| BackgroundColorectal cancer(CRC)is a common gastrointestinal malignancy,and the incidence of CRC is the third highest among all malignancies in the world.In recent years,the morbidity and mortality of CRC in China have increased significantly,and the prevalence of the population is showing a younger trend,which has become a major public health issue with the social and economic development.Therefore,it has become an urgent problem to explore the mechanism of colorectal cancer occurrence and development,search for high-quality biomarkers and explore new biological treatment methods.Long non-coding RNA is a type of non-coding ribonucleic acid with a transcript length of more than 200 nucleotides.It has been found that long non-coding RNA may play an important regulatory role in the occurrence,development,metastasis,drug resistance and other processes of a variety of neoplastic diseases,and has become one of the hotspots in tumor research.This study focused on clinical issues,explored the role of new long non-coding RNA in the development of colorectal cancer,and searched for potential tumor markers of CRC,so as to provide references for clinical and scientific research work.PurposeTo investigate the expression and its regulatory role of long non-coding RNA AGAP2-AS1 in colorectal cancer.MethodsSamples of cancerous tissue and para-cancerous tissue in this study were collected from 9 patients admitted to the first affiliated hospital of 9hengzhou university from February 2017 to April 2018.All patients received their first visit to the hospital,with complete clinical data and definite pathological diagnosis of colorectal adenocarcinoma.They did not receive preoperative neoadjuvant chemotherapy,radiotherapy,immunotherapy and traditional Chinese medicine treatment.All patients underwent radical resection of colorectal cancer in the first affiliated hospital of 9hengzhou university.After the tumor tissue was isolated from the body,an appropriate amount of the tumor tissue was placed in the cryopreservation tube and immediately placed in the refrigerator with an ultra-low temperature of minus 80 degrees for preservation.We used high-throughput sequencing to detect the expression profile of long non-coding RNA in the colorectal and combined with bioinformatics technology to screen the differentially expressed long non-coding RNA.In situ hybridization was used to detect the expression of long non-coding RNA AGAP2-AS1 in colorectal cancer patients from 76 sets of tissue samples in the tissue chip.The results of in situ hybridization were scored and compared with the clinicopathological data of the patients.The effects of AGAP2-AS1 on invasion and metastasis of colorectal cancer cell lines SW480 and HCT116 were validated by Wound-Healing Assay and Transwell(cell migration assay)experiments.SPSS v20.0 software was used for statistical analysis.The counting data were tested by chi-square test.The measurement data conform to the normal distribution analysis by t test.The data not conforming to the normal distribution were analyzed by the rank sum test.Kaplan-meier method was used to draw the survival curve.For all statistical analyses,test level=0.05 was used,and p<0.05 was considered to be statistically significant.Results1.Long non-coding RNA expression profile and potential marker screening in colorectal cancer tissuesWe used high-throughput sequencing to detect the expression of long non-coding RNA in cancer tissues and paracancer tissues of 9 patients with colorectal cancer.The results showed that a total of 7,671 long non-coding RNAs were sequenced,of which 5,625 were up-regulated and 2011 were down-regulated.We screened according to folding changes and p values,and found that long non-coding RNA AGAP2-AS1 was highly expressed in colorectal cancer tissues,with a statistically significant difference(p<0.05).2.Bioinformatics was used to predict the expression characteristics and prognosis of AGAP2-AS1 in neoplastic diseasesWe analyzed AGAP2-AS1 information in the GEPIA database.It was found that AGAP2-AS1 was highly expressed in adrenal cortical carcinoma(ACC),bladder urothelial carcinoma(BLCA),colon adenocarcinoma(COAD),bile duct carcinoma(CHOL),esophageal carcinoma(ESCA),head and neck squamous cell carcinoma(HNSC),lung squamous cell carcinoma(LUSC),rectal adenocarcinoma(READ)and gastric adenocarcinoma(STAD).And the survival curve showed that the survival condition of the group with high expression of AGAP2-AS1 was worse than that of the group with low expression.3.Expanding sample size to verify the expression characteristics and prognosis of AGAP2-AS1 in colorectal cancerWe used in situ hybridization(ISH)to detect and score tissue samples from 76 colorectal cancer patients.We found that the expression of AGAP2-AS1 in cancer tissues was significantly higher than that in paracancer tissues(p<0.001).By comparing the expression of AGAP2-AS1 with the clinicopathological data of the patients,we found that the expression of AGAP2-AS1 was significantly correlated with TNM stage,lymph node metastasis and tumor invasion depth(p<0.05).There was no significant correlation with age,sex,tumor size,general type and pathological grade(all p values were greater than 0.05).By plotting the survival curve,we found that the survival of the group with high expression of AGAP2-AS1 was significantly worse than that of the group with low expression(p<0.05).4.In vitro experiments verified the biological function of AGAP2-AS1 in regulating colorectal cancer cell linesIn order to further explore the biological function of AGAP2-AS1 in colorectal cancer,especially the effect of AGAP2-AS1 on the migration and metastasis of colorectal cancer cells,Wound-Healing Assay and Transwell migration experiments were conducted in SW480 and HCT116 cell lines.The results showed that the migration ability of colorectal cancer cell lines SW480 and HCT116 were significantly decreased after down-regulated AGAP2-AS1 expression by siRNA.ConclusionLong non-coding RNA AGAP2-AS1 was significantly highly expressed in colorectal cancer,and was significantly correlated with TNM stage,lymph node metastasis,and tumor invasion depth.Up-regulation of AGAP2-AS1 promotes migration of colorectal cancer cells.The prognosis of colorectal cancer patients with high expression of AGAP2-AS1 was worse.As a new cancer promoting molecule,AGAP2-AS1 is expected to become a new metastasis marker of colorectal cancer,providing theoretical basis for targeted drug development. |
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