M6a-modified Exosomal Pseudogene TDGF1P3 Interacts With HNRNPA2B1 Protein To Promote Liver Metastasis Of Colorectal Cancer

Objective:The incidence and mortality of colorectal cancer(CRC)are high,and liver metastasis is one of the important reasons for the poor prognosis of CRC.However,the molecular mechanism of CRC liver metastasis is not fully understood to date.Exosomes are a type of extracellular vesicles with a diameter of approximately 40-160nm.They are formed through the fusion of intracellular multivesicular bodies with the cell membrane and are released into the extracellular matrix.Exosomes play a significant role in the distant metastasis of tumors.Pseudogenes are a hot topic in the field of non-coding molecular biology research.Previous studies have found that the pseudogene TDGF1P3 is significantly increased in the serum exosomes of CRC patients with liver metastasis,but the molecular mechanism by which TDGF1P3 regulates CRC liver metastasis is not clear.Methods:1.Expression and clinical significance of pseudogene TDGF1P3 in colorectal cancer tissue:Firstly,based on the TCGA database,the expression of pseudogene TDGF1P3 in colorectal cancer tissue was initially analyzed.Secondly,the real-time fluorescence quantitative reverse transcription PCR(qRT-PCR)method was used to investigate the expression of TDGF1P3 in120 paired colorectal cancer tissues and their adjacent normal tissues.The expression of TDGF1P3 was analyzed for correlation with clinical parameters such as age,gender,degree of tissue differentiation,tumor infiltration depth,lymph node metastasis,distant metastasis,TNM staging,and carcinoembryonic antigen(CEA)levels in colorectal cancer tissue.2.Expression and clinical significance of peripheral blood exosome TDGF1P3 in CRC:A total of 37 CRC patients and 20 healthy individuals were included in this study,and peripheral blood exosomes were extracted using a kit.qRT-PCR was used to detect the expression of TDGF1P3 in peripheral blood exosomes,and its potential as a molecular marker for CRC was investigated by constructing a ROC curve and analyzing its clinical significance.Additionally,preoperative and postoperative peripheral blood exosomal TDGF1P3 levels were examined to analyze their clinical significance.3.Effects of TDGF1P3 on the proliferation,migration and invasion of CRC cells and liver metastasis in nude mice:Overexpression plasmids and small interfering RNA(si RNA)targeting TDGF1P3 were constructed and transfected into CRC cells.Changes in cell proliferation,migration,and invasion were assessed using CCK-8,scratch assay,and transwell methods,respectively.sh RNA targeting TDGF1P3 was also constructed,and a liver metastasis model was established through splenic implantation in nude mice to observe the impact of TDGF1P3 on hepatic metastasis in vivo.4.Molecular mechanism of TDGF1P3 promoting liver metastasis of CRC cells:The molecular mechanisms of TDGF1P3 were investigated using various methods,including RNA pull-down,RNA immunoprecipitation(RIP),Western blotting(WB),qRT-PCR,RNA sequencing(RNA-seq),methylated RNA immunoprecipitation(me RIP),and rescue experiments.These approaches were employed to elucidate the molecular mechanisms underlying the role of TDGF1P3 in CRC cell hepatic metastasis.Results:1.The pseudogene TDGF1P3 is overexpressed in colorectal cancer tissue and serum exosomes and is associated with liver metastasis,TNM staging,and poor prognosis.The level of peripheral blood exosome TDGF1P3 decreased significantly after colorectal cancer resection compared with that before surgery(P<0.05).2.Overexpression of TDGF1P3 promotes proliferation,migration,and invasion of CRC cells both in vitro and in vivo while inhibiting TDGF1P3 has the opposite effect.Exosomes overexpressing TDGF1P3 can promote the proliferation and invasion of co-cultured colorectal cancer cells.Compared with the control group,liver metastases in the TDGF1P3 sh RNA group were significantly reduced.3.Mechanistically,N~6-methyladenosine(m~6A)modified TDGF1P3 interacts with HNRNPA2B1 protein to promote the occurrence and development of liver metastasis of CRC.Conclusion:1.TDGF1P3 is a promising biomarker for the diagnosis and prognosis of liver metastases in colorectal cancer.2.m~6A modified TDGF1P3 in exosomes interacts with HNRNPA2B1 protein to promote liver metastasis of CRC.