| Objective: Colorectal cancer is one of the most common malignancies in humans.Because the onset of colorectal cancer is occultly,symptoms are easily negelected,usually colorectal cancer patients are diagnosed at the advanced stage.Colorectal cancer is a serious threat to human health.Therefore,it is extremely urgent to explore the molecular mechanism of the occurrence and development of colorectal cancer,and to find the biomarkers for early diagnosis and potential therapy targets.The genome sequencing project showed that protein-coding genes accounting for only about 2% of the total gene,and more than 90% of the transcripts were non-coding RNAs(ncRNAs).Long non-coding RNA(lncRNA)is a type of RNA that is longer than200 nucleotides(nucleotide,nt)but does not encode proteins.Lnc RNA participates in the biological activity of many eukaryotic cells,and can regulate gene expression in epigenetics,transcriptional and post-transcriptional levels.They play an important role in human growth and cell apoptosis and differentiation.Over the past few years,growing evidences have been shown that lncRNA may be associated with the development of multiple diseases including tumor.About 70% of the genes have corresponding antisense sequences.The natural antisense transcript is located near the 5' promoter or 3' terminal of the sense gene,and does not overlap with the sense coding gene.Among the tumor related lncRNA,RP1-13P20.6 and HNF1A-AS1 are both antisense lncRNAs.RP1-13P20.6,also called linc01527 and lnc-SPRR2D-1,is located on the antisense strand in 1q21 region with a length of 1997 nt,consisting of two exons.There isn't any evidence whether RP1-13P20.6 relates to the occurrence and development of colorectal cancer.Based on our previous microarray results,RP1-13P20.6 was found to be down-regulated in colorectal cancer tissues.This study aims to identify the expression level of RP1-13P20.6in colorectal cancer tissues,analyze its correlation with the clinicopathological features and prognosis of colorectal cancer patients,and try to explore the possible mechanisms of how RP1-13P20.6 affects the ability of proliferation and invasion of colorectal cancer cells through the experiments in vitro.Furthermore HNF1A-AS1 is an antisense lncRNAlocated on the antisense strand of the HNF1 A gene at 12q24.31.Previous studies have revealed that HNF1A-AS1 acted as an oncogene and was dysregulated in many malignant tumors like esophageal adenocarcinoma,lung adenocarcinoma,and hepatocellular carcinoma.It is worth noting that the roles and mechanisms of HNF1A-AS1 in different tumors are quite different.This study intends to identify the expression level of HNF1A-AS1 in colorectal cancer tissues,and analyze its correlation with the clinicopathological features and prognosis of colorectal cancer patients.We try to explore the possible mechanisms of how HNF1A-AS1 affects the ability of cell proliferation and invasion through the experiments in vitro.So that we can provide theoretical basis and new targets for the treatment of colorectal cancer.Methods:1.The relative expression levels of RP1-13P20.6 and HNF1A-AS1 in colorectal cancer tissues(1)Real-time Polymerase Chain Reaction was used to detect the expression levels of RP1-13P20.6 and HNF1A-AS1 in colorectal cancer tissues and normal adjacent tissues(NATs).(2)The Wilcoxon test was used to compare the expression levels of the two lncRNAs in colorectal cancer tissues and NATs.2.The correlations between the relative expression of RP1-13P20.6 and HNF1A-AS1 in cancerous tissues and the clinicopathological characteristics as well as prognosis(1)The correlations between lncRNAs relative expression and clinicopathological characteristics were tested using the Mann-Whitney U and the Krushal-Wallis H test.(2)The overall survival rates were calculated by the Kaplan-Meier method,and the differences between the survival rates of the colorectal cancer patients were tested using Log-rank test.3.Construction of RP1-13P20.6 and HNF1A-AS1 overexpression vectors(1)Extract total RNA from SW480 cell line,and identify the transcription start and end point of RP1-13P20.6 using 3' Rapid Amplification of cDNA Ends technique and 5'Rapid Amplification of cDNA Ends technique,respectively.(2)According to the results of 3'RACE and 5' RACE,the full-length of RP1-13P20.6sequence was synthesized and the overexpression vector was constructed through enzyme digestion,ligation,and transformation et al.(3)According to the previous reports,we obtained the full length of HNF1A-AS1 sequence and synthesized the overexpression vector through enzyme digestion,ligation,and transformation et al.4.The intracellular location of RP1-13P20.6 and the effects of RP1-13P20.6 and HNF1A-AS1 on the proliferation and invasion of colorectal cancer cells(1)Studies have shown that HNF1A-AS1 is located in the cytoplasm,but the location of RP1-13P20.6 in the cell is unknown.Using cell hypotonic buffer,the nucleus and cytoplasm of colorectal cancer cells were separated,and total RNA in cytoplasm and nuclei was extracted respectively.U6 is used as the nuclear reference,and ?-actin is used as cytoplasmic reference.According to the expression of RP1-13P20.6 in the nucleus and the cytoplasm,the localization of RP1-13P20.6 is identified.(2)CCK-8 test was used to detect the proliferation of RP1-13P20.6 overexpressed cells,HNF1A-AS1 overexpressed cells and corresponding Negative control(NC)cells.Compared with NC cells,the effects of overexpression of RP1-13P20.6 and overexpression of HNF1A-AS1 on the proliferation were observed and analyzed.