Expression Of LncARSR In Colorectal Cancer Tissues And Its Effects On Invasion And Metastasis Of Colorectal Cancer Cells

Background and PurposeColorectal cancer(CRC)is the third most common malignant tumors,and the third major cause of death from cancer around the world.CRC ranks third in the disease spectrum of malignant tumors in China.CRC mainly occurs in people over 50 years-old,however,in last few years,the known prevalence of CRC has been on the rise in adolescents and adults under 45,who are more likely to develop advanced tumors.In recent decades,a variety of malignancies including CRC have been extensively,deeply studied.On account of the popularity and development of screening methods,surgery,targeted and therapy,the survival rate of CRC has been greatly enhanced.However,there is still a lack of biomarkers with perfect sensitivity and accuracy in the prognosis of CRC,and the efficacy of the therapeutic options are not enough to prevent or significantly reduce the number of CRC deaths.The 5-year survival rate of this malignancy is still poor.Therefore,in order to search more effective biomarkers and comprehensive treatment,it is necessary and crucial to further explore the molecular mechanism of the occurrence,progression and metastasis of colorectal cancer.In the last several years,along with the remarkable advancement of sequencing techniques,long non-coding RNA(1ncRNA),once considered as "junk RNA",has been shown to play important roles in many diverse biological processes.LncRNAs can regulate gene expression at the epigenetic horizon(by regulating chromatin remodeling,DNA methylation,or histone modification),the transcriptional horizon(intercombination with transcription factors,enhancers,or promoters),and post-transcriptional horizon(by alternative splicing,transfer and translation of pre-mRNA,or through interactions with miRNAs).There is increasing evidence that IncRNA is involved in regulating various biological characteristics of tumors,including proliferation,apoptosis,metastasis and metabolism.LncARSR is a newly discovered lncRNA,whineich has been found to affect cholesterol synthesis,progression of alcoholic fatty liver disease and drug resistance of tumor cells by regulating miRNA or mediating related signaling pathways.Some studies also believe that IncARSR is one of cancer-promoting genes and able to promote the development of bladder,liver and ovarian cancersHowever,the role and inner mechanism of IncARSR in the onset and progression of CRC have not been reported.Therefore,the object of the research was to detect the expression of IncARSR in the organization of CRC,the correlation with clinicopathological characteristics,and the impact on migration and invasion of CRC On this basis,we further explored the regulatory mechanism of IncARSR on colorectal cancer,to look for new potential biomarkers and targets in therapies and provide important reference for personalized treatment in patients with CRCMethods1.A total of 89 patients with CRC attended at the second hospital of shandong university were involled in the study.The expression of LncARSR was tested in CRC specimens by real-time fluorescent quantitative PCR.Then,the correlational analyses between IncARSR expression and clinical features or survival time of patients suffered from colorectal cancer was done.2.The expression of lncARSR in human normal intestinal mucosal cell lines and CRC cell lines(SW480,SW620,HCT-8,HT29,Caco-2 and RKO)was detected by real-time fluorescent quantitative PCR.3.Through the application of liposome transfection technique,constructed high/low expression of IncARSR in colorectal cancer cell lines,transwell chamber technology was employed to analyze the migration and invasion ability of CRC cells after transfection.4.The effects of IncARSR on the glycolysis of CRC cells were examined by glucose consumption,lactic acid production and ATP bioluminescence assay.5.The impact of IncARSR expression on liver metastasis of CRC cells was analyzed by injecting IncARSR overexpressed Caco-2 and IncARSR silenced HCT-8 cells as well as their controls into the tail veins of nude mice.6.Bioinformatics was used to predict the binding sequence of miR-34a-5p with lncARSR and HK1,and the interaction between miRNA-34a-5p and IncARSR or HK1 mRNA were investigated by luciferase reporter assay,RNA binding protein immunoprecipitation assay,Western blot and so on7.The double luciferase reporter gene assay was used to further determine the relationship among lncARSR,miRNA and HK1,which was verified in clinical samples.8.Rescue assays in vitro/vivo proved that IncARSR promote the metastasis and glucose metabolism reprogramming of CRC cells via competing binding miR-34a-5p.9.Survival analysis was employed to examine the effects of IncARSR and HK1 expression levels on OS and DFS in 89 CRC patients.Results1.The expression of IncARSR in CRC tissues and clinical significance(1)The results of qPCR displayed that the expression of IncARSR in CRC tissues was apparently higher than that in normal structures close to the cancer.The expression of IncARSR in ? and ? CRC was significantly higher than that in ? and ? CRC.The expression of IncARSR in persons with no relapse postoperation was dramatically lower than that with relapse.(2)The expression of IncARSR was significantly correlated with colorectal cancer AJCC stage,SUVmax of PET/CT,lymph node and distant metastasis,but not with other clinical indexes.(3)The survival curve displayed persons with high expression of IncARSR had a lower overall survival time and disease free time than those with low expression of lncARSR.In patients with postoperative relapse,the overall survival of patients with high expression of IncARSR was lower than that of patients with low expression of lncARSR.In patients without relapse,the overall survival had no remarkable differenence between patients with high expression of IncARSR and patients with low expression of IncARSR2.Effects of IncARSR on the biological properties of CRC cells(1)Among 6 human CRC cell lines,the relative expression level of IncARSR in Caco-2 was the lowest,while the relative expression level in HCT-8 was the highest.We selected these two cell lines for the subsequent experiment(2)The number of cells invaded and migrated in Caco-2 cells with overexpression of IncARSR significantly increased.Meanwhile,the expression of IncARSR was suppressed in HCT-8 cells,and the invasion and migration characteristics of CRC cells were obviously weakened.(3)LncARSR overexpression promoted glucose consumption,lactic acid production and ATP production in CRC cells.(4)High expression of LncARSR enhanced liver metastasis of CRC cells in nude mice.3.Mechanistic experiments showed that lncARSR acted as a ceRNA for miR-34a-5p,which attenuated the inhibitory effect of miR-34a-5p on HK1 expression,and promoted the invasion,migration and glucose metabolism reprogramming of colorectal cancer cells in vitro and liver metastasis of colorectal cancer cells in vivo.Combination of high lncARSR-HK1 could predict tumor recurrence or metastasis and a poor survival for CRC patients.Conclusions1.LncARSR over expressed in CRC and is related to clinicopathologic features and poor outcome.2.LncARSR sponges miR-34a-5p to promote the invasion and metastasis of colorectal cancer via HK1 mediated glycolysis metabolic reprogramming,thereby enhancing liver metastasis of colorectal cancer cells.3.LncARSR may become a potential prognostic biomarker and molecular treatment target for colorectal cancer.