Effects Of Pro-protein Convertase Subtilisin/kexin 9 On Intestinal Tumor In Apc^Min/+ Mice

Backgrounds:Colorectal cancer（CRC）had become a serious threat to human life and health.According to data released by the World Health Organization in 2020,CRC was respectively the second and third most common cancer in women and men globally.In China,the incidence rate of CRC increased year by year,ranking fifth in 2015 and rising to second in 2020.At present,the treatment of CRC mainly depends on surgery,radiotherapy,chemotherapy,targeted therapy,immunization therapy,palliative therapy and so on.Unfortunately,the 5-years survival rate of patients with CRC was still very low.The occurrence and development of CRC was related to a variety of gene mutations,among which the mutation rate of adenomatous polyposis coli（Apc）gene accounted for about 85%.Mutation of Apc gene could induce colorectal adenoma,which was the source of most colorectal cancers.The transformation from adenoma to adenocarcinoma could be catalyzed by hyperlipidemia.As an endogenous protein that regulated lipid homeostasis,pro-protein convertase subtilisin/Kexin 9（PCSK9）had attracted extensive attention from researchers.In addition to its important role in regulating blood lipids,PCSK9 was also involved in many non-cholesterol-related processes,such as endothelial function,inflammation,apoptosis and platelet activation.In recent years,it had been found that PCSK9 was closely related to glioma,lung adenocarcinoma,acute lymphoblastic leukemia,prostate cancer,gastric cancer and other malignant tumors.However,the role of PCSK9 in the pathogenesis of CRC was not unclear.In conclusion,by using ApcMin/+mice model in this thesis,we systematically analyzed and demonstrated the effect of PCSK9 on the occurrence and development of intestinal tumor in mice,and then reached relevant conclusions.Methods:Male ApcMin/+ mice were bred with female C57BL/6J WT mice and female PCSK9（KI）mice,respectively.By using PCR and Southern blotting,we generated ApcMin/+mice and ApcMin/+PCSK9（KI）littermates.All the littermates were treated with Evolocumab or saline at 2 months of age,which gained ApcMin/+mice,Evolocumab-treated ApcMin/+mice（Inhibition of PCSK9）,and ApcMin/+PCSK9（KI）mice（overexpression of PCSK9）.The mice were anatomized at 6 months,then,the effects of PCSK9 on the health status and intestinal tumorigenesis of ApcMin/+ mice were determined by comparing the variation tendency of body weight,number and size of intestinal adenomas in mice of the same age.The effects of PCSK9 on the progression and malignancy of intestinal tumor in ApcMin/+ mice could be intuitively observed by observing tumor tissue sections by H.E.staining.In addition,we tested the regulatory of PCSK9 on JAK2/STAT3 signaling pathway by Western-blot analysis experiment,and also summarized that the fluctuation of expression levels of apoptosis-related proteins Bax and bcl-2 in tumor tissues was also related to PCSK9.The purpose of IHC experiment is to further verify the experimental results of Western-blot analysis and ensure the accuracy and reliability of the final data.Results:ApcMin/+PCSK9（KI）mice had higher numbers and larger sizes of adenomas than others,with 83.3%of these mice developing adenocarcinoma（vs.16.7%of ApcMin/+mice）.However,treatment with Evolocumab reduced the number and size of adenomas and prevented the development of adenocarcinomas in ApcMin/+ mice（None of the mice developed adenocarcinoma）.Further molecular mechanism studies showed that PCSK9 can regulate the activation of JAK2/STAT3/SOCS3 signaling pathway,and reduce the occurrence of apoptosis by down-regulating the Bax/bcl-2 ratio.Conclusion:PCSK9 might act as an oncogene or had an oncogenic role in ApcMin/+mice model.The occurrence and cancerization of colonic adenoma mediated by Apc gene mutations could be slowed down by inhibiting PCSK9.Research significance:This study verified the effective value and significance of PCSK9 in the course of colon cancer mediated by Apc gene mutation,and brought a new perspective for the follow-up treatment of this disease.More importantly,this study confirmed for the first time that Evolocumab could antagonize the carcinogenic activity of PCSK9,which laid a theoretical foundation for further exploration of Evolocumab in the drug therapy of CRC.