(3)The Transwell test was used to detect the invasion of RP1-13P20.6 overexpressed cells,HNF1A-AS1 overexpressed cells and corresponding NC cells.Compared with NC cells,the effects of overexpression of RP1-13P20.6 and overexpression of HNF1A-AS1 on the invasion were observed and analyzed.5.The effects of RP1-13P20.6 and HNF1A-AS1 on the cell cycle and apoptosis(1)In RP1-13P20.6 overexpressed cells,HNF1A-AS1 overexpressed cells,and corresponding NC cells,PI staining was used to detect the effect of RP1-13P20.6overexpression and HNF1A-AS1 overexpression on cell cycle.(2)In RP1-13P20.6 overexpressed cells,HNF1A-AS1 overexpressed cells,and corresponding NC cells,Annexin V-APC/PI staining was used to detect the effects of RP1-13P20.6 overexpression and HNF1A-AS1 overexpression on cell apoptosis.Results:1.The expression levels of RP1-13P20.6 and HNF1A-AS1 in colorectal cancer tissuesIn 99 colorectal cancer patients,compared with NATs,the expression of RP1-13P20.6 in colorectal cancer tissues was significantly lower in colorectal cancer,the?CT value of colorectal cancer tissues was 8.5±3.98,the ?CT value of NATs was5.10±3.18,P<0.001.In 59 colorectal cancer patients,there was no significant difference in the expression of HNF1A-AS1 in colorectal cancer tissues compared with NATs,P=0.079.2.The correlations between the expression of RP1-13P20.6 and HNF1A-AS1 in cancerous tissues and the clinicopathological characteristics as well as prognosis(1)The statistical results of nonparametric test showed that there was no significant correlation between the expression levels of RP1-13P20.6 as well as HNF1A-AS1 and the characteristics such as gender,age,tumor size,histological grades,pT grades,pN grades and TNM stage in colorectal cancer patients,P>0.05.(2)Calculate the total survival rates using the application of Kaplan-Meier method,and evaluate the difference of the two groups using Log-rank test.The results showed that there was no significant correlation between the expression level of RP1-13P20.6and the prognosis of colorectal cancer patients,P=0.466;there was no significant correlation between the expression level of HNF1A-AS1 and the prognosis of colorectal cancer patients,P=0.860.3.RP1-13P20.6 and HNF1A-AS1 overexpressed vectors construction and transfection(1)According to the results of 3 'RACE and 5' RACE,we constructed RP1-13P20.6overexpression vector and transfected in SW480 cells,and detected efficiency by Real-time PCR.The expression of RP1-13P20.6 in RP1-13P20.6 overexpressed cells was upregulated by 3275±927 times,P<0.001.(2)According to the HNF1A-AS1 sequence reported in studies,we constructed HNF1A-AS1 overexpression vector,and transfected in SW480 cells.HNF1A-AS1 in HNF1A-AS1 overexpressed cells was upregulated by 3028±382 times,P=0.008.4.RP1-13P20.6 overexpression induces S-phase arrest,promotes cell apoptosis,inhibits the proliferation,and inhibits the invasion of colorectal cancer cells(1)In RP1-13P20.6 overexpressed cells and NC cells,CCK-8 test was used to detect the proliferation of the cells.Compared with NC cells,the proliferation of RP1-13P20.6overexpressed cells was significantly decreased,P=0.007.(2)In RP1-13P20.6 overexpressed cells and NC cells,the Transwell test was used to detect the invasive ability of the cells.Compared with NC cells,the invasive ability of RP1-13P20.6 overexpressed cells was significantly decreased(119±14 vs.181±19,P<0.001).(3)In RP1-13P20.6 overexpressed and NC cells,flow cytometry showed that overexpression of RP1-13P20.6 led to S-phase arrest in colorectal cancer cells,P<0.001;Using Annexin V-APC/PI to detect cell apoptosis,the results showed that overexpression of RP1-13P20.6 promotes apoptosis of colorectal cancer cells,P<0.05.5.The effects of HNF1A-AS1 on the proliferation,invasion,cell cycle and apoptosis of colorectal cancer cells(1)In HNF1A-AS1 overexpressed cells and NC cells,Transwell test was used to detect the invasive ability of cells.Compared with NC cells,the invasion ability of HNF1A-AS1 overexpressed cells increased significantly,P=0.001.(2)In HNF1A-AS1 overexpressed cells and NC cells,CCK-8 test was used to detect cell proliferation.Compared with NC cells,overexpression of HNF1A-AS1 had no significant effect on cell proliferation,P=0.617.(3)In HNF1A-AS1 overexpressed cells and NC cells,flow cytometry showed that overexpression of HNF1A-AS1 had no obvious effect on cell cycle(P>0.05);Using Annexin V-APC/PI to detect cell apoptosis,overexpression of HNF1A-AS1 had no significant effect on colorectal cancer cell apoptosis,P>0.05.Conclusions:1.RP1-13P20.6 was abnormally down-regulated in colorectal cancer tissues.There is no significant correlation between the expression levels of RP1-13P20.6 and the pathological characteristics of the colorectal cancer patients.2.In colorectal cancer cells,the overexpression of RP1-13P20.6 leads to S-phase arrest,apoptosis and inhibits the proliferation of colorectal cancer cells.The overexpression of RP1-13P20.6 inhibits the invasion of colorectal cancer cells.3.There was no significant difference in the expression of HNF1A-AS1 in colorectal cancer tissues compared with NATs.There is no significant correlation between the expression level of HNF1A-AS1 and the pathological characteristics of the colorectalcancer patients.4.In colorectal cancer cells,the overexpression of HNF1A-AS1 promotes cell invasion,but has no significant effect on cell cycle,apoptosis and cell proliferation. |
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