The P300/YY1/miR-500a-5p/HDAC2 Signalling Axis Regulates Cell Proliferation In Human Colorectal Cancer

Background and objectiveThe incidence of colorectal cancer ranks third among malignant tumors,and the mortality rate ranks second.China is a country with a large incidence of colorectal cancer.The morbidity and mortality in China have increased in the past decade.Although the level of diagnosis and treatment have improved,most patients are in the late stages of colorectal cancer when they got confirmed diagnosis,which result in unsatisfactory treatment.Therefore,the pathogenesis and metastasis of colorectal cancer still require further study.Recently,more and more studies showed that miRNAs are involved in the development of colorectal cancer.Some studies have found that the expression of miR-500a-5p is related to the growth and invasion ability of tumor cell,but its expression and regulatory mechanism in colorectal cancer remains unclear.We found that the expression of miR-500a-5p is significantly down-regulated in colorectal cancer cells and HDAC2 is a potential target of miR-500a-5p.Three YY1 binding sites were predicted in miR-500a-5p promoter region,which indicated that YY1 may be the upstream transcription factor of miR-500a-5p.This research will be conducted at four levels:molecular interaction,cell function,animal model,and the correlation of tissue samples from patients with cororectal cancer.The purpose of this project is to investigate the effect of miR-500a-5p on the biological characteristics of colorectal cancer,explore its mechanism of action,and explore whether the transcription complex YY1/p300/HDAC2 regulates expression of miR-500a-5p,which can provide clues for early diagnosis of colorectal cancer and searching for potential targets of medication.Methods1.We detected the expression of miR-500a-5p in colorectal cancer tissues by qPCR and evaluated the correlation between miR-500a-5p and clinical pathological characteristics or prognosis of colorectal cancer patients.We transfected mimics or inhibitors into CRC cells to increase or inhibit the expression of miR-500a-5p.EdU and colony formation assay were used to observe changes of cell proliferation capacity.Flow cytometry and related cyclin protein were used to assess cell cycle process.Migration capacity of colorectal cancer cells was assessed by wound healing assay and transwell chamber migration assay.2.Bioinformatics databases,mRNA chip,dual luciferase reporter system were used to explore the potential target relationship between miR-500a-5p and HDAC2.We used colony formation assay?transwell chamber migration assay and subcutaneous tumor formation experiment to evaluate whether miR-500a-5p affect the biological behavior of cororectal cancer cells in vitro and the tumorigenicity of nude mice through HDAC2.To explore the correlation between the expression of miR-500a-5p and HDAC2 in colorectal cancer tissues,we performed qPCR with 81 pairs of colorectal cancer tissue.3.Bioinformatics database was used to predict upstream regulators of miR-500a-5p.Double luciferase reporter system and ChIP assay were performed to detect direct binding of YY1 to miR-500a-5p promoter.qPCR was used to evaluate wether the transcription factor YY1 negatively regulate the expression of miR-500a-5p and the correlation between the expression of YY1 and miR-500a-5p in colorectal cancer tissues.4.Co-IP was carried out to detect the interaction of p300?HDAC2 and YY1.We evaluated the effect of the YY1/HDAC2/p300 transcription complex on the transcriptional activity of the miR-500a-5p promoter by qPCR and dual luciferase activity tests.ChIP?re-CHIP was used to verify the binding of transcription complex and miR-500a-5p promoter.5.Flow cytometry and Western blot were used to detect whether miR-500a-5p affected the sensitivity of colorectal cancer cells to FK228 in vitro.TUNEL staining was used to evaluate the effect of combined application of miR-500a-5p and FK228 on apoptosis of transplanted tumors in vivo.Results1.The expression of miR-500a-5p is decreased in colorectal cancer cells and tissues.The expression level is related to tumor differentiation,lymph node metastasis,AJCC stage and prognosis of patients.Overexpression of miR-500a-5p inhibits the proliferation and migration ability of colorectal cancer cells and delays the cell cycle.Suppressing its expression gave the opposite result.2.miR-500a-5p targets HDAC2 to promote colorectal cancer cell proliferation,migration and tumorigenicity in vitro.3.YY1 negatively regulates the expression of miR-500a-5p and participates in the formation of transcription factor complexes.YY1/p300/HDAC2 transcription factor complex regulates the expression of miR-500a-5p through a positive feedback in colorecatal cancer cells.4.miR-500a-5p can enhance the sensitivity of colorectal cancer cells to FK228.ConclusionsmiR-500a-5p plays a role as a tumor suppressor in the development of colorectal cancer,targeting HDAC2 to inhibit the proliferation and migration of colorectal cancer cells.Transcription factor complex YY1/p300/HDAC2 regulates the expression of miR-500a-5p through a positive feedback.