptake is a very important biological process in body's metabolism and it regulates various signaling pathways and a lot of protein expression in human body. The dysfunction of glucose uptake can lead insulin resistance(IR) and other metabolic abnormalities, as well as diabetes mellitus, cancer and other varity of diseases which threaten human health. Palmitic acid(PA) is one kind of saturated fatty acid, which has been proved that can induce the dysfunction of glucose uptake and establish the model of insulin resistance in hepatocytes. Micro RNAs(mi RNAs) are a class of non-coding, small and single-stranded RNA, which can inhibit translation of the target m RNA molecule by complementary base pairing with 3'UTR of the target m RNA molecule, and then regulate various signaling pathways at the molecular level. There are many studies showing that mi RNAs can regulate the dynamic balance of glucose. Mi R-195 is one of many mi RNAs and can regulate various signaling pathways and protein expression, which has been proved that it is closely associated with the occurrence and development of diabetes mellitus, but the specific molecular mechanisms which induce the dysfunction of glucose uptake remains unclear.Objectives1.To identify whether there is abnormal expression of mi R-195 in the dysfunction of glucose uptake of L02 cell induced by PA; 2.To validate whether mi R-195 regulates the dysfunction of glucose uptake of L02 cell; 3.To screen and validate the target gene of mi RNA-195;4.To explore the specific molecular mechanisms of mi R-195 and target gene on the glucose uptake of L02 cell.Part 1 PA treatment on the expression of micro RNA-195 in L02 cellPA induce L02 cell with different concentrations(0.6 mmol/L, 1 mmol/L) for 8h and then observe the morphology of L02 cell under the inverted phase contrast microscope. Then, detect the ability of glucose uptake of L02 cell in each group by GOD-POD method and the expression of mi R-195 after the PA treatment by RT- PCR technique.Methods1. Observe the morphological change of L02 cell after the PA treatment under the inverted phase contrast microscope;2. Detect the ability of glucose uptake of L02 cell after the PA treatment by GOD-POD;3. Detect the expression change of mi R-195 after the dysfunction of glucose uptake induced by PA with Real-Time PCR.Results1. The initial observations show that with the PA treatment the morphology of L02 cell under the inverted phase contrast microscope show that cell morphology significantly blurred, there are no clear edges and corners, and the loose are without a clear line between cells and cells, with the nucleus increases; 2. The detection results of GOD-POD confirms that glucose content in the cell culture supernatant of the groups which are treated by PA with 0.6 mmol/L and 1 mmol/L is higher than that of the control group, this phenomenon suggests that the ability of glucose uptake of L02 cell decreases when it induced by PA at certain concentrations; 3. The detection results of RT-PCR show that the expression of mi R-195 in L02 cell increases after the dysfunction of glucose uptake, and the lower the ability of glucose uptake, the higher the expression of mi R-195.ConclusionPA with different concentrations can induce L02 cell to form the dysfunction of glucose uptake, and the expression of mi R-195 in L02 cell increased after the dysfunction of glucose uptake.Part 2 The effect of micro RNA-195 on the glucose uptake of L02 cellDetect the change of glucose uptake after up-regulate mi R-195 and down-regulate mi R-195 and the change of GLUT1 and GLUT4 in L02 cell with higher mi R-195 expression and lower mi R-195 expression by GOD-POD.Methods1.Detect the fluorescent intensity of L02 cell transfected by mi R-195 mimic and mi R-195 inhibitor using confocal laser scanning microscopy and then calculate the transfection efficient; 2.Detect the expression of mi R-195 in L02 cell transfected by mi R-195 mimic and mi R-195 inhibitor by RT-PCR;3.Detect the change of glucose uptake after up-regulate mi R-195 and down-regulate mi R-195 by GOD-POD; Detect the ability of glucose uptake of L02 cell with up-regulate mi R-195 and down-regulate mi R-195 after the treatment of PA by GOD-POD;4.Western-blot detect the expression of GLUT1 and GLUT4.Results1.The transfection efficient of L02 cell transfected by mi R-195 mimics and mi R-195 inhibitor reaches 40% by confocal laser scanning microscopy; 2.The detection results of real-time PCR confirms that the expression of mi R-195 increased in L02 cell which transfected by mi R-195 mimics, and the expression of mi R-195 decreased in L02 cell which transfected by mi R-195 inhibitor; 3.The detection results of GOD-POD method confirm that the ability of glucose uptake of L02 cell with over-expression of mi R-195 is lower than that of the control group, and the ability of glucose uptake of L02 cell with down-expression of mi R-195 is higher than that of the control group; and PA treatment with over-expressed of mi R-195 can increase the dysfunction of glucose uptake in L02 cell; 4.Western-blot show that the expression of GLUT1 and GLUT4 are down-regulated when the expression of mi R-195 is high.ConclusionThe ability of glucose uptake on L02 cell with over-expression of mi R-195 is lower than that of the control group, thus the ability of glucose uptake of L02 cell with low mi R-195 expression is higher than that of the control group. The ability of glucose uptake of L02 cell can been decreased by over-expressed mi R-195 and treatment of PA. GLUT1 is lower expressed while mi R-195 is over-expressed.Part 3 Predict and validate Sirt-1, the target gene of micro RNA-195Analyse and predict the target gene of mi R-195 based on varity of database, and validate whether it is the target gene of mi R-195 by Western-blot. In addition, explore the molecular mechanisms between the target gene of mi R-195 and the protein associated with glucose metabolism.Methods1.Analyse and predict the target gene of mi R-195 based on database;2.Detect the expression change of Sirt-1 after mi R-195 expression is up-regulated and down-regulated by Western-blot;3.Detect the expression change of GLUT1 and GLUT4 after down-regulate Sirt-1 by Western-blot.Results1.The target gene of mi R-195, which is analysed and predicted based on database is more likely to be Sirt-1; 2. The expression of Sirt-1 decreased after up-regulate mi R-195 and increased after down-regulate mi R-195; 3.The expression of GLUT1 and GLUT4 decreased by down-regulate Sirt-1.ConclusionThe target gene of mi R-195 is more likely to be Sirt-1, and mi R-195 can decrease the expression of GLUT1 and GLUT4 by down-regulating the expression of Sirt-1, and then regulates the glucose uptake of L02 cell. |
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