The Functions And Molecular Mechanisms Of LncRNA-TSLC8 In Colorectal Cancer

Research background and objectiveColorectal cancer(CRC)is one of the common malignant tumors in the digestive tract,and ranks the third in morbidity and the second in mortality among all malignant tumors in the world.CRC is a serious threat to people’s health and life.Studies have shown that the occurrence and development of CRC is closely related to many factors such as diet,genetics and environment.Currently,surgical resection,chemotherapy,radiotherapy and targeted therapy are commonly used in clinical treatment of CRC.With the deepening of the understanding of CRC and the continuous improvement of treatment methods,the mortality rate of CRC has been reduced,the five-year survival rate has been increased,and the quality of life of patients has been significantly improved.The occurrence site of CRC is hidden,and early diagnosis is difficult.At present,specific diagnostic markers of CRC have not been identified,and there is also a lack of effective diagnostic methods.Most patients come to see a doctor when they are already in the middle and advanced stage,and the clinical treatment effect of middle and advanced stage of CRC is not satisfactory.With the development of molecular biology and the advancement of research technology,the discussion on the molecular mechanism of tumor pathogenesis has been deepened,and the development of tumor molecular pathology has also been rapid.We have made great achievements in early diagnosis and treatment of some tumors.However,the molecular mechanism of the occurrence and development of CRC remains to be clarified.Therefore,revealing the molecular mechanism of CRC pathogenesis is the key to search for early specific diagnostic markers and new targets for clinical treatment of CRC,and it is also of great significance to improve the efficacy and the quality of life of patients.In recent years,long non-coding RNAs(lncRNAs)have attracted extensive attention due to their important regulatory roles in cell life process and tumor biology.LncRNA is a class of RNA molecules with a length of more than 200 nt.Traditionally,lncRNA is not used as a template for protein translation,but functions directly in the form of RNA.LncRNAs can participate in the biological processes of cell proliferation,differentiation and development at multiple levels including transcription,post-transcription,translation and post-translational modification,thus playing a regulatory role in epigenetics,embryonic development and the occurrence and development of tumors and other physiological activities.Studies have shown that lncRNA plays an important regulatory role in the occurrence and development of a variety of tumors.In CRC,it has been found that lncRNA can affect the Epithelial Mesenchymal Transition(EMT)or cell cycle arrest of tumor cells by participating in the regulation of a variety of signaling pathways,thus affecting the growth,apoptosis,migration and invasion of tumor cells.In addition,some lncRNAs may be involved in chemotherapy resistance and radiotherapy sensitivity of tumors.High-throughput sequencing,however,is based on system identification of the CRC of lncRNA and functional research reports are very few,this research adopts the high-throughput sequencing identification and screening of lncRNA in CRC high expression,and its biological function,molecular mechanisms and clinical significance is analyzed,in order to look for CRC early diagnostic markers and novel targets for drug treatment.In conclusion,lncRNAs,as important intracellular regulatory factors,play an important regulatory role in the occurrence and development of malignant tumors,including CRC.However,lncRNAs differentially expressed in CRC have not been widely identified,and their molecular mechanisms are still unclear.Therefore,in this study,clinical CRC tissue samples were used to systematically identify lncRNAs differentially expressed in cancer tissues.Subsequently,functional studies showed that one of the overpowering lncRNA-TSLC8(Tumor Suppressive lncRNA on Chromosome 8,TSLC8)has an important effect on the growth,proliferation,migration and invasion of CRC cells and Tumor formation.Mechanism studies have shown that the ectopic expression of TSLC8 enhances the abundance of Puma,and this effect is mediated by the binding and stabilization of TSLC8-Puma,which has certain clinical significance.The research methods Chapter 1: LncRNAs differentially expressed in colorectal cancer tissues were analyzed by RNA-seq microarray technology,so as to screen out the lncRNAs with obvious biological functions.The expression difference of lncRNA-TSLC8 in colorectal cancer and paraccancerous tissues was quantitatively analyzed by real-time quantitative PCR(RT-qPCR),and the expression of lncRNA-TSLC8 in normal colon epithelial cells FHC and five colorectal cancer cell lines(SW480,HT-29,SW-620,Caco-2,and HCT-116)was also analyzed.The nucleoplasmic localization of TSLC8 was detected by nucleoplasmic separation combined with RT-qPCR.According to the expression of lncRNA-TSLC8 in 116 samples,the correlation between lncRNA-TSLC8 and the clinical data such as tumor tissue size,TNM stage and lymph node metastasis was statistically analyzed according to the clinical information,and Kaplan-Meier survival curve was analyzed.Chapter 2: First use slow virus no-load(Control)or have loaded TSLC8 expressed sequence slow virus(TSLC8)express TSLC8,through CCK8 cell proliferation experiment,flow cytometry and Annexin V/PI tumor ball formation experiment and xenograft tumor detection after expressing TSLC8 cell proliferation of SW480 cells,cell apoptosis,tumor ball formation ability and influence on tumor formation in mice;HCT-116 cells were also transfected with TSLC8(Sh-TSLC8)control no-load(Sh-Ctrl)or interference sequence to detect the cell survival rate,proliferation ability,apoptosis,tumor ball formation and the effect on tumor formation in mice.Chapter 3: In order to determine lncRNA-TSLC8 inhibit the occurrence and development of colorectal cancer molecular mechanism,predicting the unbiased with ntra RNA confirmed its downstream targets for Puma,RNA and RNA in vitro analysis TSLC8 interaction with Puma,silent TSLC8 expression to observe the transcription of Puma,to show SW480 and HT-29 cell transfection TSLC8 and/or silent Puma,observe the Puma as TSLC8 