The Investigation Of Sumo1, Rb And Cdk6Expression In Colorectal Cancer And Their Influence Mechanism On Cell Cycle Regulation

Colorectal cancer is one of the most common malignant tumor in digestivetract, it’s indicated that the morbidity of colorectal cancer is the third highest inmalignant tumor, there’s1200thousands new patients per year. Systemicchemotherapy is paid close attention in clinical, which aiming at eliminating orrestrain the patients from residual lesions and micrometastasis, improving theprovability of cure, lengthening the survival time and improving their lifequality by systematic and local function.The exploitation and application of the molecular targeted therapy makingcolorectal carcinoma great progress, which also boosting the comprehensivetreatment of the colorectal carcinoma, the related research is the hotspot, aimingat these molecular events, a large amount of researches about kinase relatedantagonists are underway, for example, CDK inhibitor is the hotspot of thenew-style anti-neoplastic drugs whose mechanism is the CDK functioning in thecell cycle.In different types of cancers, Cyclin D-CDK4and cyclin-CDK6complexdrive the cell cycle G1-S transition by Rb phosphorylation. So the related drugsoccur. CDK4and CDK6selective inhibitor PD-0332991has past the anti-cacertests and clinical stage I/II tests. Currently, there’re no related researches aboutapplying potential in colorectal cancer, meanwhile, there’re less people payingattention to the cell cycle passage applied on colorectal cancer. It’s indicated that PD-0332991has anticancer activity, it performs RB dependence in differenttumor treatments considered as selective cell cycle protein dependent kinase. It’sunknown whether CDK4and/or CDK6need Rb phosphorylation, so PD-0332991can be CDK-Rb axis cancer trement.SUMO protein is generally expressed in the whole eucaryotae, in the body,SUMOmay influence the target protein function of stabilization positioning oractivating. SUMO participate in every cell cycle stage including mitosis, growth,differentiation, aging and decay. It’s indicated that SUMO remodeling pathwaymakes difference in tumorgenesis and development. The increase of UBC9isdetected in many cancers, and UBC9overexpression can make tumor cell grow.E3protein PIAS3(activated STAT3protein inhibitor) of SUMO overexpressesin many different kinds of cancer, SUMO E1enzyme level is related to thesurvival rate of the hepatocellular carcinoma. Besides, SUMO can participateand adjust important tumor profiling.Part I: the mechanism research of PD-0332991colorectal cancer inhibitionthrough CDK6-RB axisObjective: Study the expression condition of the CDK4, CDK6, RB etc incolorectal cancer cells, verify the function of CDK4, CDK6in cell cycleadjustment, test the inhibition function of PD-0332991in colorectal cell andresearch the mechanism. Method: application of the acid phosphatase assay tomeasure the cell growth inhibition rate, cell cycle detection through PI dyeing, extract tissue total protein, protein electrophoresis and western Blot examination,lentiviral transfection and RNAi, shRNA cell stabilization transfection etc.Result: It’s indicated through this research that compared by the normalcolorectal tissue, the cancer tissue overexpresses the CyclinD1, D2, D3, CDK4,CDK6and pRB protein in G1stage, CDK4and CDK6control the G1-Stransform, CDK6knockout rather than CDK can obviously reduce Rbphosphorylation. Therefore, CDK6makes key function on RB phosphorylationand tumor growth. In colorectal cancer cell, PD-0332991induces RBphosphorylation blocked and control cells growth through G1stage retardation.Indicate the CDK6-Rb axis make difference in cancer growth for the first time,PD-0332991can be proved as the new type colorectal cancer drugs throughCDK6-RB axis target positioning test.Part II: participation of SUMO1in decoration of CDK6-RB pathway incolorectal cancerObjective: study the SUMO1protein expression condition in colorectalcells, verify the mechanism of the participation of SUMO in cell cycle proteindecoration. Method: cell activity test through acid phosphatase assay, cell cycledetection through PI dyeing, protein electrophoresis and western Blotexamination, lentiviral transfection and RNAi, shRNA cell stabilizationtransfection, Immunoprecipitation etc. Result: it’s indicated that SUMO1pathway is activated in colorectal cells, SUMO1overexpresses, so SUMO1 knockout can depress cell cycle of the cancer cell and tumor growth,16h aftercell cycle synchronization, the time of entering S stage is obviously lengthened.,it’s indicated by IP that CDK6is decorated by two kinds of SUMO1monomer,Sumolation of CDK6can stabilize the protein, thus sustain Rb phosphorylationenzyme activated. Therefore, in the early research, we preliminary establishCDK6-SUMO1cell cycle pathway. The biological function of CDK6-SUMO1-Rb pathway may let it become one of the ideal target, and may becomehigh efficient, specific and low-toxic antitumor drugs based on the current andfollowing research.Conclusion: It’s indicated through this research that compared by thenormal colorectal tissue, the cancer tissue overexpresses the SUMO1, CDK6,CDK4. SUMO1, CDK6and CDK4overexpress in colorectal cell lines;PD-0332991reach treatment effectiveness through inhibition of CDK6-Rbpathway, PD0332991blocks Rb phosphorylation, induce G1escape, and thenblock the growth of tumor cells; COLO320of the colorectal tumor cell isdecorated by two kinds of SUMO1, SUMO1participate in decorative adjust ofCDK6-Rb pathway in COLO320of colorectal tumor cell; SUMO1, CDK6shRNA knockout obviously inhibit COLO320growth, the growth curve ofSUMO1and CDK6separate knockout make consistency.