Hsa_circ₀001875 Facilitates The Expression Of MYC Via Interacting With LPCAT1 In Colorectal Cancer

Objective: Colorectal cancer(CRC)is the second leading cause of cancer-related death.The establishment of an early cancer screening program,as well as the advancement of colonoscopy diagnosis and treatment technology,has improved the prognosis of CRC patients to some extent,and the five-year survival rate of early cancer patients has reached90%.However,because of a lack of clear early signs and screening constraints,the majority of CRC patients were classified as cancer progression at the time of detection.The prognosis for these patients remained poor,particularly for those with distant metastases,where the five-year survival rate was only 14%.As a result,further investigation and clarification of the molecular mechanism of colorectal cancer occurrence and metastasis is required in order to identify diagnostic biomarkers and therapeutic targets.Circular RNA(circ RNA)is a non-coding circular RNA with a closed covalent structure.Sanger et al.were the first to discover it in 1976.Circ RNA was originally thought to be a byproduct of abnormal splicing events.Many studies have confirmed that circ RNA plays regulatory roles in tumors,diabetes,aging,tissue development,cardiovascular disease,and other diseases.Most studies focused on its role in tumors.Many circ RNAs have been identified as tumor suppressors or oncogenes.Circ RNA is highly stable and difficult for exonucleases to degrade.Circ RNA expression patterns are also complex,involving species,time,tissue,and cell specificity.Circ RNA may regulate the occurrence and progression of tumors by binding to proteins in tumors and regulating their stability,subcellular localization,and interactions with other proteins or DNA.Lysophosphatidylcholine acyltransferase 1(LPCAT1)is the key enzyme of the Lands cycle in the lipid remodeling pathway,which is closely related to cancer.It had abnormal expression in a variety of cancers and influenced their occurrence and progression.LPCAT1 was found to be significantly overexpressed in colorectal cancer cells and tissues,which could promote the accumulation of total choline metabolites via phosphatidylcholine remodeling,altering the lipid profile and other characteristics of the disease.MYC is a proto-oncogene that encodes a nuclear phosphate protein and can form heterodimers with the related transcription factor MAX,which regulates important aspects of tumor biology such as proliferation,cell adhesion,and angiogenesis,making MYC dysfunction a sign of cancer.Studies showed that MYC is highly expressed in CRC,which can promote the occurrence and development of colorectal cancer cells.MYC can also influence the EMT pathway.Tumor cells can lose some epithelial cell characteristics through the EMT pathway to acquire interstitial cell characteristics,which reduces contact between tumor cells and surrounding cells and cell interaction.This process enhances the ability of cell migration and makes the cells obtain the characteristics of invasion and metastasis.This research studied the expression of hsa＿circ＿0001875 in CRC tissues and cell lines,the relationship with clinicopathological data,and its influence on CRC biological behaviors.This research also studied the relationship between hsa＿circ＿0001875,LPCAT1 and MYC and revealed the molecular mechanism of hsa＿circ＿0001875 promoting CRC metastasis.Hsa＿circ＿0001875 may become a potential therapeutic target that can improve the therapeutic effect and the prognosis of CRC patients.Methods: 1.Downloaded the circ RNA expression array data of GSE126094,GSE138589 and GSE142837 data sets from the GEO database,and used the limma package of R language for data preprocessing and differential analysis.Sanger sequencing was used to detect the PCR product of hsa＿circ＿0001875,and compared with its own sequence to verify its circular structure.The expression of hsa＿circ＿0001875 and its parent gene FAM120 A was detected by q PCR following RNase R treatment,and the stability of the two genes was compared.2.Collected 42 pairs of fresh CRC and adjacent tissues,and cultured CRC cell lines: RKO,HT29,SW480,SW620,and normal intestinal epithelial cells NCM460 for RNA extraction.Subsequently,the differential expression of hsa＿circ＿0001875 in colorectal cancer was detected by