Clinical Significance And Mechanism Of LOXL2,SHMT2 On Colorectal Cancer And Isolation And Identification Of TIL

Colorectal cancer(CRC)is the third most prevalent cancer in the world and ranks the fourth fatality rate in all cancers.The different stages have a great impact on the prognosis of colorectal cancer.The 5-year survival rate of early colorectal cancer was 90.1%,and the rate of lymph node metastasis was 69.2%.The 5-year survival rate of distant metastasis was only 11.7%.In addition,the molecular pathogenesis of colorectal cancer has a strong heterogeneity.The impact of different molecular mechanisms on CRC patients varies greatly,which also has a tremendous impact on treatment and prognosis.The new molecular typing based on complex mutation patterns will play a crucial role in the treatment of colorectal cancer.Therefore,the study of the molecular mechanisms of different pathogenic genes and the role of immune cells is particularly crucial.This study is divided into three parts,the first two parts of the study through the previous chip data,excavated two genes LOXL2 and SHMT2,which is important for CRC development.In the reported studies,LOXL2 is thought to affect tumor development only in the extracellular matrix(ECM).However,our study confirms that it not only plays a role in ECM but also overexpresses in colorectal cancer cells and affects the proliferation and metastasis of tumor cells.There is not much research on tumor growth in SHMT2.We found its downstream target genes by RNAseq,and the results were verified by the putback experiments.The first two parts provide a new direction for the molecular treatment of colorectal cancer.The third part,the separation of TIL,provides the technical possibility for immunotherapy and individualized treatment of colorectal cancer.Through the interconnection and relatively independent research in three parts,this article clarifies the molecular mechanisms that may affect the occurrence and development of CRC from a new perspective and provides new clues for the targeted therapy of CRC.Part Ⅰ: Study on the function and molecular mechanism of LOXL2 oncolorectal cancerLysyl oxidase 2(LOXL2)is a member of the lysyl oxidase protein family.The LOX family is crucial for the formation of connective tissue,which encodes the extracellular copper-dependent amine oxidase and catalyzes the first step in the formation of crosslinked collagen and elastin.Many studies have revealed the function of LOXL2 in fibrotic diseases.A number of other studies have also shown a link between LOXL2 and cancer progression.LOXL2 is up-regulated in many types of cancers and is associated with poor prognosis.LOXL2 has also been shown to be associated with tumor metastasis mainly due to its primary role in the covalent cross-linking of collagen and elastin catalyzed by extracellular matrix(ECM).This part of the study first using Realtime-PCR,Western blot and other methods to detect the LOXL2 expression levels of colorectal cancer specimens,we found LOXL2 in colorectal cancer tissue was significantly higher than the expression of normal intestinal glands,and by immunohistochemistry Staining confirmed that LOXL2 overexpression predicted poor prognosis.Next,we knocked down LOXL2 by shRNA in vitro.CCK8 and clonogenicity results showed that the proliferation ability of colon cancer cells was significantly inhibited.Meanwhile,Wound Healing and Transwell assays showed that the metastatic capacity of colon cancer cells was significantly decreased.Next,we performed in vivo experiments using immunodeficient mice.The knockdown of LOXL2 in colon cancer cells was significantly impaired in both the proliferative and the metastatic potential in mice.Finally,we analyzed the mechanism of LOXL2 effect on cell proliferation and metastasis by luciferase reporter assay,found LOXL2 knockdown cells,Vimentin expression was significantly decreased.Part Ⅱ: Study on the function and molecular mechanism of SHMT2on colorectal cancerSerine hydroxymethyltransferase(SHMT)is a pyridoxal phosphate(vitamin B6)-dependent enzyme.There are two isozymes SHMT,SHMT1 and SHMT2.SHMT1 mainly exists in the cytoplasm and SHMT2 exists in the mitochondria.SHMT2 plays a regulatory role as a bridge between serine catabolism and one carbon unit exchange.Serine-driven carbon monoxide metabolism was identified as an important pathway for NADPH production.It is for this reason that SHMT2 is considered as a very important gene in the development of tumors,and many kinds of tumors have been proved to be related to it.In this part,the expression of SHMT2 in clinical specimens was detected by Realtime-PCR and Western blot.The expression of SHMT2 in colorectal cancer tissues was significantly higher than that in normal intestinal glands,and the high expression level of SHMT2 in CRC tissues which was detected by immunohistochemical staining indicates poor prognosis.Next,we knocked down SHMT2 in CRC cells using shRNA and found that the proliferation of cells was significantly inhibited,and the same result was obtained in in vivo experiments.Subsequently,we sequenced knockdown SHMT2 cells using RNAseq and found several downregulated genes associated with the cell cycle.We used lentivirus to overexpress the top-ranked gene UHRF1 and put-back UHRF1 in SHMT2-knockdown cells.The proliferative ability of the cells recovered in vitro and in vivo but did not reach the initial level.As a result,we again put-back CCND1,the second most important RNAseq result,and eventually restored its ability of proliferation completely.Part Ⅲ: Isolation and identification of tumor infiltratinglymphocytesTumor-infiltrating lymphocytes(TILs)are leukocytes that leave the bloodstream and migrate into the tumor.The function of tumor-infiltrating immune cells can dynamically change throughout the tumor progression and respond to anti-cancer therapy.TIL involves killing the tumor cells,and the presence of lymphocytes in the tumor is often associated with better clinical outcome.The presence of TIL means that lymphocytes are found between tumor cells and CD3 has been used to detect lymphocytes in tumor samples.In colorectal cancer,tumor-infiltrating lymphocytes are associated with microsatellite instability cancers.At the same time,gastrointestinal cancer checkpoint inhibitor therapy,also need to be used in combination with TIL.TIL as an adoptive cell-transfer therapy for the treatment of cancer by the National Cancer Institute,Dr.Steven Rosenberg first proposed.Autologous lymphocytes are isolated from the patient’s tumor and extensively expanded in vitro.This part of the study by direct separation of colorectal cancer tissue and its matched normal intestinal gland tissue lymphocytes,to modifiy the methods published in the previous study,in order to culture and identificate TIL.Thus we can establish a system of TIL separation and identification.