Screening Of Long Non-coding RNA Associated With Prognosis Of Colorectal Cancer And Its Functional Verification

Background:Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.At present,surgical treatment and chemotherapy are still commonly used in clinical treatment.With the advent of targeted drugs,although their prognosis has improved to some extent,the survival time has not yet exceeded 30 months.Long-chain non-coding RNAs(LncRNAs)are non-coding RNAs longer than 200nt in length and have been considered transcriptional "noise".However,a number of studies in recent years have shown that abnormal expression and function of LncRNAs are related to a variety of diseases,and can lead to the occurrence of various tumors by affecting biological behaviors such as cell proliferation,apoptosis,invasion,metastasis,and drug resistance.So far,a number of CRC-related lncRNAs have been discovered,but mechanisms of many expressions and functions are not completely clear which need further research.In view of the important role of LncRNA in cell activity,research on the function of LncRNA related to the prognosis of CRC and its relationship with tumorigenesis and development,explore possible upstream and downstream effector molecules,and clarify its possible signaling pathways,which can be colorectal Bio-targeted therapies for cancer provide new therapeutic targets and may improve the prognosis of CRC.Objective:To discover new lncRNAs related to the prognosis of CRC and investigate the clinical significance of differential expression of candidate lncRNA FAM222A-AS1 in CRC by using bioinformatics.And then we analyze it's effect on proliferation,migration and invasion of CRC cells and explore its potential molecular mechanism.Methods:1.Bioinformatics analysis:861 colorectal cancer related differentially expressed lncRNA(Transcript ID)were downloaded from LncRNnome,combined with GENCODE database to complete the lncRNA gene name information,and downloaded the above 861 differentially expressed lncRNA expression data and case sample number information from the database MiTranscriptome,and compared them with the prognosis information of 268 CRC cases(196 cases of colon cancer and 72 cases of rectal cancer)from the literature library of 7256 RNA sequencing(rna-seq).Finally,we obtained 208 aberrant expressed lncRNAs related with colorectal cancer prognostic information,Maxstat software package of R is used to calculate the optimal cutoff value,and Kaplan Meier-survival analysis,finally 18 dysregulation expressed lncRNAs(11 upregulation,7 downregulation)related with colorectal cancer prognosis were selected,and 7 downregulated lncRNA were further comprehensive analyzed to selected four candidate lncRNAs expression in colorectal cancer cell line in vitro and vivo function.By comprehensive analyzed the cell biology function of candidate lncRNA to selected the purpose FAM222A-AS1.And then,analyze the effect of FAM222A-AS1 expression in colorectal cancer on the invasion and migration of colorectal cancer cells and its relationship with the prognosis of colorectal cancer;2.In vitro:The expression of FAM222A-AS1 in normal colorectal epithelial cells(FHC)and colorectal cancer cell lines(SW480,SW620,RKO,HCT116,Caco2,HT29)was analyzed by real-time fluorescent quantitative PCR(qRT-PCR).After the design of siRNA for transient transfection of target cell silencing FAM222A-AS1,the effect of FAM222A-AS1 expression reduction on the proliferation of rectal cancer cells was detected by CCK-8 cell proliferation assay.Transwell and cell scratch assay examined the effect of FAM222A-AS1 silencing on the invasion and migration of colorectal cancer cells;3.In vivo:Respectively build pINDUCER series Doxycycline(Doxycycline,Dox)induced silence and a stable FAM222A-AS1 lenti-virus vector transfection cell lines,functional verification testing through a cellular level FAM222A-AS1 on cell proliferation,invasion and migration,and will build a cell line respectively a tumor-burdened subcutaneous injected into BALB/c Nude mouse to construct nude mice model,and testing FAM222A-AS1 silence and show the influence of the animal in vivo tumor growth;4.The R&D system antibody chip was used to explore FAM222A-AS1 related phosphorylated proteins,and the related role of phosphorylated protein molecules was initially explored.The FAM222A-AS1 interacting miRNA and target genes of miRNA were predicted through RegRNA2 and miRDB prediction website,respectively;5.Statistical analysis:The software used in Statistical analysis of experimental data of this research mainly includes SPSS 22.0,R,the software GraphPad prism 7 was used for statistical drawing,ImageJ is used to count clone,2-??Ct method was used to analyze LncRNAs relative mRNA expression level,the difference was analyzed by the paired sample Student't test,the results were showed in Mean±SD.The independent sample t test analysis was used to analyzed the the same point in time differences between different groups of CCK8 cell proliferation experiment;ImageJ was used to calculate the scratch area and the cells count in the cell scratch experiment and the Transwell experiment,respectively.Kaplan-Meir was used to draw the survival curve and log-rank method was used to compare the survival curve for the difference between the groups.P<0.05 