reverse transcription and q PCR.Collected 50 pairs of paraffin sections from colorectal cancer and adjacent tissues,detected the expression of hsa＿circ＿0001875 via in situ hybridization,and analyzed the correlation between hsa＿circ＿0001875 and clinicopathological data.3.The plasmid and si RNA of hsa＿circ＿0001875 and LPCAT1 were transfected respectively into SW480 and RKO cells in order to achieve overexpression and knockdown.Wound healing and Transwell assays were used to identify the SW480 and RKO cells’ capacity for invasion and migration.4.The interaction between hsa＿circ＿0001875 and LPCAT1 was verified by RNA pulldown and RIP experiments,and their colocalization was verified by fluorescence in situ hybridization and immunofluorescence.Co-IP assays were carried out with the IP-level antibodies of LPCAT1 and MYC respectively,and the interaction between the two proteins was confirmed in both the forward and backward directions.5.Overexpressed and knocked down hsa＿circ＿0001875 in SW480 and RKO cells and extracted protein.Using Western blot to detect the changes of expression of LPCAT1,MYC,E-cadherin,N-cadherin and Vimentin.6.The rescue experiment between hsa＿circ＿0001875/LPCAT1/MYC was performed by wound healing and Transwell assays to demonstrate the necessity of LPCAT1 and MYC on hsa＿circ＿0001875 affecting the phenotypes of colorectal cancer.7.By injecting caudal veins in nude mice,the effect of hsa＿circ＿0001875 and MYC on colorectal cancer metastasis was confirmed in vivo.Results: 1.Through the intersection of the differential analysis results of three GEO data sets,the differentially expressed circ RNA was obtained and the target molecule was screened.The results of Sanger sequencing and alignment suggested that the PCR product of hsa＿circ＿0001875 crossed the back splice junction,confirming the circular structure of hsa＿circ＿0001875.The q PCR results after RNase R treatment also suggested that hsa＿circ＿0001875 was more stable than the linear gene.2.hsa＿circ＿0001875 was highly expressed in colorectal cancer tissues and cells,and its high expression level was significantly correlated with lymph node metastasis and tumor differentiation,but not with gender,age,depth of tumor invasion and distal metastasis.3.Wound healing and Transwell assays revealed that in SW480 and RKO cells,overexpression of hsa＿circ＿0001875 and LPCAT1 could promote the migration and invasion of colorectal cancer cells,whereas knockdown of hsa＿circ＿0001875 and LPCAT1 could inhibit the migration and invasion of colorectal cancer cells.4.Fluorescence in situ hybridization and immunofluorescence assays revealed that hsa＿circ＿0001875 and LPCAT1 colocalized in SW480 and RKO cells.RNA pulldown and RIP assays confirmed the interaction between hsa＿circ＿0001875 and LPCAT1.Co-IP assays proved that the interaction of LPCAT1 and MYC in SW480 and RKO cells.5.Western Blot showed that overexpression of hsa＿circ＿0001875 in SW480 and RKO cells could increase the expression level of LPCAT1,MYC,N-cadherin and Vimentin protein,and decrease the expression level of E-cadherin;Knockdown of hsa＿circ＿0001875 reduced the expression level of LPCAT1,MYC,N-cadherin and Vimentin protein,and increased the expression level of E-cadherin;Overexpression of LPCAT1 can increase the expression level of MYC,N-cadherin and Vimentin protein,and decrease the expression level of E-cadherin;The knockdown of LPCAT1 reduced the expression level of MYC,N-cadherin and Vimentin proteins and increased the expression level of E-cadherin.6.Western blot,wound healing,and Transwell assay results suggested that the effect of hsa＿circ＿0001875 on the EMT pathway,migration,and invasion ability in colorectal cancer was dependent on the roles of LPCAT1 and MYC.7.A tumor metastasis model revealed that MYC overexpression reversed the inhibitory effect of hsa＿circ＿0001875 knockdown on colorectal cancer cell metastasis in vivo.Conclusions: It was the first time to prove that hsa＿circ＿0001875 was highly expressed in colorectal cancer tissues and cell lines,and the high expression level was closely related to lymph node metastasis and tumor differentiation.Hsa＿circ＿0001875 could interact with LPCAT1 and promote the interaction of LPCAT1 and MYC,thus increasing MYC expression,activating EMT pathway and promoting the migration and invasion of colorectal cancer cells.