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

BackgroundColorectal cancer (colorectal cancer, CRC) refers to the large intestine mucosa epithelium transform to malignant lesions under the influence of a variety of genetics and environment carcinogenic factors. As one of the most common malignant tumor,colorectal cancer become the third member of the malignant tumor for its high mortality.Recently,the survival and quality of life of patients with colorectal cancer has improved greatly due to the research progress in pathogenesis of this malignant tumor in the west and North America and other developed countries.However, in China, the incidence and mortality rate of colorectal cancer patients are still increasing.At present,the pathogenesis of colorectal cancer is still unclear. According to the current data analysis,chronic inflammation,large adenomas,genetic and high fat and protein diet was significantly associated with colorectal cancer incidence.Cigarette smoking,environmental factors, such as molybdenum deficiency in the soil,schistosomiasis, a variety of causes such as pelvic radiation can also contribute to the incidence of colorectal cancer.Early diagnosis and early treatment are the gold standard for any malignant tumor, however,due to atypical early symptoms,nearly half of the patients with colorectal cancer were found with distant organ metastasis,such as liver metastasis.The gold standard screening for colorectal cancer is electronic colonoscopy examination,but it is hard for its popularization for its invasion and thus poor patient compliance.Therefore,early diagnosis and early treatment for colorectal cancer are very difficult.Besiedes,there are reports that in patients with colorectal cancer with no distant metastases before the operation,there are still more than a third of the patients relapsed,which indicates that effective early diagnosis and treatment are particularly important.Tumor occurrence and development are complicated multi-factor and multi-step process, gene mutation and abnormal expression in tumor occurrence and development play an important role. In recent years, the role of oncogenes and tumor suppressor genes related with the large intestine cancer and its influence factors such as DNA methylation modification, non-coding RNA,histone modification and chromatin remodeling factors in the occurrence and development of colorectal cancer are becoming the research center.Long chain noncoding RNA (LncRNA) is a kind of RNA in the length between 200-100000nt without the ability to encode proteins but regulates the expression of genes in epigenetic.transcriptional and post-transcriptional aspects.Reports in recent years have prompted that lncRNA expression in normal tissue and tumor tissue are different.LncRNA abnormal expression in tumor becomes an important marker in cancer diagnosis and prognosis and therapeutic evaluation.In recent years,it is suggested that lncRNA may participate in malignant tumor development in a number of ways,such as chromatin remodeling,DNA methylation regulation,histone modification,and as a precursor of microRNAs.At present,more and more reports about lncRNA in colorectal cancer appeared. Studies have shown that expression of lncRNA MALAT1 in colorectal cancer tissue increased,its high expression is closely related to tumor patients’ overall survival and disease-free survival which prompts lncRNA MALAT1 as a predictor of colorectal cancer patient’s overall survival and disease-free survival.Yin,etc reports that lncRNA GAS5 was low expressed in colorectal cancer,and was closely related to the tumor size, histological grade and TNM stage. Further experiments showed that lncRNA GAS5 can restrain the proliferation of colorectal cancer in invasion ability. Chen et al,found that lncRNA FEZF1-AS1 disorders participates in the occurrence and development of colorectal cancer (proliferation, migration, invasion, etc.), through the induction of FEZF1. Xiang, etal, found that lncRNA CCAT1-1 is located in the upstream of MYC gene and plays a MYC transcriptional regulatory role as well as promoting the long-distance chromatin circulation. DANCR, one of the important members of the lncRNA family, its expression in colorectal cancer and effects on colorectal cancer proliferation and development are still unclear.LncRNA DANCR is a recently discovered type of lncRNA which plays an important role in the development of tumor.Yuan,the earliest identifier of lncRNA DANCR in the liver cancer,reports that it is overexpressed in HCC stem cell sample and it can be used as the prognosis biomarker for patients with HCC.On the contrary,the low expression DANCR could reduce the stem cell properties,and can lead to tumor cell vigor,tumor shrinkage, etc. Further study found DANCR is increased by inhibiting CTNNB1 stem cell function. Zhang and other researchers have shown that Sox4 can enhance cartilage cell differentiation and synovial source sex cells proliferation by activating lncRNA DANCR.Chen et al,showed that lncRNA DANCR could inhibit of human dental pulp cells differentiation by inhibiting Wnt/ β-catenin pathway. Tong et al,found that lncRNA DANCR in human circulation monocytes could be used as postmenopausal osteoporosis related biomarkers. At present, research about DANCR in the tumor is still less, and there is no report on DANCR and colorectal cancer development.This research consists of the following three parts:(1) the quantitative polymerase chain reaction (PCR) to detect lncRNA-DANCR expression in 104 cases of colorectal cancer tissues and normal tissues adjacent to carcinoma and colorectal cancer cell line SW480, HCT116, LS174T, CaCo, SW620, SW1116, HT 29 and LoVo; analysis of the correlation of the expression and clinicopathological features of colorectal cancer;evaluate lncRNA prognostic significance in patients with colorectal cancer; (2) establish lncRNA-DANCR over-and low-expressed colorectal cancer cell lines, further studies on lncRNA DANCR biological function in colorectal