Study On Expression And Functional Study Of Long Non-coding RNA FOXD3-AS1 In Colorectal Cancer

Objective:lncRNAs expressions in the serum of patients with colorectal cancer were screened by high-throughput IncRNA microrrays.The expressions of lncRNAs were detected in colorectal cancer,and preliminary functional studies were also performed,so that new highly specific and sensitive colorectal cancer potential diagnostic markers can,be found,and provide new experimental basis for the occurrence,development,diagnosis,treatment and prognosis of colorectal cancer.Methods:LncRNA microarray technique was used to detect the expression of lncRNAs in the serum of 6 colorectal cancer patients and 4 healthy volunteers.The differentially expressed genes were used to construct a co-expression network to search for possible IncRNAs with biological functions,highly expressed candidate lncRNA in colorectal cancer patients’ serum were determined.The qRT-PCR was used to detect the expression levels of candidate lncRNA in the serum of 36 colorectal cancer patients and 30 healthy volunteers.The correlation between their expression and the clinicopathological features was evaluated,the specificity and the sensitivity as diagnostic markers were also estimated.Lentivirus-mediated siRNA technology was used to interfere with the expression level of candidate lncRNAs in colorectal cancer cell lines for screening cell lines with the highest expression levels and siRNAs with the highest interference efficiency.CCK-8 experiments,scratch experiments,the detection of cell cycle,apoptosis and Matrigel invasion assays were performed to study the effects of candidate lncRNAs on biological function of colorectal cancer cell lines.Results:After IncRNA microarray analysis,differentially expressed 1540 lncRNAs and 556 mRNAs were achieved(FC≥2 and P<0.05).Among these lncRNAs,412 lncRNAs were upregulated in colorectal cancer serum,and 1128 were down regulated.The construction of lncRNA-miRNA co-expression network revealed that long non-coding RNA FOXD3 antisense RNA1(lncRNA FOXD3-AS1)highly expressed in serum of colorectal cancer which might affect the biological function of colorectal cancer cells,and thus used as an lncRNA for subsequent experiments.The expressions of lncRNA FOXD3-AS1 detected by qRT-PCR in serum and HCT116SW480、SW620、LoVo cell lines of colorectal cancer were up-regulated(P<0.05).The expression level of IncRNA FOXD3-AS1 was related to lymph node metastasis,and TNM stage(P<0.001),yet not related to gender,age,tumor size,depth of invasion(P>0.05).Subject area under the ROC(AUC)was 0.91(95%CI= 0.83-0.99,P<0.001)with a specificity of 96.67%and a sensitivity of 86.11%.In vitro experiments showed that after the expression of interfering IncRNA FOXD3-AS1,the proliferative capacity of colorectal cancer cells was significantly decreased,the proportion of apoptosis was significantly increased,and the movement and invasion of cells were significantly inhibited.The growth of cells was arrested in the early stage of DNA synthesis.Conclusion:The main conclusions are listed as follow:1 Through IncRNA microarray detection,a large number of IncRNAs that are differentially expressed in serum of colorectal cancer have been found.These lncRNAs may be related to the diagnosis,occurrence,development and treatment of colorectal cancer,which provide a new basis for further research.2 The long non-coding RNA FOXD3-AS1 is obviously up-regulated in serum of colorectal cancer,which can better distinguish between colorectal cancer and healthy people.It may be a potential molecular marker of colorectal cancer,and perhaps it has a clinical significance for the diagnosis of colorectal cancer.3 Long non-coding RNA FOXD3-AS1 can regulate proliferation by regulating the growth cycle and apoptosis of colorectal cancer cell,and also playan important role in colorectal cancer cell movement and invasion.