Study Of Expression And Functional Role Of Long Non-coding RNA GAPLINC In Colorectal Cancer

BackgroundColorectal cancer(CRC)refers to the epithelial cells of colorectal mucosa transforming into malignant cells under multiple pathogenic factors,such as diet,environment or gene.The incidence and mortality rates of CRC are third and fourth worldwide respectively.In developed countries.CRC is the second most common malignant tumor.When CRC is confined to the intestinal wall,about 70%to 80%of patients can be cured by surgical resection of the lesion and the five-year survival rate is about 90%.But for patients with lymph node metastasis,the five-year survival rate dropped to about 60%.At present,due to the early diagnosis of CRC is still difficult for lacking of typical symptoms and valuable biomarkers,a considerable number of patients are diagnosed at an advanced stage.Therefore,the early diagnosis and treatment of CRC is of great clinical significance.The formation and development of tumor is a very complex process,involving multiple factors.Gene mutation and abnormal expression play an important regulatory role in tumor formation and progression.Because of the research on the development of CRC related oncogenes,tumor suppressors and mechanisms of action,people gradually realized that non-coding RNA,DNA methylation.histone modification and the change of chromatin location and structure play important roles in the formation and progression of CRC.Long non-coding RNAs(LncRNAs)are a kind of RNA molecules with a length of 200-100000 base pairs.In earlier studies,lncRNAs were ignored by scientists and regarded as meaningless transcription fragments.But with the advent of high-throughput gene sequencing technology,the function of IncRNAs has been gradually revealed.Through some specific research and analysis on the specific expression of IncRNAs,scientists found that IncRNAs play important roles in regulating gene expression.Current studies suggest that IncRNAs mainly derive from the next 5 sources.First,the mRNAs with the function of protein coding have lost their normal coding function due to the mutation of the gene,thus forming the IncRNAs with the function of regulating gene expression.Second,a number of distant gene fragments close to each other to form new IncRNAs due to chromosomal rearrangements.Third,in the retrotransposition process,the gene fragments overlap.Fourth,the gene fragments are repeated in the process of replication.Fifth,due to gene insertion,thus form a new IncRNA.At present,scientists have found that IncRNAs play a regulatory role in their adjacent genes(justice regulation),but also in their distant genes(anti-regulation).It is of great significance to study the function of lncRNAs.LncRNAs play an essential role in the occurrence and development of tumors.Researchers have come to realize that IncRNAs may play an important role in the diagnosis and treatment of tumors.In terms of diagnosis,the abnormal expression of IncRNAs in tumor tissue makes it possible to be the tumor markers.Especially in the early stage of some tumors,the traditional imaging methods are difficult to find the tumors because of their small sizes.Through detecting the level of lncRNAs expression in the blood,it becomes much more convenient to predict the possibility of tumor occurrence.In the aspect of treatment,the study on the role of IncRNAs in the tumor pathogenesis provides a new idea for the selection of new tumor therapeutic targets and research of new anticancer drugs with high efficiency,precision,low toxicity and side effects.At present,the research of lncRNAs is still in the primary stage,and the study on the function mechanism of lncRNAs is still under exploration.Over the past few years,lncRNAs have become a hot spot in cancer research,numerous abnormally expressed IncRNAs have been found in a variety of human tumors continuously.Scientists found that the expression of HOTAIR in tumor tissues and plasma of breast cancer patients was increased,and the level of HOTAIR in plasma was significantly correlated with the content of estrogen receptor(ER)and lymph node metastasis.Further studies had shown that plasma HOTAIR level may be a potential biomarker for breast cancer diagnosis.Zheng et al.showed that IncRNA CCAT2 is highly expressed in prostate cancer and is associated with poor prognosis in prostate cancer.Further studies had shown that lncRNA CCAT2 can affect the epithelial mesenchymal transition of prostate cancer and thus promote tumor metastasis.Wu et al.showed that the expression of GAS5-AS1 in non-small cell lung cancer was significantly lower than that in adjacent normal lung tissues.Low expression of GAS5-AS1 was significantly correlated with larger tumor size,advanced TNM stage and lymph node metastasis.The abnormal expression of GAS5-AS1 had no effect on cell cycle,apoptosis and proliferation,but could significantly affect the migration and invasion of non-small cell lung cancer cells.Over-expression of GAS5-AS1 can reduce the expression of ZEB1,N-cadherin,vimentin,SNAIL1 in non-small cell lung cancer cells,which could affect the epithelial mesenchymal transition.In recent years,more and more studies about the role of lncRNAs in CRC have been reported.Han et al.found the expression of IncRNA AFAP1-AS1 was obviously increased in CRC tissues and HCT116 and SW480 cell lines.Inhibition of AFAP1-AS1 expression could inhibit the proliferation,colony formation,migration and invasion of SW480 cells.In addition,interference of AFAP1-AS1 could inhibit the expression of EMT related genes.Li et al.showed that SNHG20 expression was significantly up-regulated in CRC.and the high expression of SNHG20 was obviously related to TNM stage.Multivariate analysis indicated that high expression of SNHG20 was an independent prognostic factor for overall survival in patients with CRC.Further studies showed that SNHG20 expression could inhibit cell proliferation,invasion and migration,and affect the cell cycle progression of CRC cells.In addition,SNHG20 regulates cell growth by regulating a number