Effect Of Novel Gene KLF14Overexpression On Glucose Metabolism And Insulin Resistance In Hepatoma Cell

PART ONECONSTRUCTION AND IDENTIFICATION OF THEEUKARYOTIC OVER-EXPRESSION VECTOR OFMOUSE KLF14GENEObjective: To construct and identify the mouse recombinant plasmidpIRES2-EGFP-KLF14.Methods: First of all, RNA was extracted from mouse tissues andDNA was obtained by the method of reverse transcript-polymerase chainreaction (RT-PCR). Then the recombinant plasmid pIRES2-EGFP-KLF14was constructed after gel extraction, purification, double enzymes digestionand ligation with the vector fragment of DNA fragment. At last, therecombinant plasmid was identified by double enzymes digestion and DNAsequence analysis.Results: By agarose gel electrophoresis, the enzyme digestionproducts of the recombinant plasmid were presented with two bands. Thelarge fragment with5.3kb is accordance with the plasmid vector pIRES2-EGFP. The small fragment with1000bp is identical with theKLF14cDNA products. This is a preliminary experiment that has provedthe recombinant plasmid has been constructed successfully. Sequenceresults were analyzed by DNA Blast, and its nucleotide sequence seem tobe the same with the CDS code sequence of mouse KLF14genecompletely in Genbank. This is a further experiment that proves theconstruction successful.Conclusion: The recombinant plasmid of mouse KLF14has beenconstructed successfully, which will be helpful for the subsequent study onthe effect of KLF14over-expression on glucose metabolism and insulinresistance. PART TWOMRNA EXPRESSION AND DISTRIBUTION OF KLF14GENE INDIFFERENT TISSUES OF MICEObjective: To observe mRNA expression and distribution of KLF14gene in different tissues of healthy C57BL/6J mice.Methods: The mice were killed by cervical dislocation. Varioustissues, such as heart, liver, kidney, brain, speech, lung, stomach, intestine, epididymis, muscle and fat, of C57BL/6J mice were obtained. Then totalRNA were extracted from each different tissues and reversed transcriptioninto cDNA. Real-time fluorescent quantitative PCR was performed tomeasure the mRNA level of KLF14in different tissues. The experimentwas repeated three times.Results: The KLF14gene is ubiquitously expressed in mice. Therelative mRNA expression level of KLF14from high to low in order isheart, muscle, liver, fat, intestine, kidney, brain, lung, stomach, speech andepididymis.Conclusion: The KLF14gene is ubiquitously expressed in mice. Itmeans that KLF14may be involved in maintaining the normalphysiological regulation of the body. What’s more, there is a relative highlevel of KLF14mRNA in liver, so hepatocyte can be selected as the objectof study in vitro, which offers experimental model for further research onthe physiological function of KLF14gene. PART THREEEFFECT OF KLF14GENE OVER-EXPRESSION ON KEYSIGNALING MOLECULES IN THE RELATED SIGNAL PATHWAYObjective: To observe the effect of KLF14gene over-expression onthe key signaling molecules in InsR/IRS/AKT/GSK-3β andAMPK/TORC2/PEPCK signal pathway. To investigate the molecularbiological mechanism involved in the effect of KLF14on glucosemetabolism and insulin resistance.Methods: The research model of KLF14gene over-expression hepaticcell in vitro was established. The experiment was divided into four groups,which contained black control group, insulin treatment group (INS),KLF14transfection group and insulin and KLF14co-treatment group(INS+KLF14). And each group was given corresponding treatmentrespectively. Then the protein expression level of key signaling moleculeswas detected to evaluate the function of KLF14gene. The glucose uptakewas determined by2-Deoxy-D-[3H]-glucose assay in hepatoma cells.Results: Compared with KLF14un-transfection group, thephosphorylation of IRS, AKT, AMPK, TORC2were significantly enhancedafter KLF14transfection and insulin treatment (P<0.01), While theexpression of p-InsR protein was not changed. In addition, the expressionof p-GSK3β protein was also elevated while the PEPCK was decreased(P<0.01). The glucose uptake rate was also increased after KLF14transfection.Conclusion: KLF14gene over-expression may improve hepaticinsulin resistance by activating the classical pathway of insulin. Meanwhile, it can ameliorate hepatic glucose metabolism by inhibiting gluconeogenesisand promoting glucose uptake and glycogen synthesis.