The Role And Mechanism Of Long Non-coding RNA FEZF1-AS1in The Metastasis Of Colorectal Carcinoma

BACKGROUND&OBJECTIVEColorectal cancer (CRC) is one of the most malignant tumorworldwide.The incidence rate and mortality rate have risen rapidly in rencentyears.Micrometastasis often occurred before radical operation in most of colorectal cancer patients,which is the main cause leading to death.Therefore, it is crucial important to improve the prediction abilites, diagnostic level and early intervention for colorectal cancer metastasis.In that way,understanding the molecular mechanisms underlying CRC metastasis, identificating molecules which involved in CRC metastasis has becoming important than any time.As we all know that cell’s growth,proliferation,differtiation and die are a vary sequential process.Conversely,tumorigenesis is a complicated process which depengs largely on the interaction of different genes in both temporal and spatial.Recent year’s study shows that besides protein-coding genes,non-coding RNA are regarded as a newplayer in cancer.General speaking,non-coding includes small non-coding RNAs(such as micro RNA) and long/large non-coding RNAs which more than200nt and non protein-coding function.lt has been revealed that long non-coding RNA can exert epigenetic,transcriptional and posttranscriptional regulation in gene networks.Meanwhile,the deregulation of long non-coding RNAs is associated with a variety of cancers.In our previous studies,FEZF1-ASl,one of the discriminating genes,was up-regulated in the high metastasis potential cell line, which tested by lncRNAs Microarray in different transfer potential cells.This finding shows that the disorder or dysfunction of FEZF1-AS1gene may has close relationship with colorectal cancer.However,the molecular role and mechanism of FEZF1-AS in development and progression of colorectal cancer is not clear.In this study,we aim to to clarify the possible role and mechanism of FEZF1-AS1gene in the proliferation,ivasion and metastasis of colorectal cancer.METHODS1. Analyze disparity expression spectra in differential metastasis potential CRC cells using Arraystar LncRNA microarray.The expression of differential expresssion lncRNAs in colorectal carcinoma cell lines and small sample CRC clinical specimens and their pair normal colorectal mucosa were detected by Real-Time PCR.Then the long non-coding RNA FEZF1-AS1was futher determinded as a lncRNA related to metastasis.The expression of FEZF1-AS1was examined in specimens of CRC with follow-up visit information by in sity hybridization(ISH).The relationship of FEZF1-AS1expression and clinical pathological factors,prognosis was analyzed by SPSS13.0software.2. Stable cell lines were established by retroviral expression system.FEZFl-AS1silenced cell lines (HCT116-Ai/HCT116-Ci and M5-Ai/M5-Ci) and control groups (HCT116-NC and M5-NC).3. The biological function of FEZF1-AS1A in cell proliferation ability wainvestigated by CCK8assay,plate colony formation assay.Cell cycle was detected by cell cycle esperiment.Cell invasion ability was tested by transwell invasion assay,scratch wound healing assay.The in vivo tumorigenesis and metastasis abilities were measured by tumorigenesis assay,tail vein injection of nude mice.4. The expression of FEZF1,which transcripts from the opposite strand of FEZF1-AS1, in stable FEZF1-AS1silenced cells above and colorectal cancer tissues and paired adjuvant non-cancerou tissue samples were detected by immunohistochemical(S-P) method.Meanwhile, we used Western blot to detecte the expression of FEZF1in stable FEZF1-AS1knocked down cells.The relationship of FEZF1expression and FEZF1-ASlwas analyzed by SPSS13.0software.The structural relationships of FEZF1and FEZF1-AS1was predicted by bioinformatics analysis.RESULTS1. LncRNAs disparity expression spectra in differential metastasis potential CRC cells.In our previous study,we founded some differential expression lncRNAs in differential metastasis potential CRC cell lines(SW480and M5) using Arraystar Human LncRNA Microarry.2. LncRNAs related to CRC metastasis.The expression of LncRNAs founded in microarray above were validated in SW480and M5cell lines by Real-Time polymerase chain reaction (qRT-PCR).Results showed that FEZF1-AS1was up-regulated in high metastasis potential cell lines (M5) than SW480. Next, FEZF1-AS1was also founded overexpression in M5,HCT116,Lovo and SW620than in SW480,Caco2,HT29,DLD1.Besides,data showed that the expression of FEZF1-ASlwas pretty higher in CRC tissues than in paired adjuvant non-cancerous tissues in small samples. Meanwhile,it was oviously that FEZF1-AS1was quite higher in metastasis samples than that without metastasis (P<0.05).Then it was chosed for subsequently study.3. Analysis the relationship of FEZF1-AS1expression with clinical pathology factors and prognosis.The expression of FEZF1-AS1at mRNA levels was higher in34CRC fresh samples than corresponding paired normal colorectal mucosa by Real-Time PCR (P=0.002).The results of in situ hybridization(ISH) in