The Role Of Long Non-coding RNA TRERNA1 In The Development Of Colorectal Cancer

Colorectal cancer(CRC)is one of the most common malignant tumors all over the word.It threatens human healthy seriously due to its third highest incidence and second highest mortality in the world.In China,the incidence and mortality of colorectal cancer rank fifth and forth in malignant tumors respectively.The pathogenesis of colorectal cancer is a complex multi-factor procedure.There are many risks that play important role in the development of colorectal cancer including unhealthy living habits,environmental factors,genetic factors and abnormal gene expression.The early prognosis of patients with colorectal cancer can be improved by comprehensive treatments such as surgical resection,chemotherapy,radiation therapy,and molecular targeted therapy at early stage.However,patients diagnosed with advanced colorectal cancer have high mortality due to unsatisfactory treatment.Thus,early detection and diagnosis play an important role in the prognosis of patients with colorectal cancer.Therefore,it is necessary to find biomarkers and potential target molecules related to the diagnosis and treatment of colorectal cancer.Abnormal gene expression may affect non-coding RNA and protein expressions directly or indirectly,including microRNA(miRNA),long non-coding RNA(LncRNA),and Circular non-coding RNA(circRNA),thereby further affecting the biological functions that these molecules are involved in,such as cell proliferation,apoptosis,senescence,migration,invasion etc.At present,a large number of studies focusing on non-coding RNAs have demonstrated that a variety of non-coding RNAs play important roles in cell proliferation,invasion and migration.Long non-coding RNA is a ribonucleotide sequence,which is more than 200 nt in length but cannot be translated into a protein.Many studies have shown that LncRNAs have complex biological functions and play an important role in the development of cancer,neurodegenerative diseases and cardiovascular diseases.In addition,LncRNA can regulate a certain signal pathway through endogenous competitive adsorption of miRNA or binding protein as a cofactor of protein to affect the development of various diseases.At present,a large number of studies on non-coding RNAs have demonstrated that a variety of non-coding RNAs are involved in cell proliferation,invasion and migration.In some tumors,LncRNA can act as a tumor suppressor molecule.For example,the expression of long non-coding RNA SLC25A5-AS1 decreased in gastric cancer cells,with the effects of inhibiting cell proliferation,promoting apoptosis and relating to tumor volume,TNM stage and lymph node metastasis.In some tumors,LncRNA plays a role in promoting the development of cancer,such as increased expression of AGAP2-AS1 in pancreatic cancer cells.AGAP2-AS1 is associated with tumor volume and pathological stage of pancreatic cancer,and can also affect pancreatic cancer cell proliferation,apoptosis,invasion and metastasis.At present,many experiments have shown that long non-coding RNA is closely related to the development of colorectal cancer.For example,long non-coding RNA GAS5 can inhibit cell migration and invasion,and long non-coding RNA BDNF-AS can inhibit cell proliferation and migration in colorectal cancer.However,the role of many long non-coding RNAs in colorectal cancer remains unclear.The Human Cancer Pathway Finder RT2 lncRNA PCR Array of Qiagen summarizes 84 LncRNAs that are currently closely related to the development of human cancers.We used the PCR ARRAY and randomly selected three pairs of colorectal cancer tissues and their corresponding non-cancerous adjacent tissues to detect the differential expression of LncRNA related to cancer,and carry out subsequent verification experiments.LncRNA TRERNA1(Translation Regulatory Long non-coding RNA 1)gene is located near SNAI1 and is highly expressed in gastric cancer and liver cancer,and promotes metastasis of gastric cancer cells and liver cancer cells by promoting epithelial-mesenchymal transition(EMT)pathway.However,the role of TRERNA1 in colorectal cancer is still unclear.Therefore,this study was designed to investigate the expression of TRERNA1 in colorectal cancer and its role in the development of colorectal cancer.ObjectiveThe study was designed to look for LncRNAs that are abnormally expressed in colorectal cancer and verified at the tissue and cell level.Analyze the correlation between TRERNA and the development of colorectal cancer.And find markers and potential target molecules associated with the diagnosis and treatment of colorectal cancer.Method1.Detection of abnormally expressed LncRNA in colorectal cancer using PCR ARRAYThree pairs of colorectal cancer tissues and their corresponding non-tumorous adjacent tissues were randomly selected and operated according to the Human Cancer Pathway Finder RT2 LncRNA PCR Array Kit operating manual to detect abnormally expressed LncRNA in colorectal cancer by qRT-PCR.2.Validation of abnormally expressed LncRNA in tissues using qRT-PCRBased on the results of PCR ARRAY,we selected LncRNA with significant changes(*P<0.05)for subsequent experiments.We selected 130 pairs of colorectal cancer and its non-tumorous adjacent tissues to verify the results of PCR ARRAY by qRT-PCR.Then we analyzed the correlation between the expression level of TRERNA1 and the pathological parameters of colorectal cancer patients,and evaluated whether it could