The Role And Mechanism Of LINC01606 On Biological Function Of Colon Cancer Cells By SCD1-Wnt/β-catenin-TFE3 Signaling

Purpose:To investigate the effect and mechanism of lnc RNA LINC01606 on the biological function of colon cancer cells.Method:1)The expression of LINC01606 in colon cancer was analyzed by online TCGA database Driver DBv3,and the relationship between LINC01606 expression level and prognosis in colon cancer was analyzed.Quantitative real-time PCR（qRT-PCR）was used to further detect the expression level of LINC01606 in 83 pairs of colon cancer tissues and adjacent normal tissues,and the relationship between LINC01606expression level and clinicopathological features of colon cancer patients was analyzed,combining with clinicopathological data.The clinical diagnostic value was analyzed by the receiver operating characteristic（ROC）curve.QRT-PCR was used to detect the expression level of LINC01606 in 5 colon cancer cell lines and normal colon epithelial cell line.2)LINC01606 interference and overexpression vectors were constructed by lentivirus and stable cell lines of LINC01606 interference and overexpression vector and corresponding control vector were established.After lentiviral vector infection,the proportion of fluorescent cells was observed by fluorescence microscope,and the expression level of LINC01606 was detected by qRT-PCR to verify the infection efficiency.After interference and overexpression of LINC01606,the effects of LINC01606 on the proliferation,invasion,migration and growth of colon cancer cells were detected by CCK-8 assay,transwell migration and invasion assay,clone formation assay and nude mice subcutaneous tumorigenesis assay.3)The expression level of LINC01606 in spheroid cells and adherent cells was detected by qRT-PCR.The CD44⁺CD133⁺cells and CD44^-CD133^-cells were sorted by flow cytometry,and the expression level of LINC01606 in CD44⁺CD133⁺cells and CD44^-CD133^-cells was detected by qRT-PCR.After interference and overexpression of LINC01606,the effect of LINC01606 on the ability of sphere formation was detected by sphere formation assay,the proportion of CD44⁺CD133⁺cells and CD44^-CD133^-cells was analyzed by flow cytometry,the expression levels of stem markers CD44,CD133,Nanog,Sox2,Epcam,OCT4 and Lgr5 were detected by qRT-PCR,the sensitivity of LINC01606 on 5-fluorouracil（5-Fu）,a classical chemotherapy agent for colon cancer,was determined by CCK-8 assay and flow cytometry.4)After the cells were treated with ferroptosis inducer or inhibitor,the morphological changes of cells were observed by transmission electron microscope（TEM）and the cell activity changes were detected by CCK-8.After interference and overexpression of LINC01606 and treated with ferroptosis inducer,the changes of cell morphology,cell activity,intracellular Fe²⁺and total iron,mitochondrial superoxide,lipid ROS and mitochondrial membrane potential were observed.5)The expression and distribution of LINC01606 in SW480 and HT29 cells were detected by fluorescence in situ hybridization（FISH）assay.Biological information analysis showed that miR-423-5p may bind to LINC01606,SCD1 and Wnt3a.Luciferase assay was used to detect the binding of LINC01606,SCD1 and Wnt3a to miR-423-5p.RNA pull down and Ago2-RIP assay were used to detect the direct binding relationship between miR-423-5p and LINC01606 and SCD1.The expression levels of SCD1 and LINC01606 were detected after interference and overexpression of LINC01606 or miR-423-5p.6)After interference and overexpression of LINC01606 or miR-423-5p,the expression levels of Wnt signaling molecules including Wnt3a,β-catenin,EP300,TCF7,LEF1 and c-Myc were detected.The relationship between LINC01606 and Wnt/β-catenin signaling was detected by Topflash/Fopflash assay.The expression level of LINC01606,SCD1and Wnt signaling molecules were detected after the cells were treated with Wnt signal inhibitor C59 and activator BML-284 or combined with interference and overexpression of LINC01606.7)LINC01606 promoter DNA pull down assay was used to screen the transcription factor TFE3 that related to Wnt signaling.The expression level of TFE3 in colon cancer was analyzed in TCGA database and 83 pairs of colon cancer tissues.The expression level of LINC01606 was detected after overexpression of TFE3.Jaspar database was used to predict the binding site of TFE3 and LINC01606 promoter,and wild-type and mutant luciferase vectors were constructed according to the binding site.Luciferase assay was used to verify the binding relationship between TFE3and LINC01606 promoter region.The enrichment signal values of TFE3 in LINC01606 promoter region were analyzed by ENCODE database and chip-seq assay.8)After the cells were treated with ferroptosis inducer combination with Wnt signaling inhibitor and agonist,and overexpressed LINC01606,the concentrations of intracellular Fe²⁺,total iron,mitochondrial superoxide,lipid ROS and the changes of mitochondrial membrane potential were detected.After CD44⁺CD133⁺cells and CD44^-CD133^-cells were treated with ferroptosis inducer,the concentrations of Fe²⁺,total iron,mitochondrial superoxide,lipid ROS and the changes of mitochondrial membrane potential were detected.9)After the cells were treated with RSL3 and interfered with LINC01606,gas chromatography-mass spectrometry（GC-MS）was used to detect the concentration changes of 40 kinds of medium and long chain fatty acids and analyzed the change concentration ratio of saturated fatty acids,monounsaturated fatty acids and polyunsaturated fatty acids.Results:1)The expression of LINC01606 was increased in TCGA database and 83 pairs of colon cancer tissues,and LINC01606 expression level was significantly correlated with the depth of tumor invasion,lymph node metastasis,TNM stage