targets in colorectal cancer cell lines in the biological function;In addition,we noted a potential FOXO1 recognition element(FRE)upstream of the TSLC8 transcription start site(TSS),a transcription factor that plays a key role in tumor inhibition.To confirm whether FOXO1 regulates TSLC8 expression,We studied the expression of Fox O1 and tslc8 in tissues.TSLC8 observed in samples due to the large intestine cancer cells and expression of a significant loss,we have the original generation of colon cells from healthy donors WGBS,and compare the results with the results of the analysis of CRC cell line,by no CpGLess TSLC8 promoter build body is inserted into the carrier,and in vitro induced by the building body is Sss I methylation,observe whether TSLC8 silence is caused by DNA methylation modification.The results of the study Chapter 1: LncRNA microarray sequencing analysis was performed between colorectal cancer tissues and normal paracancerous tissues,and a total of 6598 lncRNAs were screened for differential expression.Results showed that 218 genes were significantly cut,discovered three new transcript,and choose the one with the highest lncRNA lower multiples transcript ensg00000272128.1,its located on chromosome 8(GRCh38: CM000670.2 Ch8,38099471-38099931)is located in the former chain,we call it on chromosome 8 tumor inhibitory lncRNA(TSLC8),the total length of 461 nt transcription protein coding with minimum potential;Northern hybridization further detected the endogenous high expression of lncRNA-TSLC8 in normal FHC cells and significantly low expression in CRC cell lines.RT-qPCR validation also showed that TSLC8 was significantly down-regulated in various CRC cell lines and colorectal cancer samples.The expression of TSLC8 was significantly correlated with TNM stage,metastatic state and tumor size,but not with age,sex or location.Nucleoplasmic analysis showed that TSLC8 was mainly located in the cytoplasm.Fluorescence in situ hybridization further showed that the distribution was mainly cytoplasmic.These data suggest that TSLC8 is significantly down-regulated in colorectal cancer,which may be related to the development of colorectal cancer.Chapter 2: Since the expression of TSLC8 in SW480 cells was significantly reduced,we selected SW480 cells for analysis.We found that the expression of TSLC8 was significantly up-regulated in SW480 cells transfected with PWPXL-TSLC8 overexpressing vector.Overexpression of TSLC8 significantly inhibited the viability of CRC cells and the formation of tumor spheres,and promoted cell apoptosis.Next,we investigated whether TSLC8 had an effect on the tumorigenesis of colorectal cancer cells in vivo.The results showed that overexpression of TSLC8 significantly reduced the growth and tumor weight of xenograft tumors.These results suggest that TSLC8 can significantly inhibit the pathological process of colorectal cancer in vivo and in vitro.We noted that HCT-116 cells have relatively high TSLC8 expression compared to other colorectal cancer cell lines,so we selected HCT-116 cells to inhibit TSLC8 expression and evaluate the related effects.Interfering the expression of TSLC8 significantly promoted the survival of CRC cells and the formation of tumor spheres.However,if the expression of TSLC8 is restored,the viability and migration ability of CRC cells will be significantly inhibited,while the apoptosis rate will be significantly alleviated.In vivo xenograft tumor models,interference with TSLC8 significantly increased tumor weight and xenograft tumor growth.These data provide further evidence that inhibition of TSLC8 expression may promote tumorigenicity in colorectal cancer cells.Chapter 3: In vitro RNA-RNA analysis confirmed that TSLC8 interacts with Puma.Overexpression of TSLC8 significantly increased the mRNA level of Puma and promoted the expression of Puma protein in SW480 and HT-29 cells.Cell function experiments confirmed that the tumor suppressive effect of TSLC8 overexpression was counteracted by the accompanying Puma silencing.Therefore,TSLC8 may inhibit the expression of Puma by stabilizing Puma transcripts in CRC cells,thereby promoting Puma expression.We found a significant correlation between FOXO1 transcripts and TSLC8,and ecotopic expression of FOXO1 promoted endogenous TSLC8 expression.However,when TSLC8 was overexpressed,no concomitant increase in FOXO1 levels was observed,suggesting that the regulation was unidirectional.When we knocked out the expression of FOXO1,we observed a significant decrease in both FOXO1 and TSLC8.In addition,compared with blank control group,overexpression of FOXO1 stimulated TSLC8 promoter activity.We conclude that FOXO1 may regulate the expression of TSLC8 in colorectal cancer cells.To determine the regulatory mechanism of TSLC8 silencing.WGBS results were compared with CRC cell line analysis results.We found that colorectal cancer cells and tumor tissues were hypermethylated,and the methylation state was also negatively correlated with the expression of TSLC8.Subsequently,we found that after DNMT3 A inhibitor treatment,the methylation of CpG island near the TSLC8 promoter region was significantly inhibited.In addition,DNMT3 A levels were negatively correlated with TSLC8 expression.The research conclusion In our current work,we first identified a novel lncRNA named TSLC8,which has a potential tumor suppressor function in colorectal cancer.TSLC8 was significantly down-regulated in various CRC cell lines and tumor specimens.We also noted that overexpression of TSLC8 in CRC cells inhibited cell viability,tumor ball formation,and promoted cell apoptosis.In CRC cells,the functional silencing of TSLC8 severely aggravates the malignant phenotype,while apoptosis is consistently inhibited.The mechanism studies showed that TSLC8 could effectively stabilize the transcript of Puma.FOXO1 is responsible for the induction of TSLC8,and hypermethylation of TSLC8 may interfere with the expression of TSLC8 in CRC,and the increased expression of DNMT3 A may be related to the observed hypermethylation.The enzyme activity of DNMTs and its role in the progression of colorectal cancer still need to be further explored.In this study,lncRNA-TSLC8 is expected to be used as a new target for the diagnosis and treatment of colorectal cancer,providing a new idea for the treatment of colorectal cancer and improving the prognosis of patients.