be used as a potential diagnostic marker for colorectal cancer.3.Analysis of the correlation between LncRNA TRERNA1 and clinical pathologyAnalyzed the relationship between TRERNA1 and the prognosis and survival rate of colorectal cancer patients by Kaplan-Meier,to determine whether TRERNA1 could be used as a target molecule for the prognosis and treatment of colorectal cancer.4.Effect of TRERNA1 on cell function of colorectal cancer cellsThe small interfering RNA(siRNA)of TRERNA1 was designed and synthesized.The siRNA and negative control were transfected into colorectal cancer cell lines HCT-8 and HCT-116.Its interference effect on TRERNA1 gene expression was detected by qRT-PCR.The effect of down-regulating TRERNA1 on the migration ability of colorectal cancer cells was detected by transwell migration assay.The effect of TRERNA1 on the invasion of colorectal cancer cells was examined by transwell Matrigel invasion assay.5.Preliminary study on the molecular mechanism of TRERNA1 regulating colorectal cancer metastasisThe correlation between TRERNA1 and tumor markers related to tumor metastasis was detected.After knocking down TRERNA1,qRT-PCR and Western blot were used to measure the expression of invasion and metastasis-related molecules in HCT-8 and HCT-116 cells.And bioinformatics analysis preliminary studies the molecular mechanism of TRERNA1 regulating the metastasis of CRC.6.Bioinformatics analysis of the molecular mechanism of TRERNA1 regulating Snail expressionBioinformatics software were used to find miRNAs that can be combined with both TRERNA1 and Snail,and we analyze the function of miRNAs from the literature,and to investigate whether TRERNA1 regulates Snail expression through endogenous competitive effects.Results1.Select differentially expressed LncRNA in CRCUsing PCR ARRY analysis of the data,the results showed that LncRNA TRERNA1 expression was significantly different in colorectal cancer tissues and adjacent non-cancerous tissues,the change factor was 21.88(*P=0.045341).In addition,the results also showed a significant difference in LncRNA BANCR expression,with a fold change of 21.14(*P=0.005813).Therefore,based on the analysis results,we selected LncRNA TRERNA1 as a research object in colorectal cancer to further explore its related role in colorectal cancer.2.LncRNA TRERNA1 is highly expressed in colorectal cancer tissuesUsing qRT-PCR to detect 130 pairs of colorectal cancer tissues and adjacent non-cancerous tissues,the results showed that TRERNNA1 expression was significantly increased in colorectal cancer tissues compared with non-cancer adjacent tissues(**P=0.008).3.LncRNA TRERNA expression and clinicopathological correlationIn patients with CRC,we found that the expression of LncRNA TRERNA1 was not associated with age,gender or tumor growth site,tumor differentiation,lymph node metastasis,but was related with tumor growth,stage,distal metastasis and neurological invasion.4.Knockdown of LncRNA TRERNA expression inhibits invasion and migration of HCT-8 cellsThe expression of TRERNA1 was knocked down in HCT-8 cells by small interfering RNA,and the effect of TRERNA 1 on invasion and migration of colorectal cancer cells was detected by transwell assay and transwell Matrigel invasion assay.The results showed that compared with the control group,the number of cells in the lower compartment of the experimental group was significantly reduced,and the migration ability of HCT-8 cells was significantly inhibited(**P<0.01).Transwell Matrigel invasion assay showed that compared with the control group,the number of cells in the lower compartment of the experimental group was significantly reduced,and the invasion ability of HCT-8 cells was significantly inhibited(**P<0.01).5.LncRNA TRERNA1 is associated with prognosis in patients with colorectal cancerAccording to the expression level of LncRNA TRERNA1 in colorectal cancer tissues,130 patients were divided into two subgroups(median as a standard),which were high expression group and low level group,respectively.Correlation between LncRNA TRERNA1 expression levels and overall survival in patients with CRC was investigated by Kaplan-Meier analysis.The results of the survival curves show that the overall survival rate of colorectal cancer patients with higher LncRNA TRERNA1 expression levels is significantly shorter than patients with low LncRNA TRERNA1 expression levels.In addition,multivariate analysis showed that high expression of LncRNA TRERNA1 was associated with poor prognosis in patients with CRC,except for metastatic distance and lymph node metastasis.6.TRERNA1 may affect colorectal metastasis by regulating snail expressionAccording to qRT-PCR results,the expression of TRERNA1 in colorectal cancer was positively correlated with the expression of snail(***P<0.001).After knocking down TRERNA1,mRNA and protein and levels of snail in HCT-8 and HCT-116 cells decreased(**P<0.01,***P<0.001),and the protein level of snail is increased while the protein level of E-cadherin is decreased.Bioinformatics analysis finds that TRERNA1 and snail can jointly bind miR-1207-5p.Therefore,TRERNA1 may affect the epithelial-mesenchymal transition pathway to regulate the metastasis of CRC through endogenous competitive effects in combination with miR-1207-5p and thus regulating snail expression.ConclusionIn colorectal cancer,LncRNA TRERNA1 could promote the development of disease and is associated with the prognosis of patients with colorectal cancer.