Investigating The Roles Of MiR-155 In Hepatocarcinogenesis And Glucose&Lipid Metabolism,and Related Mechanisms By MiR-155 Transgenic Mice

Chapter One—1 Generation of transgenic mice for conditional overexpression of mouse miR-155 by Cre/lox P switching expression systemmiR-155 frequently up-regulates in various solid cancers[i.e.,such as nasopharyngeal carcinoma(NPC),breast cancer,lung cancer,colon cancer,cervical cancer,etc],suggesting that it might play a significant role in the process of carcinogenesis,but the a direct evidence that miR-155 as oncomiR could also cause the mentioned-above solid tumors has not been obtained experimentally in transgenic mice.Therefore,we want to produce the Rm155LG transgenic mice for the conditional overexpression of miR-155 transgene mediated by Cre/lox P system.The Rm155LG construct used for generating Rm155LG transgenic mice contained the human cytomegalovirus enhancer and chicken ?-actin promoter(CAG promoter),and a floxed monomeric red fluorescence protein(mRFP)cassette to direct ubiquitous expression of mRFP,while the downstream miR-155 and luciferase(Luc)transgenes were activated by Cre-mediated excision of lox P-flanked mRFP sequence.Three Rm155LG transgenic founders,as determined by mRFP assay and PCR-based genotyping,were attained.Red fluorescence was detected in tissue/organ samples including heart,liver,spleen,lung,kidney,stomach,brain,testis,intestine,muscle,thymus,skin,eye and pancreas of Rm155LG transgenic mice.Additionally,the homozygous Rm155LG transgenic mice were visually,rapidly and readily determined immediately after birth by whole-body(newborn)fluorescence imaging.More importantly,miR-155 and Luc transgenes were successfully activated in a liver-restricted pattern in Rml55LG/Alb-Cre double transgenic mice,demonstrating that Rml55LG conditional transgenic system worked in a Cre-dependent manner.The combined use of this conditional miR-155 transgenic mouse line and various cell/tissue-specific Cre mouse lines will be a valuable in vivo tool to fully dissect the roles of miR-155 as oncomiR in initiating solid tumors.Chapter One—2 Simple and rapid determination of homozygous transgenic mice by in vivo fluorescence imagingBackground:Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from heterozygous transgenic animals.The combinational use of fluorescence reporter transgene and small animal in-vivo imaging system might allow us to visually and rapidly determine transgenic mice homozygous for transgene by in vivo fluorescence imaging.Results:RLG,RCLG,Rmi17LG,Rm21LG and Rm155LG transgenic mice ubiquitously express red fluorescent protein(RFP).To identify homozygous RLG transgenic mice,whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice(F1-generation)derived from the same transgenic founder was performed,followed by the immediate data analysis of in vivo qualitative and quantitative fluorescence imaging,which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s)with strong fluorescence from the rest of F2-generation littermates,and further determine this/these RLG individual(s)showing strong fluorescence to be homozygous,as strongly confirmed by mouse mating.Additionally,homozygous RCLG,Rmi17LG,Rm21LG and Rm155LG transgenic mice were also easily,rapidly and precisely distinguished by the above-mentioned optical approach.This approach allowed us within the shortest time period to obtain transgenic mice homozygous for RLG,RCLG,Rmi17LG,Rm21LG and Rm155LG transgene,respectively,as verified by mouse mating,indicating the practicality and reliability of this method.Conclusions:Taken together,these findings fully demonstrate that in vivo qualitative and quantitative fluorescence imaging offers a visual,simple,rapid and reliable alternative method to the traditional approaches(i.e.,mouse mating and real-time quantitative PCR)in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.Chapter Two Investigating the roles of miR-155 in hepatocarcinogenesis and hepatic lipid metabolism by miR-155 transgenic miceObjective:To explore the roles of miR-155 in lipid metabolism by miR-155 transgenic mice.Methods:The liver-specific overexpression transgenic mice(Rm155LG/Alb-Cre)were generated by mating RL-m155 mice with Alb-Cre mice,and the serum lipid of Rm155LG/Alb-Cre mice was evaluated.The different levels of the liver mRNAs involved in the regulation of glucose metabolism were compared by Gene Chip and qRT-PCR between the Rm155LG/Alb-Cre mice and control mice.Results:Rm155LG/Alb-Cre mice showed significantly higher miR-155 expression than control mice(P<0.05),and exhibited lower serum triglyceride(TG),total cholesterol(TC),and high density lipoprotein cholesterol(HDL-c)than control mice.Rm 155LG/Alb-Cre mice demonstrated decreased hepatic expression of genes involved in triglyceride synthesis(Dgat2,Pcsk9,Ppap2a)and cholesterol biosynthesis(Cnbp,Cyb5r3,Dhcr24),and increased genes expression in PPAR signaling pathway(Cyp4a14?Acaa1b?Fabp2)than mice.Conclusion:miR-155 regulated the lipid synthesis and metabolism and could reduce serum lipd.It is suggested that miR-155 might be valuable to the ameliorated hyperlipidemia.Chapter Three Investigating the effects of miR-155 on glucose metabolism and related mechanisms by miR-155 transgenic miceObjective:To investigate the effects of miR-155 on glucose metabolism and related mechanisms by miR-155 transgenic mice.Methods:The mRNAs from miR-155 transgenic mice involved in the regulation of glucose metabolism and insulin sensitivity were analyzed by Gene Chip and qRT-PCR.Intraperitoneal glucose tolerance test(IPGTT),insulin tolerance test(ITT),and pyruvate tolerance tests(PTT)were used to evaluate the glucose disposal and insulin sensitivity.In addition,the effect of miR-155 on streptozotocin(STZ)-induced diabetic mice was explored in vitro.Results:miR-155 promoted the hepatic gluconeogenesis by targeting the genes that regulate glucose metabolism and insulin sensitivity.In vivo,the overexpression of miR-155 could improve the IPGTT,ITT,and PTT in mice.Moreover,STZ-induced diabetic miR-155 transgenic mice showed ameliorated serum glucose level than control mice.Conclusions:miR-155 modulates the glucose metabolism and insulin sensitivity.It is suggested that miR-155 may contribute to the glucose homeostasis and could be a potential target in diabetes.