Studies On The Biological Functions Of Human Disease-related GenesDHTKD1、Kif18A And Rig-Ⅰ

PART I. The Role of DHTKD1in Mitochondrial Biogenesis andFunction MaintenanceThe functional integrity of mitochondria is crucial for overall cell activities, such asnormal cell functions, cellular signal transduction and intracellular traffic.Mitochondrial dysfunctions could change energy metabolism and in many cases areassociated with neurodegenerative diseases and neurological diseases. We havereported an autosomal dominant pedigree of Charcot-Marie-Tooth disease type2(CMT2), in which, we found that the patients carried a heterozygous nonsensemutation in dehydrogenase E1and transketolase domain containing1(DHTKD1)through genome-wide scan and linkage analysis. The product of this gene is amitochondrial protein and is similar to E1subunit of α-ketoglutarate dehydrogenasecomplex. Till now, no information about the function of this protein could be found.We tried to study its role in mitochondrial energy metabolism through biochemicaland molecular biological methods, uncovering the relationship of DHTKD1mutationand pathogenesis of CMT2. We found that the expression level of DHTKD1wasassociated with intracellular adenosine triphosphate (ATP) content and oxidativephosphorylation activities, indicating that DHTKD1plays an important role in energymetabolism in the mitochondria. Further studies showed that the expression ofmitochondrial biogenesis genes was down-regulated after DHTKD1silencing, that is,DHTKD1silencing caused mitochondrial biogenesis defect, further led to increasedintracellular reactive oxygen species (ROS) and ultimately affected cell proliferationand apoptosis. Our study showed that DHTKD1plays an important role in mitochondrial biogenesis and function maintenance. Mitochondrial dysfunctioncaused by DHTKD1gnen mutation and toxic effects of intracellular accumulation ofROS may contribute to the pathogenesis of CMT2. PART II. The Role of Kif18a in Colitis-associated ColorectalTumorigenesisThe mitotic spindle is a validated target in cancer chemotherapy and a variety ofanti-mitotic drugs, such as taxanes and vinca alkaloids, have been successfully used inthe clinic. However, these spindle poisons have certain limitations because tumor cellsmay become resistant to these drugs. Thus, there is a significant effort to find otherways of targeting the mitotic spindle, which could potentially overcome some of thetoxicities and mechanisms of resistance. In recent years, the kinesin family of motorproteins has gained significant attention, being crucial for mitosis and thus emergingas a target for chemotherapeutic intervention. Kinesins are microtubule-based motorproteins that function at different stages of cell division, intracellular vesicle andorganelle transport, and the movement of microtubules. Several kinesins have beenimplicated in tumorigenesis.Kif18a is a member of the kinesin-8family, and it is a molecular motor proteinthat uses adenosine triphosphate (ATP) hydrolysis to produce force and movementalong microtubules, regulating microtubule dynamics and cell division. In this study,we found that Kif18a expression is up-regulated in AOM/DSS-induced colorectaltumors. Kif18a deficiency affected tumor cell proliferation and apoptosis, because ofreduced Akt phosphorylation in tumor cell. These results demonstrate the significanceof Kif18a in colitis-associated colorectal (CAC) progression. Thus, Kif18A couldbecome a target for CAC therapeutic intervention and could be used as a CACdiagnostic biomarker. Fortunately, a small molecule BTB-1has been reported toinhibit Kif18a activity, which is worth further exploration and development. PART III. The Role of Rig-I in Intestinal Microbiome Regulation andCAC CarcinogenesisRetinoic acid-inducible gene-I (Rig-I) is an intracellular viral RNA receptor, whichspecifically recognizes double-stranded viral RNA initiating antiviral innate immunity.In this process, Rig-I recruits a specific adaptor protein, mitochondrial antiviralsignaling protein (MAVS; also named Cardif, IPS-1or VISA), and activatesdownstream IFN regulatory factor3(IRF3) and NF-κB signaling, producing type Iinterferon and inflammatory cytokines to resist virus invasion. In fact, Rig-I wasoriginally identified as being up-regulated obviously in the all trans-retinoic acid(ATRA)-induced acute promyelocytic leukemia (APL) cell line NB4differentiation.In our previous studies, we found that Rig-I knock-out mice were susceptible to colitis,which was similar with human inflammatory bowel disease (IBD), accompanied withdecreased expression of G protein subunit α-i2(Gαi-2) and defects in T cell activation.Rig-I knock-out mice were also found to be more susceptible to infection with E. colias compared to wild-type mice due to decreased phagocytosis of bacteria.Furthermore, Rig-I-/-mice were susceptible to spontaneous infection with acommensal bacterium, S. xylosus, in the skin around eyes and neck, accompanied withdefects in B cell development and specific IgG3immunoglobulin class switchrecombination (CSR). These indicated that the regulatory functions of Rig-I arestrikingly broad. It plays a crucial role not only in antiviral responses but also inantibacterial responses. Additionally, Rig-I was down-regulated in intestinal epithelialcompartment of IBD patients, accompanied with apparent disorder of intestinal flora.And18%of IBD patients may eventually develop into colorectal cancer. Now, westudied intestinal microbiome of genotype-different mice through high-throughputsequencing. We found that the gut flora of Rig-I-/-mice were very different from thatof WT mice, and the gut flora in Rig-I-/-mice were more abundant and diversified.The difference between wild type (WT) and knock out (KO) mice were found when they were two weeks old, and by the age of eight weeks, the gut flora in Rig-I-/-micehad stabilized, but it still slow changed in WT mice. We suppose that intestinal floraof WT and Rig-I-/-mice developed to different directions after birth, and Rig-I-/-micereached a relatively stable state when they were4-8weeks old, earlier than WT mice.Results from induced CAC model showed that Rig-I-/-mice were more susceptible toCAC, and the tumors were more severe than that in WT mice. These demonstrate thatRig-I has an important role in the regulation of intestinal flora and development ofcolorectal cancer, and there may be a definite link between the two.