and expression abundance of Ki-67.Survival analysis showed that overall survival（OS）,progression free interval（PFI）,and disease free interval（DFI）in colon cancer patients with high expression of LINC01606 was significantly lower than that of colon cancer patients with low LINC01606 expression.ROC curve analysis showed that LINC01606 was a diagnostic marker in colon cancer with an area of 0.725,and a sensitivity of 54.22%and a specificity of 84.34%.The expression level of LINC01606 in colon cancer cells was significantly higher than that in normal intestinal epithelial cells,and the expression level of LINC01606was the highest in HT29 and SW480.2)The proportion of green fluorescence cells in each group was more than 90%.After interference of LINC01606,the ability of proliferation,invasion,migration and growth were significantly decreased in colon cancer cells.After overexpression of LINC01606,the ability of proliferation,invasion,migration and growth were significantly increased in colon cancer cells.3)The expression level of LINC01606 in spheroid cells was higher than that in adherent cells,and in CD44⁺CD133⁺cells was significantly higher than that in CD44^-CD133^-cells.After interference of LINC01606,the ability of sphere formation,the proportion of CD44⁺CD133⁺cells,the tolerance to 5-Fu and the expression of stem markers CD44,CD133,Nanog,Sox2,Epcam,and Lgr5 were decreased;After overexpression of LINC01606,the proportion of CD44⁺CD133⁺cells,the tolerance to 5-Fu and the expression of stem markers CD44,CD133,Nanog,Sox2,Epcam and Lgr5 were increased in colon cancer cells.4)Ferroptosis inducers Erastin and RSL3 induced the typical morphological changes of ferroptotic cell death and significantly inhibited cell activity in colon cancer cells.Interference of LINC01606 could increase cell damage,decrease cell activity,increase intracellular concentrations of Fe²⁺,total iron,mitochondrial superoxide and lipid ROS,and reduce mitochondrial membrane potential.Overexpression of LINC01606 could reduce cell damage,increase cell activity,decrease intracellular concentrations of Fe²⁺,total iron,mitochondrial superoxide and lipid ROS,and increase mitochondrial membrane potential.5)LINC1606 was distributed in both cytoplasm and nucleus,and was highly expressed in cytoplasm.Luciferase assay indicated that miR-423-5p could directly regulate LINC01606 and SCD1,but not Wnt3a.RNA pull down and Ago2-RIP assays further verified that miR-423-5p could directly bind LINC01606 and SCD1.Interference of LINC01606 downregulated the expression of SCD1,overexpression of LINC01606 upregulated the expression of SCD1.Overexpression of miR-423-5p downregulated the expression of SCD1 and LINC01606.Interference of miR-423-5p upregulated the expression of SCD1 and LINC01606.6)Interference of LINC01606 or overexpression of miR-423-5p decreased the activity of Wnt/β-catenin signaling,while overexpression of LINC01606 or interference of miR-423-5p increased the activity of Wnt/β-catenin signaling.Topflash/Fopflash assay suggested that LINC01606 could regulate TCF/LEF transcriptional activity byβ-catenin-mediated regulation.The Wnt signaling inhibitor C59 could down-regulate the expression levels of LINC01606,SCD1 and Wnt/β-catenin signaling molecules.The Wnt signaling agonist BML-284increased the expression of LINC01606,SCD1 and Wnt/β-catenin signaling molecules.Interference of LINC01606 could inhibit the activation of Wnt signaling by BML-284,while overexpression of LINC01606 could alleviate the inhibition of Wnt signal by C59.7)LINC01606 promoter DNA pull down indicated that 116nucleoproteins were binding to LINC01606 promoter region,and the transcription factor TFE3 related to Wnt signal was screened out from it.The expression of TFE3 in colon cancer was positively correlated with that of LINC01606.Overexpression of TFE3 could significantly increase the expression level of LINC01606.Luciferase assay and chip-seq assay confirmed that TFE3 could act on the promoter of LINC01606.8)The Wnt signaling inhibitor C59 could increase RSL3-induced intracellular concentrations of Fe²⁺,total iron,mitochondrial superoxide and lipid ROS,and decrease mitochondrial membrane potential.The Wnt signaling agonist BML-284 could alleviate RSL3-induced the concentration of Fe²⁺,total iron,mitochondrial superoxide and lipid ROS,and increase mitochondrial membrane potential.Overexpression of LINC01606 could decrease the concentration of Fe²⁺,total iron,mitochondrial superoxide and lipid ROS induced by the Wnt signaling inhibitor C59,and the increase of mitochondrial membrane potential.In CD44⁺CD133⁺cells,the concentration of Erastin and RSL3-induced intracellular Fe²⁺,total iron,mitochondrial superoxide and lipid ROS were lower than those in CD44^-CD133^-cells,and mitochondrial membrane potential was higher than that in CD44^-CD133^-cells.9)After interference of LINC01606 in SW480 and HT29 cells,a total of 17 fatty acids were changed,among which 9 fatty acids were changed more than 1μg/10⁷,of which 7 fatty acids increased and 2 fatty acids decreased.Interference of LINC01606 could increase the concentration of saturated fatty acids and polyunsaturated fatty acids,and decrease the concentration of monounsaturated fatty acids.In addition,LINC01606 could increase the concentration of palmitic acid（C16:0）and stearic acid（C18:0）and decrease the concentration of palmitic acid（C16:1n7）and oleic acid（C18:1n9）,and increase the ratio of C16:0/C16:1n7 and C18:0/C18:1n9.Conclusion:The results of this study suggest that LINC01606 may inhibit ferroptosis in colon cancer cells through SCD1-Wnt/β-catenin-TFE3positive feedback signaling,thereby promoting the stemness of cancer cells and facilitating the development and progression of colon cancer.