a total of153paired paraffinembedded noncancerous and CRC tissues showed that FEZF-1AS1was predominently stained in the cytoplasm of carcinoma cells.FEZF1-AS1expression was significantly associated with Dukes stage (P<0.001),Lymph node metastasis (P<0.001),distant metastasis (p<0.001) but no relationship with age,gender,carcinoma site and size (P>0.05).The difference between the survival curves which FEZF1-AS1expressed highly or lowly was analyzed by Kaplan-Meier and was determined by Log-Rank test.The mean survival time was53.525months,which was lower than in low FEZF1-AS1expression group (79.404months).Patients with high FEZF1-AS1levels exhibited shorter survival time (P=0.002).Multivariate analysis suggested that FEZF1-AS1was a risk factor for prognosis in CRC (P=0.035).The relative risk of FEZF1-AS1>1suggested that higher expression of FEZF1-AS1predicted poor prognosis of CRC patients examined (P=0.035).4. Downregulation of FEZF1-AS1decreased the proliferation, invasion and migratin activities of HCT116and M5cells.We transfected human colorectal carcinoma cell lines,HCT116and M5with FEZF1-AS1-shRNA (also named as "Ai/Ci") which was constructed into pGLV3/Hl/GFP+Puro Vector as well as control (Scramble shRNA) by retroviral expression system.Then selected stable transfectants by Fluorescence Activated Cell Sorter (FACS).The FEZF1-AS1expression in transfectants was confirmed by Real-Time PCR.HCT116-shRNA(-Ai,-Ci) and M5-shRNA (-Ai,-Ci) showed a significantly decreased proliferation abilities compaired with the HCT116-Scramble cells and M5-Scramble cells,respectively,as determined by in vitro CCK8assay (FHCT116=91.25, P<0.001; Fm5=183.833, P<0.001), plate colony formation assay (FHCT116=34.609,P=0.001;FM5=8.875,P=0.016). Ttranswell ssays (FHCT116=95.128, P<0.001;FM5=238.472, P<0.001),boyden chamber assays(FHCT116=91.477, P<0.001;FM5=174.358, P<0.001), and wound healing assay showed FEZF1-AS1downregulation decreased the ability of migration and invasion of HCT116and M5CRC cells.HCT116-sh RNA(-Ai) cells exhibited decreased tumorigenesis ability in vivo as tested by subcutaneous injection in nude mice (F=37.381, P<0.001).Knocked down of FEZFl-AS1decreased metastasis ability which was evaluated by injecting HCT116-sh RNA (-Ai) cells and HCT116-Scramble(-NC) cells into tail veins of nude mice (P=0.036).5. The expression of FEZF1,which transcript from the opposite strand of FEZF1-AS1,in CRC clinical samplesReal-Time PCR datsa showed that the expression of FEZF1was higher in30freshly frozen CRC samples than adjacent noncancerous specimes (P=0.049). Immunohistochemical(IHC) results indicated that the positive incidence was92.1%(141/153) in a total of153paired paraffinembedded noncancerous and CRC tissues. Kaplan-Meier and Log-Rank test were used to analyzed the survival curves between patients with high expression of FEZF1and lower group.It was founded that patients with low FEZF1levels exhibited higher five-year survival rate (73%,P=0.031)6. The relationship between FEZF1-AS1and FEZF1We found that the expression of FEZF1at protein level was remarkablely declined in CRC cells which downregulated of FEZF1-AS1compared with control cells.The expression trend was consistent with FEZF1-AS1.Besides,Spearman linear correlation analyze indicated a significant correlation between the expression of FEZF1-AS and FEZFl in20paired CRC clinical specimes (r=0.741,P<0.001). Bioinformatics analysis using ViennaRNA Package (RNAfold and RNAcofold) suggested that the minimum free energy of combination was far less than that of the two separate formation (Co-RNA:-2703.54; FEZF1-AS1:-1037.42; FEZF1:-755.31).In other words,it is easy to combine between the two RNAs and get more stability.CONCLUSION1. FEZF1-AS1was upregulated in CRC and significantly associated with Lymph node metastasis,Dukes stage,distant metastasis,differentiated degree.FEZFl-AS1may be used as an independent indicator to evaluate prognosis of CRC patients.Higher expression of FEZF1-AS predicted poorer prognosis of colorectal carcinoma patients.2. FEZF1-AS1plays an important role in proliferation, migration and invasion abilities in colorectal carcinoma cells.3. FEZF1-AS1may interact with FEZF1which plays an important role in proliferation,miagration and invasion of colorecatal carcinoma.4. FEZF1-AS1may as a new target for metastasis diagnostic markers and novel therapeutic strategies.INNOVATIONS1. FEZF1-AS1was a novel long non-coding RNA which detected from the lncRNAs Microarray in our previous work.Its function and molecular mechanism in colorectal cancer has not been reported. The role of FEZF1-AS1was revealed though in vitro and in vivo experiments that with original innovation.2. The correlation of FEZF1-AS1and FEZF1was validated.Meanwhile,the two RNAs may jointly forms a secondary structure which leading to more stable that based on the analysis of bio informatics.3. All of this study try to provides novel basis and ideas for metastasis mechanism in colorectal cancer from lncRNAthis new field of vision.