The Role Of Circular RNA CircPPP1R12A In The Progression Of Colon Cancer

Objective:Colon cancer is one of the most common malignancies worldwide,ranking third in morbidity and mortality.The 5-year survival rate is only 31%in China,principally due to the late diagnosis and recurrence/metastasis of tumor cells.Therefore,it is of great clinical significance to further study and reveal the unknown biological mechanism of colon cancer malignancy and to find new targets for colon cancer treatment.With the rapid development of new generation sequencing technology,more and more previously unknown transcripts have been discovered.Non-coding RNAs（ncRNAs）,like most of the above transcripts,cannot be translated into proteins due to the lack of obvious open reading frames（ORFs）in organisms.Circular RNAs（circRNAs）are circRNAs composed of upstream splicing receptors and downstream splicing donors.Due to the abundance and stability of circRNAs,some circRNAs can regulate gene transcription and translation by acting as endogenous competing endogenous RNAs（ceRNA）or protein binding RNAs of miRNAs,thus extensively regulating biological activities.Recently,it has been found that a variety of circRNAs encode proteins that regulate tumor progression.However,the existence of protein-encoded circRNAs in the development of colon cancer is still unknown,and its functional products are still unclear.In this study,circRNAs microarray was used to analyze the expression profile of circRNAs in colon cancer and adjacent tissues,screen differentially expressed circRNAs,and explore the potential peptide expression function of circRNAs and the molecular regulation mechanism in the occurrence and development of colon cancer,so as to provide new ideas for targeted therapy of colon cancer.Methods:Human Arraystar circRNA microarray was used to screen 10 pairs of circRNAs with significantly different expression in recently collected colon cancer tissues and adjacent tissues,and quantitative real-time PCR（qRT-PCR）was used to verify the difference of 10 significantly up-regulated circRNAs in colon cancer and adjacent cancer tissues.Subsequently,qRT-PCR and in situ hybridization（ISH）were used to further verify the expression differences of the most significantly up-regulated circRNAs in colon cancer tissues and colon cancer cell lines.The subcellular distribution of circRNA was then verified by component qRT-PCR and fluorescence in situ hybridization（FISH）.Colon cancer cell lines with stable overexpression and down-regulation of circRNA were constructed through lentivirus-mediated stable up-regulation and down-regulation.The effect of circRNA on the biological function of colon cancer cell lines was evaluated by CCK8,wound healing assay and invasive cell assay.The peptide coding ability of circRNA was evaluated by bioinformatics analysis and further verified by Flag labeling and LC-MS.Further in vitro and in vivo experiments were performed to verify the effect of circRNA-encoded peptides on the biological function of colon cancer cell lines by constructing circRNA overexpression vectors with the polypeptide coding site mutation.Illumina RNA-seq expression profile analysis was performed to further analyze the effect of circRNA-encoded peptides on the transcriptional expression profile of colon cancer cells,and to select possible signaling pathways for further verification.The effect of circRNA-encoded peptides on tumor-associated macrophages（TAMs）cell polarization was evaluated by co-culture of colon cancer cells with TAMs.To evaluate the differential expression of circRNA-encoded peptides in the serum of colon cancer patients and their clinical significance,antibodies and ELISA plates were prepared to specifically recognize the peptides encoded by circRNA.Results:Among 10 pairs of colon cancer tissues and adjacent tissues,the circRNAs with the most significant differential expression between cancer and adjacent tissues were selected（110 up-regulated circRNAs and 16 down-regulated circRNAs,fold change>1.5,P<0.05）.Hsa_circ₀000423（circPPP1R12A）was most significantly upregulation in cancer tissues.The presence of circPPP1R12A in colon cancer cells was confirmed by the Convergent and Divergent primers and the anti-RNase R enzyme degradation assay.CircPPP1R12A was significantly higher expressed in colon cancer tissues than in normal tissues,and the prognosis was worse in patients with higher circPPP1R12A level.CircPPP1R12A was found mainly in the cytoplasm of colon cancer by nuclear/cytoplasmic component separation and fluorescence in situ hybridization（FISH）.Moreover,it was found that circPPP1R12A overexpressed in colon cancer cells promoted the proliferation and clonal formation of colon cancer cells,and significantly promoted the migration and invasion of colon cancer cells.On the other hand,circPPP1R12A silencing significantly inhibited the proliferation,clone formation,migration and invasion of colon cancer cells.Further studies have shown that circPPP1R12A encodes a protein with the size of 73 amino acids（circPPP1R12A-73aa）,and in vitro and in vivo studies have demonstrated that circPPP1R12A regulates the biological functions of colon cancer cell proliferation,migration and invasion via encoding circPPP1R12A-73aa and activating the Hippo-YAP1 signaling pathway.In addition,we found that circPPP1R12A-73aa not only intracellularly regulated the function of colon cancer cells,but also secreted out of the colon cancer cells.The circPPPlR12A-73aa was also involved in the regulation of M2-type polarization of TAMs in the immune microenvironment of colon cancer,and maintained the tumor-suppresive immune microenvironment.Finally,by comparing the serum circPPP1R12A-73aa levels in colon cancer patients with healthy volunteers,we found that the serum circPPP1R12A-73aa levels in colon cancer patients were significantly higher than those in the control group.Moreover,the serum circPPPlR12A-73aa,as a molecular marker,was highly sensitive and specific in the diagnosis of colon cancer,and was significantly positively correlated with the expression level of serum CEA.Conclusions:In this study,we demonstrated that circPPP1R12A,on the one hand,activates the Hippo-YAP1 signaling pathway by encoding the protein circPPP1R12A-73aa,which promotes the proliferation and metastasis of colon cancer cells.On the other hand,circPPP1R12A-73aa regulates M2-type polarization of TAMs in the immune microenvironment of colon cancer,maintaining the tumor-suppresive immune microenvironment.Furthermore,serum circPPP1R12A-73aa serves as a potential novel diagnostic marker,providing new strategies and approaches for screening,diagnosis and treatment of colon cancer.