cancer were determined by CCK8 experiment, cell apoptosis, cell cycle experiment as well as migration and invasion experiments. (3) Combined with literature, exploring lncRNA-DANCR inducing downstream signaling pathways, in vitro experiment was also performed to prove that lncRNA-DANCR could mediate cell proliferation and invasion ability by regulating the ERK/MAPK pathway, which could clarify lncRNA-DANCR signal pathway in promoting growth and development of colorectal cancer.Part I The expression and clinical significance of lncRNA DANCR in colorectal cancerObjectivesTo investigate the expression of lncRNA DANCR and its correlation with clinicopathologic charactistics and prognosis of patients with colorectal cancer(CRC).MethodsThe expression of lncRNA DANCR was detected by quantitative real-time PCR, (qRT-PCR) in 104 CRC specimens and cell lines. Standard statistical analysis was used to evaluate correlation between lncRNA DANCR and clinicopathological features and its prognostic significance in CRC.ResultslncRNA DANCR 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 DANCR expression was correlated with histological grade,lymphnode metastasis and TNM stage(P<0.05). Kaplan-Meier analysis showed that CRC patients with high DANCR expression had a shorter overall survival (OS) and disease-free survival (DFS) than low DANCR expression group (P<0.05). Moreover,in multivariate Cox model analysis, our results showed that DANCR expression was an independent poor prognostic factor for both OS and DFS in CRC patients.ConclusionsLncRNA DANCR might be a potentially novel biomarker for CRC prognosis.Part Ⅱ Effect of lncRNA DANCR expression on biological behaviours of colorectal cancer cellsObjectivesInvestigate the role of lncRNA DANCR in the proliferation,migration and invasion of colorectal cancer cells.Methodssi-DANCR and pcDNA-DANCR were transfected into HT29 and SW480 cells. Realtime PCR was used to detect lncRNA DANCR expression.Cell proliferation was detected by CCK8 assay,cell cycle and apotosis were investigated by FCM,wound healing assay and Matrigel invasion assay were used to measure the effects of lncRNA DANCR on cell migration and invasion in CRC, respectively.ResultsExpression of lncRNA DANCR were downregulated in siRNA-group,compared with control-group.LncRNA DANCR was increased in pcDNA-group compared with control-group.CCK8 assay demonstrated that lncRNA DANCR knockdown decreased the cell proliferation.In contrast,overexpression of lncRNA DANCR increased the cell proliferation.FCM showed that lncRNA DANCR knockdown induced G0/G1 phase arrest, Incontrast,overexpression of lncRNA DANCR resulted in a significant decrease in the cellular populationin G0/G1 phase but a sharp increase in S phase.Cell apoptosis assay indicated that si-DANCR noticeably promoted apoptosis of CRC cells,whereas, overexpression of lncRNA DANCR significantly inhibited apoptosis of CRC cells.Wound healing assay and Matrigel invasion assay suggested that downregulation of lncRNA DANCR inhibited the cell migration and invasion ability,whereas,overexpression of lncRNA DANCR promoted CRC cell migration and invasion ability.ConclusionsLncRNA DANCR could promote cell proliferation,migration and invasion in CRC cells, which indicated that DANCR may serve as an oncogene in the progression of colorectal cancer.Part III lncRNA DANCR promoted proliferation and invasion of colorectal cancer via MAPK pathwaysObjectivesInvestigate the mechanism of lncRNA DANCR in mediating the proliferation, migration and invasion in colorectal cancer cells.MethodsWe predicted and determined the targets of DANCR by gene mapping analysis and bioinformatics prediction methods. We speculated that ZAK (sterile alpha motif and leucine zipper containing kinase AZK) might be one of the target genes of DANCR. qRT-PCR was used to examine the expression of ZAK in CRC tissues, and then qRT-PCR and Western blot were used to determine the mRNA and protein expression of ZAK while DANCR was down-regulated. CCK8 assay and Western blot analysis were used to investigate the role of DANCR on ZAK in HT29 cell that had been transfected with si-DANCR, si-ZAK, or co-transfected with si-ZAK and si-DANCR.Furthermore,Western-blot was used to examine expression changes of the key proteins in MAPK signaling pathway.ResultsGene mapping analysis revealed that ZAK was located near DANCR,suggesting DANCR may regulate the expression of gene in a close genomic proximity (cis-acting regulation) via a variety of mechanisms.We found that ZAK level was significantly down-regulated in CRC tissues compared with normal controls.A negative correlation was observed between DANCR level and ZAK mRNA level in CRC tissues.Transfection of HT29 cells with si-DANCR increased the ZAK mRNA level and relative protein expression compared with the normal controls,suggesting that ZAK acted as a potential target for DANCR and DANCR negatively regulated the expression of ZAK.Through CCK8 assay,we demonstrated that the effects of DANCR knockdown on cell proliferation were partially prevented by the presence of siRNA targeting ZAK in HT29 cells.Based on these findings, we concluded that ZAK mediated the biological effects of DANCR on cell proliferation in human CRC.In addition,Western blot indicated that ERK1/2,JNK and p38 kinase phosphorylation was decreased in DANCR knockdown cells in contrast to the control cells, while DANCR-silencing plus ZAK-silencing increased the expression levels of phosphorylated ERK1/2,phosphorylated JNK and p38,which were lower than ZAK-silencing group,indicating that DANCR may regulate cell growth by inhibiting ZAK expression and activation the MAPK signaling pathway.ConclusionsDANCR promoted cell proliferation and migration by negatively regulating ZAK via activation of MAPK pathway.Therefore,the DANCR-ZAK-MAPK pathway may participate in CRC cell proliferation and migration,and may provide new ideas for studying the molecular mechanism of CRC and develop a new therapeutic strategy for CRC in the future.