of cell cycle related genes.Wu et al.showed that IncRNA ucoo2kmd.1 regulated the growth of CRC cells via CD44 by competing with miR-211-3p.As one of the important members of lncRNAs family,the function of GAPLINC in CRC remains unclear.GAPLINC is a long non-coding RNA with a length of about 924bp.The expression of GAPLINC in gastric carcinoma was higher than that in control group,and correlation analysis showed that the expression of GAPLINC was positively correlated with tumor size and lymph node metastasis.Survival analysis showed that patients with high expression of GAPLINC had a lower survival rate than those with low expression.Further studies showed that GAPLINC could promote the proliferation and invasion of gastric cancer cells,and this effect is achieved through the regulation of CD44.Further studies had shown that GAPLINC regulated CD44 through becoming a ceRNA of miR-211-3P.However,there are few studies on GAPLINC in other tumors such as CRC.Investigations on the expression of GAPLINC in CRC and its effect on the phenotype of CRC cells and its mechanism have very important clinical significance.We studied the function of GAPLINC in CRC by the following 3 parts:(1)Quantitative real-time PCR was used to study the expression of IncRNA GAPLINC in CRC,adjacent tissues and CRC cell lines.The clinical data of patients with CRC was analyzed to evaluate the correlation between GAPLINC expression and clinicopathological features,and the effect of GAPLINC expression on the prognosis of CRC patients was also assessed.(2)CRC cell lines with high expression of lncRNA GAPLINC were selected by qRT-PCR assay.Then siRNAs which specific silenced GAPLINC were designed and respectively transfected into the selected CRC cell lines in order to select the siRNA with highest interference efficiency for subsequent experiments.(3)Exploring the effects of GAPLINC on the proliferation.migration,invasion,cell cycle and apoptosis of human CRC cell lines after lncRNA GAPLINC interference.The effects of GAPLINC on the biological behavior of human CRC cells by activating Wnt/beta-catenin signaling pathway were verified using Western-Blot and immunofluorescence,which further illuminated the mechanism of lncRNA GAPLINC in promoting the growth and progression of CRC.Part ⅠThe expression and clinical significance of lncRNA GAPLINC inCRCObjectives:To investigate the expression of IncRNA GAPLINC in CRC and CRC cell lines and its correlation with clinicopathologic features and prognosis of CRC patients.Methods:The expression of IncRNA GAPLINC in 64 CRC tissues and cell lines was detected by qRT-PCR.Chi-square test and fisher exact test were used to analyse the correlation between the expression of IncRNA GAPLINC and clinicopathological features of CRC patients.Survival curves were plotted using Kaplan-Meier method and survival rate was analyzed by log-rank test.Multivariate analysis of the prognostic factors was performed with Cox regression model.Results:LncRNA GAPLINC expression was increased in CRC tissues and cell lines compared with adjacent non-tumor tissues and normal human intestinal epithelial cell line FHC(P<0.05).In addition.high GAPLINC expression was correlated with invasion,lymph node metastasis and TNM stage of CRC patients(P<0.05).Kaplan-Meier analysis showed that CRC patients with high GAPLINC expression had a shorter overall survival(OS)than low(GAPLINC expression group(P<0.05).Moreover,multivariate Cox model analysis showed that GAPLINC expression was an independent risk factor for poor OS in CRC patients.Conclusions:LncRNA GAPLINC expression was increased in CRC and was closely related to tumor progression and prognosis.LncRNA GAPLINC might be a novel prognostic biomarker and therapeutic target for CRC.Part ⅡSiRNA suppresses lncRNA GAPLINC expression in CRC cells Objectives:Select the CRC cell lines with higher lncRNA GAPLINC expression level.Design and select the siRNA which has the highest interference efficiency to GAPLINC.Methods:The expression of IncRNA GAPLINC in three CRC cell lines was detected by qRT-PCR.Three different interference sequences were designed and the expression of lncRNA GAPLINC was measured after transfecting with the siRNAs by qRT-PCR.Results:LncRNA GAPLINC expression was higher in HCT116 and HT29 cells compared to other CRC cells(P<0.05).Moreover,si-GAPLINC-2(GCCAUCUCCAUAACAUAAATT)had the highest suppression efficiency(P<0.05).Conclusions:CRC cell lines with higher expression level of IncRNA GAPLINC were successfully selected and the siRNA with satisfying interference efficiency was synthesized for the following experiments.Part ⅢEffect and underlying mechanism of lncRNA GAPLINCexpression on biological behaviours of CRC cells Objectives:To explore the role of lncRNA GAPLINC expression in CRC cells and underlying mechanism was also investigated.Methods:Cell proliferation was detected by CCK8 assay.Wound healing assay and Matrigel invasion assay were used to measure the effect of lncRNA GAPLINC on cell migration and invasion respectively.Cell cycle and apotosis were investigated by FCM.Furthermore,western blot and immunofluorescence were used to explore the effect of si-GAPLINC by activating Wnt/beta-catenin signaling pathway.Results:CCK8 assay showed that decreased expression of GAPLINC inhibited the cell proliferation(P<0.05).Wound healing assay and Matrigel invasion assay suggested that downregulation of lncRNA GAPLINC inhibited the cell migration and invasion ability(P<0.05).FCM indicated that downregulated expression of GAPLINC induced G0/G1 phase arrest,and a sharp decrease in S phase(P<0.05).Cell apoptosis assay suggested that si-GAPLINC significantly promoted apoptosis of’CRC cells(P<0.05).Furthermore,western blot and immunofluorescence showed that GAPLINC could activate the Wnt/β-catenin pathway in CRC cells.Conclusions:LncRNA GAPLINC could enhance the abilities of proliferation.migration and invasion of CRC cell lines and significantly inhibit cell apoptosis.LncRNA GAPLINC might contribute to the development of CRC by activating the Wit/β-catenin pathway.