indicated that the difference was statistically significant.Results:1.Through the analysis of multiple bioinformatics databases,18 lncRNAs related to the prognosis of colorectal cancer were preliminarily screened,among which 11 were down-regulated and 7 were up-regulated.Finally,4 candidate lncRNAs with up-regulated differentially expressed lncrnas were selected for further study(all FDR were less than 0.001),which were FEZF1-AS1(Tumor/Normal tissue expression multiple:T/N 52.63),FAM83H-AS1(T/N 7.69),FAM222A-AS1(T/N 7.69)and LINC00265(T/N 2.38);2.Through analysis of UCSC Xena database,it was found that FAM222A-AS1 expression in 308 cases of normal colorectal tissues was 0.831±0.046,FAM222A-AS1 expression in 41 paracancer tissues was 1.733±0.151,and FAM222A-AS1 expression in 288 colorectal cancer tissues was 4.796±0.085.FAM222A-AS1 expression in colorectal cancer tissues was significantly higher than that in normal colorectal tissues(P<0.0001).In addition,FEZF1-AS1 was expressed in 308 normal colorectal tissues and 288 colorectal cancer tissues,respectively(0.679±0.043 vs.5.85±0.181,P<0.0001).The expression levels of FAM83H-AS1 were(3.916±0.162 vs.10.380±0.055,P<0.0001).LINC00265 expression was(7.339±0.055 vs.8.195±0.060,P<0.0001).The expression levels of 4 lncRNAs in colorectal cancer tissues were all higher than those in normal and paracancer tissues,with statistically significant differences(P<0.05).Moreover,the patients with high expression of FAM222A-AS1 had worse prognosis than those with low expression,the 5-year survival rate of high-expression patients and low-expression patients were 35.3%and 56.3%(P=0.0432),respectively.Similarly,patients with high expression of FEZF1-AS1(0%vs 71.2%)FAM83H-AS1(42.0%vs 58.8%)and LINC00265(74.5%vs 86.7%)had poor prognosis compared with those with low expression.After comprehensive consideration of the research depth and heat of lncRNA,it was finally decided to conduct in-depth research and mechanism exploration on the newly discovered lncRNA FAM222A-AS 1;3.The results of qRT-PCR indicated that the expression levels of FAM222A-AS1 FEZF1-AS1,FAM83H-AS1 and LINC00265 in colorectal cancer cell lines were both higher than that of normal colonic epithelial cells(P<0.05).Overexpression of FAM222A-AS1 in colorectal cancer cell lines Caco2 and HT29 with low expression of FAM222A-AS1 significantly promoted the proliferation,invasion and migration of colorectal cancer cells.In vivo experimental results also showed that inhibition of FAM222A-AS1 expression could inhibit tumor growth in vivo,and high expression of FAM222A-AS1 promoted tumor growth.In addition,the predicted results of bioinformatics website lncLATAS and FISH cell localization experiments demonstrated that FAM222A-AS1 was distributed in cytoplasm and nucleus,but mainly expressed in the cytoplasm of colorectal cancer;4.Phosphorylated antibody chip results showed that FAM222A-AS1 was overexpressed in colorectal cancer cell line Caco-2,and phosphorylated protein molecules HSP60,AKT1/2/3(S473),WNK1(T60),GSK3?/?(S21/S9),STAT3(S727),EGFR(Y1086),ERK1(T202/Y204),p53(S392),p53(S46)and AKT1/2/3(T308)were significantly increased,with a difference of in gray scale1131.682,1087.364,993.036,822.036,649.632,599.854,559.632,518.193,475.683 and 446.925,and in the colorectal cancer cell line HCT116,FAM222A-AS1 was knocked down,and the expression levels of the phosphorylated protein molecule GSK3?/?(S21/S9),HSP60,PRAS40(T246),STAT3(Y705),AKT1/2/3(S473),YES(Y426),PLC-yl(Y783),ERK1/2(T202/Y204,T185/Y187),MSK1/2(S376/S360)and AMPKal(T183)were significantly decreased,and the difference in gray values was 2075.384,1916.586,551.051,509.657,497.85,443.829,427.484,398.435,393.728,379.636 and 318.414,among which,the phosphorylated protein molecules with changes in both knockdown and high expression of FAM222A-AS1 included HSP60,AKT1/2/3(S473),GSK3?/?(S21/S9),ERK1/2(T202/Y204).Western-blot validation showed that AKT(S473)and p53 did change with FAM222A-AS 1,indicating that these signaling protein molecules may participate in the regulatory mechanism of FAM222A-AS1 on colorectal cancer;5.Bioinformatics website prediction results found FAM222A-AS1 the only interaction of micrornas was hsa-4638-miR-5p,and hsa-4638-miR-5p target gene prediction results show PTPRA in 5 strains of colorectal cancer cells express quantity are highest,namely PTPRA hsa-4638-miR-5p is most relevant to the target gene in colorectal cancer cells.The results illustrate the potential through FAM222A-AS 1-microRNAs-PTPRA pathways promote colorectal cancer cell proliferation,invasion and migration.Conclusion:1.FEZF1-AS1,FAM83H-AS1,LINC00265 and FAM222A-AS1 were all significantly up-regulated in CRC,and their high expression was associated with malignant pathological features and poor prognosis of CRC;2.FAM222A-AS1 promote proliferation,invasion and migration of colorectal cancer cell;3.LncRNA FAM222A-AS1 can promote tumor growth in animals;4.LncRNA FAM222A-AS1 interacts with hsa-miR-4638-5p,and hsa-miR-4638-5p is closely related to PTPRA in colorectal cancer cells.Moreover,phosphorylated antibody chip results showed that FAM222A-AS1 and HSP60,AKT,GSK3?/?signaling proteins changed simultaneously,indicating that FAM222A-AS1 signaling pathway may be involved in these phosphorylated protein molecules.