LncRNA CCAT1 Affects Mitochondrial Function By Regulating Mitochondrial Genome Transcription In Colon Cancer Cells

Background:In recent years,there has been an alarming rise in the incidence of colon cancer in China,making it one of the leading types of digestive system tumors.Uncontrolled cell proliferation and metastasis are major obstacles that hinder effective clinical treatment and contribute to the high mortality rates in cancer patients.Therefore,it is crucial to explore key molecules in tumor progression to identify early tumor biomarkers and create new,effective therapeutic drugs.Mitochondria are important organelles within cells.Studies have indicated that impaired mitochondrial function may play a significant role in the initiation and progression of tumors.When the mitochondrial function is disrupted,it may hinder the production of intracellular ATP,consequently affecting the normal function and metabolism of cells.This could increase the chances of cell mutation and the development of cancer.In addition,the abnormal mitochondrial function could amplify oxidative stress,leading to DNA damage,gene mutation,and lessened apoptosis.These factors combined promote tumor growth and occurrence.Mitochondria play a crucial role in regulating cell apoptosis.Any disruption or damage to mitochondria can unbalance cell apoptosis and enable cancer cells to avoid programmed cell death,thus increasing the risk of tumorigenesis.Exploring the correlation between mitochondrial dysfunction and tumor development can enhance our understanding of the mechanisms behind tumorigenesis and pave the way for novel approaches to cancer prevention and treatment.Long non-coding RNA(lncRNA)plays an important regulatory role in cell biology,and its impact is particularly evident in mitochondrial function.Recent studies have revealed the intricate ways in which specific lncRNAs are involved in regulating mitochondrial function through multiple pathways.As part of our comparative analysis of RNA expression in the mitochondria of normal intestinal epithelial cells and colon cancer cells,our research group identified lncRNA CCAT1 as one of the most significantly differentially expressed lncRNAs.However,CCAT1 is a long non-coding RNA that originates from the nucleus.The role in regulating mitochondrial function,and the potential impact on colon cancer prognosis remain largely unknown and require further investigation.Objectives:1.This study aimed to clarify whether the nuclear-encoded lncRNA CCAT1 is enriched in the mitochondria of colon cancer and to investigate its effect on mitochondrial function.2.This study aimed to investigate the specific mechanism of how lncRNA CCAT1 affects mitochondrial function.3.This study aimed to clarify the effect of lncRNA CCAT1 on the biological function of colon cancer cells.Methods:1.Using RNA fluorescence in situ hybridization(FISH)and RT-q PCR to determine the enrichment of lncRNA CCAT1 in the mitochondria of colon cancer cells;2.Using shRNA technology to knock down CCAT1 in colon cancer cell lines;Designing and establishing the Lwa Cas13a-BN-MLS system to knock down CCAT1 in the mitochondria of colon cancer cells;3.Using ATP detection,Seahorse detection,Mito SOX fluorescent probe,TMRM fluorescent dye,and transmission electron microscopy techniques to assess the impact of CCAT1 on mitochondrial function;4.Employing the function detection kits of the respiratory chain complexes to explore the influence of CCAT1 on the functions of mitochondrial respiratory chain;5.The RT-qPCR method was used to investigate alterations in the copy number and transcripts of the mitochondrial genome following the knockdown of CCAT1;And using the WB method,the expression changes of mitochondrial genome encoded proteins were observed;6.The mitochondria genes combined with CCAT1 were detected in purified mitochondria by CHIRP assay,and the effect of CCAT1 on mitochondrial genome transcription was verified by Luciferase assay;7.Using the RNA-protein binding prediction website to find transcription factors that may interact with CCAT1;And verifing the combination of CCAT1 and TFAM through RNA chromosome precipitation(RIP)and RNA PULL DOWN techniques;8.Using the CLIP experiment to find the fragment of CCAT1 that combines with TFAM,and using the luciferase experiment to determine the functional site of CCAT1 that combines with TFAM;9.The effects of CCAT1 on the biological function of colon cancer cells were explored through CCK-8 experiment,transwell experiment,scratch experiment,clone formation experiment and flow cytometry.Results:1.This study found that the long non-coding RNA encoded by the nuclear genome,CCAT1,was highly enriched in the mitochondria of colon cancer cells;2.This study demonstrated that silencing CCAT1 significantly impacts mitochondrial functions.This was manifest in several ways,including changes in mitochondrial morphology,decreased ATP production,reduced ROS production,and alterations in mitochondrial membrane potential and reduced oxidative phosphorylation levels;3.After CCAT1 knockdown,the functions of mitochondrial respiratory chain complexes were impaired;4.After CCAT1 knockdown,the transcription level of mitochondrial DNA and the expression of protein encoded by mitochondrial genome decreased significantly;5.CCAT1 binding to the heavy strand promoter 1(HSP1)region of mitochondrial genome affected the methylation level of the promoter region and positively regulated the transcription of the mitochondrial genome;6.CCAT1 binding to the transcription factor TFAM through the 925-1040 base region positively regulated the transcription of the mitochondrial genome;7.CCAT1 positively regulated the proliferation ability of the colon cancer cell lines.Conclusions:1.The nuclear-encoded lncRNA CCAT1 is enriched in colon cancer mitochondria;2.CCAT1 regulates the function of mitochondrial oxidative phosphorylation,affects mitochondrial morphology,ATP synthesis,ROS generation,and maintenance of membrane potential;3.As a molecular backbone,CCAT1 binds to the mitochondrial heavy chain promoter and the transcription factor TFAM to regulate the transcription of the mitochondrial genome;4.CCAT1 in the mitochondrial affects the proliferation ability of colon cancer cells.